Ensemble docking to difficult targets in early-stage drug discovery: Methodology and application to fibroblast growth factor 23.

PubMed

Velazquez, Hector A; Riccardi, Demian; Xiao, Zhousheng; Quarles, Leigh Darryl; Yates, Charless Ryan; Baudry, Jerome; Smith, Jeremy C

2018-02-01

Ensemble docking is now commonly used in early-stage in silico drug discovery and can be used to attack difficult problems such as finding lead compounds which can disrupt protein-protein interactions. We give an example of this methodology here, as applied to fibroblast growth factor 23 (FGF23), a protein hormone that is responsible for regulating phosphate homeostasis. The first small-molecule antagonists of FGF23 were recently discovered by combining ensemble docking with extensive experimental target validation data (Science Signaling, 9, 2016, ra113). Here, we provide a detailed account of how ensemble-based high-throughput virtual screening was used to identify the antagonist compounds discovered in reference (Science Signaling, 9, 2016, ra113). Moreover, we perform further calculations, redocking those antagonist compounds identified in reference (Science Signaling, 9, 2016, ra113) that performed well on drug-likeness filters, to predict possible binding regions. These predicted binding modes are rescored with the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) approach to calculate the most likely binding site. Our findings suggest that the antagonist compounds antagonize FGF23 through the disruption of protein-protein interactions between FGF23 and fibroblast growth factor receptor (FGFR). © 2017 John Wiley & Sons A/S.

Antagonist-perturbation mechanism for activation function-2 fixed motifs: active conformation and docking mode of retinoid X receptor antagonists

NASA Astrophysics Data System (ADS)

Tsuji, Motonori

2017-06-01

HX531, which contains a dibenzodiazepine skeleton, is one of the first retinoid X receptor (RXR) antagonists. Functioning via RXR-PPARγ heterodimer, this compound is receiving a lot of attention as a therapeutic drug candidate for diabetic disease controlling differentiation of adipose tissue. However, the active conformation of HX531 for RXRs is not well established. In the present study, quantum mechanics calculations and molecular mechanical docking simulations were carried out to precisely study the docking mode of HX531 with the human RXRα ligand-binding domain, as well as to provide a new approach to drug design using a structure-based perspective. It was suggested that HX531, which has the R configuration for the bent dibenzodiazepine plane together with the equatorial configuration for the N-methyl group attached to the nitrogen atom in the seven-membered diazepine ring, is a typical activation function-2 (AF-2) fixed motif perturbation type antagonist, which destabilizes the formation of AF-2 fixed motifs. On the other hand, the docking simulations supported the experimental result that LG100754 is an RXR homodimer antagonist and an RXR heterodimer agonist.

Estrogen Receptor α L543A,L544A Mutation Changes Antagonists to Agonists, Correlating with the Ligand Binding Domain Dimerization Associated with DNA Binding Activity*

PubMed Central

Arao, Yukitomo; Hamilton, Katherine J.; Coons, Laurel A.; Korach, Kenneth S.

2013-01-01

A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα. PMID:23733188

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

PubMed

Yang, Zhenlin; Han, Shuo; Keller, Max; Kaiser, Anette; Bender, Brian J; Bosse, Mathias; Burkert, Kerstin; Kögler, Lisa M; Wifling, David; Bernhardt, Guenther; Plank, Nicole; Littmann, Timo; Schmidt, Peter; Yi, Cuiying; Li, Beibei; Ye, Sheng; Zhang, Rongguang; Xu, Bo; Larhammar, Dan; Stevens, Raymond C; Huster, Daniel; Meiler, Jens; Zhao, Qiang; Beck-Sickinger, Annette G; Buschauer, Armin; Wu, Beili

2018-04-01

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y 1 , Y 2 , Y 4 and Y 5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y 1 receptor (Y 1 R) 4 . A number of peptides and small-molecule compounds have been characterized as Y 1 R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y 1 R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y 1 R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y 1 R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y 1 R can enable structure-based drug discovery that targets NPY receptors.

Structure-based Understanding of Binding Affinity and Mode ...

EPA Pesticide Factsheets

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interactions and specific hydrogen bonds with the ligand. Here we present a framework for quantitative analysis of the steric and electronic features of the human ERα-ligand complex using three dimensional (3D) protein-ligand interaction description combined with 3D-QSAR approach. An empirical hydrophobicity density field is applied to account for hydrophobic contacts of ligand within the LBP. The obtained 3D-QSAR model revealed that hydrophobic contacts primarily determine binding affinity and govern binding mode with hydrogen bonds. Several residues of the LBP appear to be quite flexible and adopt a spectrum of conformations in various ERα-ligand complexes, in particular His524. The 3D-QSAR was combined with molecular docking based on three receptor conformations to accommodate receptor flexibility. The model indicates that the dynamic character of the LBP allows accommodation and stable binding of structurally diverse ligands, and proper representation of the protein flexibility is critical for reasonable description of binding of the ligands. Our results provide a quantitative and mechanistic understanding of binding affinity and mode of ERα agonists and antagonists that may be applicab

GPR17: molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors.

PubMed

Parravicini, Chiara; Ranghino, Graziella; Abbracchio, Maria P; Fantucci, Piercarlo

2008-06-04

GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor. Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.

A Cyclic Tetrapeptide (“Cyclodal”) and Its Mirror-Image Isomer Are Both High-Affinity μ Opioid Receptor Antagonists

PubMed Central

Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wood, JodiAnne; Ma, Xiaoyu; Guo, Jason; Wilkes, Brian C.; Ge, Yang; Laferrière, André; Coderre, Terence J.; Schiller, Peter W.

2016-01-01

Head-to-tail cyclization of the μ opioid receptor (MOR) agonist [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2 (9; Dmt = 2′,6′-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c[-d-Arg-Phe-Lys-Dmt-] (1) (“cyclodal”), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp127 and Glu229 receptor residues. Cyclodal showed high plasma stability and was able to cross the blood–brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c[-Arg-d-Phe-d-Lys-d-Dmt-] (2), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c[-d-Arg-Phe-LysΨ[CH2NH]Dmt-] (8), with MOR agonist activity. PMID:27676089

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor

PubMed Central

Bradley, ME; Bond, ME; Manini, J; Brown, Z; Charlton, SJ

2009-01-01

Background and purpose: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. Experimental approach: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [35S]-GTPγS binding approaches in addition to chemotaxis of human neutrophils. Key results: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [125I]-interleukin-8 ([125I]-IL-8) without affecting the Kd. In contrast, IL-8 was unable to prevent binding of [3H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene α, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [35S]-GTPγS binding in this preparation. Conclusions and implications: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling. PMID:19422399

Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands.

PubMed

Wysoczynski-Horita, Christina L; Boursier, Michelle E; Hill, Ryan; Hansen, Kirk; Blackwell, Helen E; Churchill, Mair E A

2018-05-01

Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems. © 2018 John Wiley & Sons Ltd.

Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information

NASA Astrophysics Data System (ADS)

Paoletta, Silvia; Sabbadin, Davide; von Kügelgen, Ivar; Hinz, Sonja; Katritch, Vsevolod; Hoffmann, Kristina; Abdelrahman, Aliaa; Straßburger, Jens; Baqi, Younis; Zhao, Qiang; Stevens, Raymond C.; Moro, Stefano; Müller, Christa E.; Jacobson, Kenneth A.

2015-08-01

The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

Structure-based virtual screening and characterization of a novel IL-6 antagonistic compound from synthetic compound database.

PubMed

Wang, Jing; Qiao, Chunxia; Xiao, He; Lin, Zhou; Li, Yan; Zhang, Jiyan; Shen, Beifen; Fu, Tinghuan; Feng, Jiannan

2016-01-01

According to the three-dimensional (3D) complex structure of (hIL-6⋅hIL-6R⋅gp 130) 2 and the binding orientation of hIL-6, three compounds with high affinity to hIL-6R and bioactivity to block hIL-6 in vitro were screened theoretically from the chemical databases, including 3D-Available Chemicals Directory (ACD) and MDL Drug Data Report (MDDR), by means of the computer-guided virtual screening method. Using distance geometry, molecular modeling and molecular dynamics trajectory analysis methods, the binding mode and binding energy of the three compounds were evaluated theoretically. Enzyme-linked immunosorbent assay analysis demonstrated that all the three compounds could block IL-6 binding to IL-6R specifically. However, only compound 1 could effectively antagonize the function of hIL-6 and inhibit the proliferation of XG-7 cells in a dose-dependent manner, whereas it showed no cytotoxicity to SP2/0 or L929 cells. These data demonstrated that the compound 1 could be a promising candidate of hIL-6 antagonist.

Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

PubMed

Journigan, V Blair; Polgar, Willma E; Tuan, Edward W; Lu, James; Daga, Pankaj R; Zaveri, Nurulain T

2017-10-16

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

Application of binding free energy calculations to prediction of binding modes and affinities of MDM2 and MDMX inhibitors.

PubMed

Lee, Hui Sun; Jo, Sunhwan; Lim, Hyun-Suk; Im, Wonpil

2012-07-23

Molecular docking is widely used to obtain binding modes and binding affinities of a molecule to a given target protein. Despite considerable efforts, however, prediction of both properties by docking remains challenging mainly due to protein's structural flexibility and inaccuracy of scoring functions. Here, an integrated approach has been developed to improve the accuracy of binding mode and affinity prediction and tested for small molecule MDM2 and MDMX antagonists. In this approach, initial candidate models selected from docking are subjected to equilibration MD simulations to further filter the models. Free energy perturbation molecular dynamics (FEP/MD) simulations are then applied to the filtered ligand models to enhance the ability in predicting the near-native ligand conformation. The calculated binding free energies for MDM2 complexes are overestimated compared to experimental measurements mainly due to the difficulties in sampling highly flexible apo-MDM2. Nonetheless, the FEP/MD binding free energy calculations are more promising for discriminating binders from nonbinders than docking scores. In particular, the comparison between the MDM2 and MDMX results suggests that apo-MDMX has lower flexibility than apo-MDM2. In addition, the FEP/MD calculations provide detailed information on the different energetic contributions to ligand binding, leading to a better understanding of the sensitivity and specificity of protein-ligand interactions.

Discovery of a Series of Indazole TRPA1 Antagonists

PubMed Central

2017-01-01

A series of TRPA1 antagonists is described which has as its core structure an indazole moiety. The physical properties and in vitro DMPK profiles are discussed. Good in vivo exposure was obtained with several analogs, allowing efficacy to be assessed in rodent models of inflammatory pain. Two compounds showed significant activity in these models when administered either systemically or topically. Protein chimeras were constructed to indicate compounds from the series bound in the S5 region of the channel, and a computational docking model was used to propose a binding mode for example compounds. PMID:28626530

The 3D Structure of the Binding Pocket of the Human Oxytocin Receptor for Benzoxazine Antagonists, Determined by Molecular Docking, Scoring Functions and 3D-QSAR Methods

NASA Astrophysics Data System (ADS)

Jójárt, Balázs; Martinek, Tamás A.; Márki, Árpád

2005-05-01

Molecular docking and 3D-QSAR studies were performed to determine the binding mode for a series of benzoxazine oxytocin antagonists taken from the literature. Structural hypotheses were generated by docking the most active molecule to the rigid receptor by means of AutoDock 3.05. The cluster analysis yielded seven possible binding conformations. These structures were refined by using constrained simulated annealing, and the further ligands were aligned in the refined receptor by molecular docking. A good correlation was found between the estimated Δ G bind and the p K i values for complex F. The Connolly-surface analysis, CoMFA and CoMSIA models q CoMFA 2 = 0.653, q CoMSA 2 = 0.630 and r pred,CoMFA 2 = 0.852 , r pred,CoMSIA 2 = 0.815) confirmed the scoring function results. The structural features of the receptor-ligand complex and the CoMFA and CoMSIA fields are in closely connected. These results suggest that receptor-ligand complex F is the most likely binding hypothesis for the studied benzoxazine analogs.

Substituted 2-Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins.

PubMed

Kaczanowska, Katarzyna; Camacho Hernandez, Gisela Andrea; Bendiks, Larissa; Kohs, Larissa; Cornejo-Bravo, Jose Manuel; Harel, Michal; Finn, M G; Taylor, Palmer

2017-03-15

Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4β2-nAChR, and a serotonin receptor (5-HT 3A R), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 μM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC 50 values of 70 nM and K d values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4β2-nAChR or 5-HT3AR at concentrations up to 10 μM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.

Molecular docking, molecular modeling, and molecular dynamics studies of azaisoflavone as dual COX-2 inhibitors and TP receptor antagonists.

PubMed

Hadianawala, Murtuza; Mahapatra, Amarjyoti Das; Yadav, Jitender K; Datta, Bhaskar

2018-02-26

Designed multi-target ligand (DML) is an emerging strategy for the development of new drugs and involves the engagement of multiple targets with the same moiety. In the context of NSAIDs it has been suggested that targeting the thromboxane prostanoid (TP) receptor along with cyclooxygenase-2 (COX-2) may help to overcome cardiovascular (CVS) complications associated with COXIBs. In the present work, azaisoflavones were studied for their COX-2 and TP receptor binding activities using structure based drug design (SBDD) techniques. Flavonoids were selected as a starting point based on their known COX-2 inhibitory and TP receptor antagonist activity. Iterative design and docking studies resulted in the evolution of a new class scaffold replacing the benzopyran-4-one ring of flavonoids with quinolin-4-one. The docking and binding parameters of these new compounds are found to be promising in comparison to those of selective COX-2 inhibitors, such as SC-558 and celecoxib. Owing to the lack of structural information, a model for the TP receptor was generated using a threading base alignment method with loop optimization performed using an ab initio method. The model generated was validated against known antagonists for TP receptor using docking/MMGBSA. Finally, the molecules that were designed for selective COX-2 inhibition were docked into the active site of the TP receptor. Iterative structural modifications and docking on these molecules generated a series which displays optimum docking scores and binding interaction for both targets. Molecular dynamics studies on a known TP receptor antagonist and a designed molecule show that both molecules remain in contact with protein throughout the simulation and interact in similar binding modes. Graphical abstract ᅟ.

Antagonism of human CC-chemokine receptor 4 can be achieved through three distinct binding sites on the receptor

PubMed Central

Slack, Robert J; Russell, Linda J; Barton, Nick P; Weston, Cathryn; Nalesso, Giovanna; Thompson, Sally-Anne; Allen, Morven; Chen, Yu Hua; Barnes, Ashley; Hodgson, Simon T; Hall, David A

2013-01-01

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low-molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17- and CCL22-induced responses of human CCR4+ T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration-ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low-molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4. PMID:25505571

``In silico'' study of the binding of two novel antagonists to the nociceptin receptor

NASA Astrophysics Data System (ADS)

Della Longa, Stefano; Arcovito, Alessandro

2018-02-01

Antagonists of the nociceptin receptor (NOP) are raising interest for their possible clinical use as antidepressant drugs. Recently, the structure of NOP in complex with some piperidine-based antagonists has been revealed by X-ray crystallography. In this study, a multi-flexible docking (MF-docking) procedure, i.e. docking to multiple receptor conformations extracted by preliminary molecular dynamics trajectories, together with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations have been carried out to provide the binding mode of two novel NOP antagonists, one of them selective (BTRX-246040, formerly named LY-2940094) and one non selective (AT-076), i.e. able to inactivate NOP as well as the classical µ- k- and δ-opioid receptors (MOP KOP and DOP). According to our results, the pivotal role of residue D1303,32 (upper indexes are Ballesteros-Weinstein notations) is analogous to that enlighten by the already known X-ray structures of opioid receptors: binding of the molecules are predicted to require a slight readjustment of the hydrophobic pocket (residues Y1313,33, M1343,36, I2195,43, Q2806,52 and V2836,55) in the orthosteric site of NOP, accommodating either the pyridine-pyrazole (BTRX-246040) or the isoquinoline (AT-076) moiety of the ligand, in turn allowing the protonated piperidine nitrogen to maximize interaction (salt-bridge) with residue D1303,32 of the NOP, and the aromatic head to be sandwiched in optimal π-stacking between Y1313,33 and M1343,36. The QM/MM optimization after the MF-docking procedure has provided the more likely conformations for the binding to the NOP receptor of BTRX-246040 and AT-076, based on different pharmacophores and exhibiting different selectivity profiles. While the high selectivity for NOP of BTRX-246040 can be explained by interactions with NOP specific residues, the lack of selectivity of AT-076 could be associated to its ability to penetrate into the deep hydrophobic pocket of NOP, while retaining a conformation very similar to the one assumed by the antagonist JDTic into the K-opioid receptor. The proposed binding geometries fit better the binding pocket environment providing clues for experimental studies aimed to design selective or multifunctional opioid drugs.

Minireview: Mode of action of meta-diamide insecticides.

PubMed

Nakao, Toshifumi; Banba, Shinichi

2015-06-01

Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor noncompetitive antagonists showing high insecticidal activity against Spodoptera litura. The mode of action of the meta-diamides was demonstrated to be distinct from that of conventional noncompetitive antagonists (NCAs) such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. It was suggested that meta-diamides act at or near G336 in the M3 region of the Drosophila RDL GABA receptor. Although the site of action of the meta-diamides appears to overlap with that of macrocyclic lactones including avermectins and milbemycins, differential effects of mutations on the actions of the meta-diamides and the macrocyclic lactones were observed. Molecular modeling studies revealed that the meta-diamides may bind to an inter-subunit pocket near G336 in the Drosophila RDL GABA receptor better when in the closed state, which is distinct from the NCA-binding site, which is in a channel formed by M2s. In contrast, the macrocyclic lactones were suggested to bind to an inter-subunit pocket near G336 in the Drosophila RDL GABA receptor when in the open state. Furthermore, mechanisms underlying the high selectivity of meta-diamides are discussed. This minireview highlights the unique features of novel meta-diamide insecticides and demonstrates why meta-diamides are anticipated to become prominent insecticides that are effective against pests resistant to cyclodienes and fipronil. Copyright © 2014 Elsevier Inc. All rights reserved.

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Yi; Qin, Ling; Zacarías, Natalia V. Ortiz

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, heremore » we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.« less

Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors

PubMed Central

Huang, Xi-Ping; Nagy, Peter I; Williams, Frederick E; Peseckis, Steven M; Messer, William S

1999-01-01

Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala.HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors.The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors.Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor. PMID:10188986

Structure of CC Chemokine Receptor 2 with Orthosteric and Allosteric Antagonists

PubMed Central

Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel, Tracy M.

2016-01-01

Summary CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human Class A G protein-coupled receptors (GPCRs). CCR2 is expressed on monocytes, immature dendritic cells and T cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL21. CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see ClinicalTrials.gov) in search of therapies that target the CCR2:chemokine axis. To aid drug discovery efforts5, we solved a structure of CCR2 in a ternary complex with an orthosteric (BMS-6816) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in Class A GPCRs to date; this site spatially overlaps the G protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive GPCR structures solved to date. Like other protein:protein interactions, receptor:chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome drug design obstacles. PMID:27926736

Broflanilide: A meta-diamide insecticide with a novel mode of action.

PubMed

Nakao, Toshifumi; Banba, Shinichi

2016-02-01

Broflanilide is a meta-diamide [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamide] that exhibits high larvicidal activity against Spodoptera litura. It has been suggested that broflanilide is metabolized to desmethyl-broflanilide and that it acts as a noncompetitive resistant-to-dieldrin (RDL) γ-aminobutyric acid (GABA) receptor antagonist. The binding site of desmethyl-broflanilide was demonstrated to be distinct from that of conventional noncompetitive antagonists such as fipronil. It has been proposed that the site of action for desmethyl-broflanilide is close to G336 in the M3 region of the Drosophila RDL GABA receptor. However, although the site of action for desmethyl-broflanilide appears to overlap with that of macrocyclic lactones, different modes of actions have been demonstrated for desmethyl-broflanilide and the macrocyclic lactones. The mechanisms underlying the high selectivity of meta-diamides are also discussed in this review. Broflanilide is expected to become a prominent insecticide because it is effective against pests with resistance to cyclodienes and fipronil. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

PubMed

Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

2017-11-22

We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

Highly Increased 125I-JR11 Antagonist Binding In Vitro Reveals Novel Indications for sst2 Targeting in Human Cancers.

PubMed

Reubi, Jean Claude; Waser, Beatrice; Mäcke, Helmut; Rivier, Jean

2017-02-01

There is recent in vitro and in vivo evidence that somatostatin receptor subtype 2 (sst 2 ) antagonists are better tools to target neuroendocrine tumors (NETs) than sst 2 agonists. Indeed, antagonists bind to a greater number of sst 2 sites than agonists. Whether sst 2 antagonists could be used successfully to target non-NETs, expressing low sst 2 density, is unknown. Here, we compare quantitatively 125 I-JR11 sst 2 antagonist binding in vitro with that of the sst 2 agonist 125 I-Tyr 3 -octreotide in large varieties of non-NET and NET. In vitro receptor autoradiography was performed with 125 I-JR11 and 125 I-Tyr 3 -octreotide in cancers from prostate, breast, colon, kidney, thyroid, and lymphoid tissues as well as NETs as reference. In general, 125 I-JR11 binds to many more sst 2 sites than 125 I-Tyr 3 -octreotide. In 13 breast cancers, 8 had a low binding (mean density, 844 ± 168 dpm/mg of tissue) with the agonist whereas 12 had a high binding (mean density, 4,447 ± 1,128 dpm/mg of tissue) with the antagonist. All 12 renal cell cancers showed a low binding of sst 2 with the agonist (mean density, 348 ± 49 dpm/mg of tissue) whereas all cases had a high sst 2 binding with the antagonist (mean density, 3,777 ± 582 dpm/mg of tissue). One of 5 medullary thyroid cancers was positive with the agonist, whereas 5 of 5 were positive with the antagonist. In 15 non-Hodgkin lymphomas, many more sst 2 sites were labeled with the antagonist than with the agonist. In 14 prostate cancers, none had sst 2 binding with the agonist and only 4 had a weak binding with the antagonist. None of 17 colon cancers showed sst 2 sites with the agonist, and only 3 cases were weakly positive with the antagonist. In the various tumor types, adjacent sst 2 -expressing tissues such as vessels, lymphocytes, nerves, mucosa, or stroma were more strongly labeled with the antagonist than with the agonist. The reference NET cases, incubated with a smaller amount of tracer, were also found to have many more sst 2 sites measured with the antagonist. All renal cell cancers and most breast cancers, non-Hodgkin lymphomas, and medullary thyroid cancers represent novel indications for the in vivo radiopeptide targeting of sst 2 by sst 2 antagonists, comparable to NET radiotargeting with sst 2 agonists. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Profiling the interaction mechanism of quinoline/quinazoline derivatives as MCHR1 antagonists: an in silico method.

PubMed

Wu, Mingwei; Li, Yan; Fu, Xinmei; Wang, Jinghui; Zhang, Shuwei; Yang, Ling

2014-09-01

Melanin concentrating hormone receptor 1 (MCHR1), a crucial regulator of energy homeostasis involved in the control of feeding and energy metabolism, is a promising target for treatment of obesity. In the present work, the up-to-date largest set of 181 quinoline/quinazoline derivatives as MCHR1 antagonists was subjected to both ligand- and receptor-based three-dimensional quantitative structure-activity (3D-QSAR) analysis applying comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The optimal predictable CoMSIA model exhibited significant validity with the cross-validated correlation coefficient (Q²) = 0.509, non-cross-validated correlation coefficient (R²(ncv)) = 0.841 and the predicted correlation coefficient (R²(pred)) = 0.745. In addition, docking studies and molecular dynamics (MD) simulations were carried out for further elucidation of the binding modes of MCHR1 antagonists. MD simulations in both water and lipid bilayer systems were performed. We hope that the obtained models and information may help to provide an insight into the interaction mechanism of MCHR1 antagonists and facilitate the design and optimization of novel antagonists as anti-obesity agents.

A novel BLyS antagonist peptide designed based on the 3-D complex structure of BCMA and BLyS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Jian; Feng Jiannan; Li Yan

2006-08-11

B lymphocyte stimulator (BLyS) is a member of tumor necrosis factor (TNF) family. Because of its roles in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren syndrome (SS), BLyS antagonists have been tested to treat SLE- and RA-like symptoms in mice and obtained optimistic results. So far, reported BLyS antagonists were mostly decoyed BLyS receptors or anti-BLyS antibodies. In this study, a novel BLyS antagonist peptide, PT, was designed based on the modeling 3-D complex structure of BCMA and BLyS. The interaction mode of PT with BLyS was analyzed theoretically. The results of competitive ELISAmore » demonstrated that PT could inhibit the binding of BCMA-Fc and anti-BLyS antibody to BLyS in vitro. In addition, PT could partly block the proliferating activity of BLyS on mice splenocytes. The BLyS antagonizing activity of PT was significant (p < 0.05). This study highlights the possibility of using BLyS antagonist peptide to neutralize BLyS activity. Further optimization of PT with computer-guided molecular design method to enhance its biopotency may be useful in developing new BLyS antagonists to treat BLyS-related autoimmune diseases.« less

Integrated Model of Chemical Perturbations of a Biological ...

EPA Pesticide Factsheets

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“”assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 52 (2.8%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in v

Identification of a Potent Tryptophan-based TRPM8 Antagonist With in vivo Analgesic Activity.

PubMed

Bertamino, Alessia; Iraci, Nunzio; Ostacolo, Carmine; Ambrosino, Paolo; Musella, Simona; Di Sarno, Veronica; Ciaglia, Tania; Pepe, Giacomo; Sala, Marina; Soldovieri, Maria Virginia; Mosca, Ilaria; Gonzalez-Rodriguez, Sara; Fernández-Carvajal, Asia; Ferrer-Montiel, Antonio; Novellino, Ettore; Taglialatela, Maurizio; Campiglia, Pietro; Gomez-Monterrey, Isabel M

2018-06-25

TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N,N'-dibenzyl tryptamine 1, a potent TRPM8 antagonist, were prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably-transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC 50 0.2±0.2 nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an icilin-induced WDS (at 1-30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1-1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1-4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain.

Contributions of different modes of TRPV1 activation to TRPV1 antagonist-induced hyperthermia.

PubMed

Garami, Andras; Shimansky, Yury P; Pakai, Eszter; Oliveira, Daniela L; Gavva, Narender R; Romanovsky, Andrej A

2010-01-27

Transient receptor potential vanilloid-1 (TRPV1) antagonists are widely viewed as next-generation pain therapeutics. However, these compounds cause hyperthermia, a serious side effect. TRPV1 antagonists differentially block three modes of TRPV1 activation: by heat, protons, and chemical ligands (e.g., capsaicin). We asked what combination of potencies in these three modes of TRPV1 activation corresponds to the lowest potency of a TRPV1 antagonist to cause hyperthermia. We studied hyperthermic responses of rats, mice, and guinea pigs to eight TRPV1 antagonists with different pharmacological profiles and used mathematical modeling to find a relative contribution of the blockade of each activation mode to the development of hyperthermia. We found that the hyperthermic effect has the highest sensitivity to the extent of TRPV1 blockade in the proton mode (0.43 to 0.65) with no to moderate sensitivity in the capsaicin mode (-0.01 to 0.34) and no sensitivity in the heat mode (0.00 to 0.01). We conclude that hyperthermia-free TRPV1 antagonists do not block TRPV1 activation by protons, even if they are potent blockers of the heat mode, and that decreasing the potency to block the capsaicin mode may further decrease the potency to cause hyperthermia.

Contributions of different modes of TRPV1 activation to TRPV1 antagonist-induced hyperthermia

PubMed Central

Garami, Andras; Shimansky, Yury P.; Pakai, Eszter; Oliveira, Daniela L.; Gavva, Narender R.; Romanovsky, Andrej A.

2010-01-01

Transient receptor potential vanilloid-1 (TRPV1) antagonists are widely viewed as next-generation pain therapeutics. However, these compounds cause hyperthermia, a serious side effect. TRPV1 antagonists differentially block three modes of TRPV1 activation: by heat, protons, and chemical ligands (e.g., capsaicin). We asked what combination of potencies in these three modes of TRPV1 activation corresponds to the lowest potency of a TRPV1 antagonist to cause hyperthermia. We studied hyperthermic responses of rats, mice, and guinea pigs to eight TRPV1 antagonists with different pharmacological profiles and used mathematical modeling to find a relative contribution of the blockade of each activation mode to the development of hyperthermia. We have found that the hyperthermic effect has the highest sensitivity to the extent of TRPV1 blockade in the proton mode (0.43 to 0.65) with no to moderate sensitivity in the capsaicin mode (-0.01 to 0.34) and no sensitivity in the heat mode (0.00 to 0.01). We conclude that hyperthermia-free TRPV1 antagonists do not block TRPV1 activation by protons, even if they are potent blockers of the heat mode, and that decreasing the potency to block the capsaicin mode may further decrease the potency to cause hyperthermia. PMID:20107070

Structural Changes Due to Antagonist Binding in Ligand Binding Pocket of Androgen Receptor Elucidated Through Molecular Dynamics Simulations.

PubMed

Sakkiah, Sugunadevi; Kusko, Rebecca; Pan, Bohu; Guo, Wenjing; Ge, Weigong; Tong, Weida; Hong, Huixiao

2018-01-01

When a small molecule binds to the androgen receptor (AR), a conformational change can occur which impacts subsequent binding of co-regulator proteins and DNA. In order to accurately study this mechanism, the scientific community needs a crystal structure of the Wild type AR (WT-AR) ligand binding domain, bound with antagonist. To address this open need, we leveraged molecular docking and molecular dynamics (MD) simulations to construct a structure of the WT-AR ligand binding domain bound with antagonist bicalutamide. The structure of mutant AR (Mut-AR) bound with this same antagonist informed this study. After molecular docking analysis pinpointed the suitable binding orientation of a ligand in AR, the model was further optimized through 1 μs of MD simulations. Using this approach, three molecular systems were studied: (1) WT-AR bound with agonist R1881, (2) WT-AR bound with antagonist bicalutamide, and (3) Mut-AR bound with bicalutamide. Our structures were very similar to the experimentally determined structures of both WT-AR with R1881 and Mut-AR with bicalutamide, demonstrating the trustworthiness of this approach. In our model, when WT-AR is bound with bicalutamide, Val716/Lys720/Gln733, or Met734/Gln738/Glu897 move and thus disturb the positive and negative charge clumps of the AF2 site. This disruption of the AF2 site is key for understanding the impact of antagonist binding on subsequent co-regulator binding. In conclusion, the antagonist induced structural changes in WT-AR detailed in this study will enable further AR research and will facilitate AR targeting drug discovery.

Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

PubMed

Qin, M; Oie, S

1994-11-01

The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

Conserved Binding Mode of Human [beta subscript 2] Adrenergic Receptor Inverse Agonists and Antagonist Revealed by X-ray Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wacker, Daniel; Fenalti, Gustavo; Brown, Monica A.

2010-11-15

G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the {beta}{sub 2} adrenergic receptor ({beta}{sub 2}AR) is one of the most extensively studied. Previously, the X-ray crystal structure of {beta}{sub 2}AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered {beta}{sub 2}AR construct in complex with two inverse agonists: ICI 118,551 (2.8 {angstrom}),more » a recently described compound (2.8 {angstrom}) (Kolb et al, 2009), and the antagonist alprenolol (3.1 {angstrom}). The structures show the same overall fold observed for the previous {beta}{sub 2}AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with {beta}{sub 2}AR. Furthermore, receptor ligand cross-docking experiments revealed that a single {beta}{sub 2}AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.« less

Structural Modifications to Tetrahydropyridine-3-Carboxylate Esters en route to the Discovery of M5-Preferring Muscarinic Receptor Orthosteric Antagonists

PubMed Central

Zheng, Guangrong; Smith, Andrew M.; Huang, Xiaoqin; Subramanian, Karunai L.; Siripurapu, Kiran B.; Deaciuc, Agripina; Zhan, Chang-Guo; Dwoskin, Linda P.

2013-01-01

The M5 muscarinic acetylcholine receptor is suggested to be a potential pharmacotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series of M5-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3-carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold selectivity for the M5 over M1 receptor, and shows little activity at M2–M4. This compound, although exhibiting modest affinity (Ki = 2.24 μM) for the [3H]N-methylscopolamine binding site on the M5 receptor, is potent (IC50 = 0.45 nM) in inhibiting oxotremorine-evoked [3H]DA release from rat striatal slices. Further, a homology model of human M5 receptor based on the crystal structure of the rat M3 receptor was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding mode of these novel analogues to the receptor. PMID:23379472

Structure-Activity Relationship Study of Ionotropic Glutamate Receptor Antagonist (2S,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid.

PubMed

Krogsgaard-Larsen, Niels; Storgaard, Morten; Møller, Charlotte; Demmer, Charles S; Hansen, Jeanette; Han, Liwei; Monrad, Rune N; Nielsen, Birgitte; Tapken, Daniel; Pickering, Darryl S; Kastrup, Jette S; Frydenvang, Karla; Bunch, Lennart

2015-08-13

Herein we describe the first structure-activity relationship study of the broad-range iGluR antagonist (2S,3R)-3-(3-carboxyphenyl)pyrrolidine-2-carboxylic acid (1) by exploring the pharmacological effect of substituents in the 4, 4', or 5' positions and the bioisosteric substitution of the distal carboxylic acid for a phosphonic acid moiety. Of particular interest is a hydroxyl group in the 4' position 2a which induced a preference in binding affinity for homomeric GluK3 over GluK1 (Ki = 0.87 and 4.8 μM, respectively). Two X-ray structures of ligand binding domains were obtained: 2e in GluA2-LBD and 2f in GluK1-LBD, both at 1.9 Å resolution. Compound 2e induces a D1-D2 domain opening in GluA2-LBD of 17.3-18.8° and 2f a domain opening in GluK1-LBD of 17.0-17.5° relative to the structures with glutamate. The pyrrolidine-2-carboxylate moiety of 2e and 2f shows a similar binding mode as kainate. The 3-carboxyphenyl ring of 2e and 2f forms contacts comparable to those of the distal carboxylate in kainate.

Search for β2 Adrenergic Receptor Ligands by Virtual Screening via Grid Computing and Investigation of Binding Modes by Docking and Molecular Dynamics Simulations

PubMed Central

Bai, Qifeng; Shao, Yonghua; Pan, Dabo; Zhang, Yang; Liu, Huanxiang; Yao, Xiaojun

2014-01-01

We designed a program called MolGridCal that can be used to screen small molecule database in grid computing on basis of JPPF grid environment. Based on MolGridCal program, we proposed an integrated strategy for virtual screening and binding mode investigation by combining molecular docking, molecular dynamics (MD) simulations and free energy calculations. To test the effectiveness of MolGridCal, we screened potential ligands for β2 adrenergic receptor (β2AR) from a database containing 50,000 small molecules. MolGridCal can not only send tasks to the grid server automatically, but also can distribute tasks using the screensaver function. As for the results of virtual screening, the known agonist BI-167107 of β2AR is ranked among the top 2% of the screened candidates, indicating MolGridCal program can give reasonable results. To further study the binding mode and refine the results of MolGridCal, more accurate docking and scoring methods are used to estimate the binding affinity for the top three molecules (agonist BI-167107, neutral antagonist alprenolol and inverse agonist ICI 118,551). The results indicate agonist BI-167107 has the best binding affinity. MD simulation and free energy calculation are employed to investigate the dynamic interaction mechanism between the ligands and β2AR. The results show that the agonist BI-167107 also has the lowest binding free energy. This study can provide a new way to perform virtual screening effectively through integrating molecular docking based on grid computing, MD simulations and free energy calculations. The source codes of MolGridCal are freely available at http://molgridcal.codeplex.com. PMID:25229694

Synthesis and biological evaluation of guanylhydrazone coactivator binding inhibitors for the estrogen receptor.

PubMed

LaFrate, Andrew L; Gunther, Jillian R; Carlson, Kathryn E; Katzenellenbogen, John A

2008-12-01

Most patients with hormone-responsive breast cancer eventually develop resistance to traditional antiestrogens such as tamoxifen, and this has become a major obstacle in their treatment. We prepared and characterized the activity of a series of 16 guanylhydrazone small molecules that are designed to block estrogen receptor (ER) activity through a non-traditional mechanism, by directly interfering with coactivator binding to agonist-liganded ER. The inhibitory activity of these compounds was determined in cell-based transcription assays using ER-responsive reporter gene and mammalian two-hybrid assays. Several of the compounds gave IC(50) values in the low micromolar range. Two secondary assays were used to confirm that these compounds were acting through the proposed non-traditional mode of estrogen inhibitory action and not as conventional antagonists at the ligand binding site.

Sodium ion modulates D2 receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina

PubMed Central

Makman, Maynard H.; Dvorkin, B.; Klein, Patrice N.

1982-01-01

Sodium ion (Na+) influences binding of both dopamine agonists and antagonists to D2 receptors in striatum and retina. Also, Na+ markedly potentiates the loss of high-affinity agonist binding due to the GTP analogue p[NH]ppG. 2-Amino-6, 7-dihydroxy-1,2,3,4-tetrahydro[5,8-3H]naphthalene ([3H]ADTN) binds exclusively to an agonist conformation of D2 receptor in both striatum and retina, distinct from the antagonist conformation labeled by [3H]spiroperidol or [3H]domperidone in striatum or by [3H]spiroperidol in retina. Na+ is not required for interaction of [3H]ADTN or antagonist radioligand sites with the selective D2 agonist LY-141865, the D2 antagonist domperidone, or nonselective dopamine agonists or antagonists; however, Na+ is necessary for high affinity interaction of those radioligand sites with the D2 antagonists molindone and metoclopramide. With Na+ present, striatal sites for [3H]ADTN, [3H]spiroperidol, and [3H]domperidone have similar affinities for antagonists but only [3H]ADTN sites have high affinity for agonists. Na+ further decreases the low affinity of dopamine agonists for [3H]spiroperidol binding sites. Also, Na+ enhances [3H]spiroperidol and decreases [3H]ADTN binding. Na+ alone causes bound [3H]ADTN to dissociate from at least 30% of striatal and 50% of retinal sites, and with Na+ present [3H]ADTN rapidly dissociates from the remaining sites upon addition of p[NH]ppG. It is proposed that D2 receptors in striatum and retina exist in distinct but interconvertible conformational states, with different properties depending on the presence or absence of Na+ and of guanine nucleotide. PMID:6213964

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

PubMed

Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A

2016-04-15

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of a D-amino acid decapeptide HIV-1 entry inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boggiano, Cesar; Jiang Shibo; Lu Hong

2006-09-08

Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide DC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While DC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonalmore » antibody and chemokine SDF-1{alpha} to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of DC13 implies additional mode(s) of action. These results suggest that DC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors.« less

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

PubMed Central

Ajram, Laura; Begg, Malcolm; Slack, Robert; Cryan, Jenni; Hall, David; Hodgson, Simon; Ford, Alison; Barnes, Ashley; Swieboda, Dawid; Mousnier, Aurelie; Solari, Roberto

2014-01-01

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target. PMID:24534492

SSTR-Mediated Imaging in Breast Cancer: Is There a Role for Radiolabeled Somatostatin Receptor Antagonists?

PubMed

Dalm, Simone U; Haeck, Joost; Doeswijk, Gabriela N; de Blois, Erik; de Jong, Marion; van Deurzen, Carolien H M

2017-10-01

Recent studies have shown enhanced tumor targeting by novel somatostatin receptor (SSTR) antagonists compared with clinically widely used agonists. However, these results have been obtained mostly in neuroendocrine tumors, and only limited data are available for cancer types with lower SSTR expression, including breast cancer (BC). To date, two studies have reported higher binding of the antagonist than the agonist in BC, but in both studies only a limited number of cases were evaluated. In this preclinical study, we further investigated whether the application of an SSTR antagonist can improve SSTR-mediated BC imaging in a large panel of BC specimens. We also generated an in vivo BC mouse model and performed SPECT/MRI and biodistribution studies. Methods: Binding of 111 In-DOTA-Tyr 3 -octreotate (SSTR agonist) and 111 In-DOTA-JR11 (SSTR antagonist) to 40 human BC specimens was compared using in vitro autoradiography. SSTR2 immunostaining was performed to confirm SSTR2 expression of the tumor cells. Furthermore, binding of the radiolabeled SSTR agonist and antagonist was analyzed in tissue material from 6 patient-derived xenografts. One patient-derived xenograft, the estrogen receptor-positive model T126, was chosen to generate in vivo mouse models containing orthotopic breast tumors for in vivo SPECT/MRI and biodistribution studies after injection with 177 Lu-DOTA-Tyr 3 -octreotate or 177 Lu-DOTA-JR11. Results: 111 In-DOTA-JR11 binding to human BC tissue was significantly higher than 111 In-DOTA-Tyr 3 -octreotate binding ( P < 0.001). The median ratio of antagonist binding versus agonist binding was 3.39 (interquartile range, 2-5). SSTR2 immunostaining confirmed SSTR2 expression on the tumor cells. SPECT/MRI of the mouse model found better tumor visualization with the antagonist. This result was in line with the significantly higher tumor uptake of the radiolabeled antagonist than of the agonist as measured in biodistribution studies 285 min after radiotracer injection (percentage injected dose per gram of tissue: 1.92 ± 0.43 vs. 0.90 ± 0.17; P = 0.002). Conclusion: SSTR antagonists are promising candidates for BC imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Selective mode of action of guanidine-containing non-peptides at human NPFF receptors.

PubMed

Findeisen, Maria; Würker, Cäcilia; Rathmann, Daniel; Meier, René; Meiler, Jens; Olsson, Roger; Beck-Sickinger, Annette G

2012-07-12

The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By use of four nonpeptidic compounds and the peptide mimetics RF9 and BIBP3226, agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated. The binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF(1) but not in the NPFF(2) receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system.

Selective mode of action of guanidine-containing non-peptides at human NPFF receptors

PubMed Central

Findeisen, Maria; Würker, Cäcilia; Rathmann, Daniel; Meier, René; Meiler, Jens; Olsson, Roger; Beck-Sickinger, Annette G.

2012-01-01

The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By using four non-peptidic compounds and the peptide mimetics RF9 and BIBP3226 agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated, the binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII, and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF1 but not in the NPFF2 receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system. PMID:22708927

Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

PubMed

Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

2016-06-01

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABAA-ρ1 Receptors

PubMed Central

Xie, An; Yan, Jun; Yue, Lan; Feng, Feng; Mir, Fozia; Abdel-Halim, Heba; Chebib, Mary; Le Breton, Guy C.; Standaert, Robert F.; Qian, Haohua

2011-01-01

2-Aminoethyl methylphosphonate (2-AEMP), an analog of GABA, has been found to exhibit antagonist activity at GABAA-ρ1 (also known as ρ1 GABAC) receptors. The present study was undertaken to elucidate 2-AEMP's action and to test the activities of 2-AEMP analogs. Whole-cell patch-clamp techniques were used to record membrane currents in neuroblastoma cells stably transfected with human GABAA-ρ1 receptors. The action of 2-AEMP was compared with that of 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), a commonly used GABAA-ρ1 antagonist. With 10 μM GABA, 2-AEMP's IC50 (18 μM) differed by less than 2.5-fold from that of TPMPA (7 μM), and results obtained were consistent with a primarily competitive mode of inhibition by 2-AEMP. Terminating the presentation of 2-AEMP or TPMPA in the presence of GABA produced a release from inhibition. However, the rate of inhibition release upon the termination of 2-AEMP considerably exceeded that determined with termination of TPMPA. Moreover, when presented at concentrations near their respective IC50 values, the preincubation period associated with 2-AEMP's onset of inhibition was much shorter than that for TPMPA. Analogs of 2-AEMP possessing a benzyl or n-butyl rather than a methyl substituent at the phosphorus atom, as well as analogs bearing a C-methyl substituent on the aminoethyl side chain, exhibited reduced potency relative to 2-AEMP. Of these analogs, only (R)-2-aminopropyl methylphosphonate significantly diminished the response to 10 μM GABA. Structure-activity relationships are discussed in the context of molecular modeling of ligand binding to the antagonist binding site of the GABAA-ρ1 receptor. PMID:21810922

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells

PubMed Central

Tahara, Atsuo; Tsukada, Junko; Tomura, Yuichi; Wada, Koh-ichi; Kusayama, Toshiyuki; Ishii, Noe; Yatsu, Takeyuki; Uchida, Wataru; Tanaka, Akihiro

2000-01-01

[3H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [3H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (Kd) of 0.76 nM and a maximum receptor density (Bmax) of 153 fmol mg−1 protein. The Hill coefficient (nH) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [3H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [3H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu1,6]-oxytocin>AVP= atosiban>d(CH2)5Tyr(Me)AVP>[Thr4,Gly7]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca2+]i increase and hyperplasia. In contrast, the V1A receptor selective antagonist, SR 49059, and the V2 receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca2+]i increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca2+]i increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [3H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca2+]i increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. PMID:10694212

The novel isoxazoline ectoparasiticide lotilaner (Credelio™): a non-competitive antagonist specific to invertebrates γ-aminobutyric acid-gated chloride channels (GABACls).

PubMed

Rufener, Lucien; Danelli, Vanessa; Bertrand, Daniel; Sager, Heinz

2017-11-01

The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties. In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets. In these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABA A receptor was observed up to a concentration of 10 μM. Together, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate's γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.

Anti-endotoxic activity and structural basis for human MD-2·TLR4 antagonism of tetraacylated lipid A mimetics based on βGlcN(1↔1)αGlcN scaffold

PubMed Central

Garate, Jose Antonio; Stöckl, Johannes; del Carmen Fernández-Alonso, María; Artner, Daniel; Haegman, Mira; Oostenbrink, Chris; Jiménez-Barbero, Jesús; Beyaert, Rudi; Heine, Holger; Kosma, Paul

2015-01-01

Interfering with LPS binding by the co-receptor protein myeloid differentiation factor 2 (MD-2) represents a useful approach for down-regulation of MD-2·TLR4-mediated innate immune signaling, which is implicated in the pathogenesis of a variety of human diseases, including sepsis syndrome. The antagonistic activity of a series of novel synthetic tetraacylated bis-phosphorylated glycolipids based on the βGlcN(1↔1)αGlcN scaffold was assessed in human monocytic macrophage-like cell line THP-1, dendritic cells and human epithelial cells. Two compounds were shown to inhibit efficiently the LPS-induced inflammatory signaling by down-regulation of the expression of TNF-α, IL-6, IL-8, IL-10 and IL-12 to background levels. The binding of the tetraacylated by (R)-3-hydroxy-fatty acids (2 × C12, 2 × C14), 4,4′-bisphosphorylated βGlcN(1↔1)αGlcN-based lipid A mimetic DA193 to human MD-2 was calculated to be 20-fold stronger than that of Escherichia coli lipid A. Potent antagonistic activity was related to a specific molecular shape induced by the β,α(1↔1)-diglucosamine backbone. ‘Co-planar’ relative arrangement of the GlcN rings was inflicted by the double exo-anomeric conformation around both glycosidic torsions in the rigid β,α(1↔1) linkage, which was ascertained using NOESY NMR experiments and confirmed by molecular dynamics simulation. In contrast to the native lipid A ligands, the binding affinity of βGlcN(1↔1)αGlcN-based lipid A mimetics to human MD-2 was independent on the orientation of the diglucosamine backbone of the synthetic antagonist within the binding pocket of hMD-2 (rotation by 180°) allowing for two equally efficient binding modes as shown by molecular dynamics simulation. PMID:25394365

Predicting the relative binding affinity of mineralocorticoid receptor antagonists by density functional methods

NASA Astrophysics Data System (ADS)

Roos, Katarina; Hogner, Anders; Ogg, Derek; Packer, Martin J.; Hansson, Eva; Granberg, Kenneth L.; Evertsson, Emma; Nordqvist, Anneli

2015-12-01

In drug discovery, prediction of binding affinity ahead of synthesis to aid compound prioritization is still hampered by the low throughput of the more accurate methods and the lack of general pertinence of one method that fits all systems. Here we show the applicability of a method based on density functional theory using core fragments and a protein model with only the first shell residues surrounding the core, to predict relative binding affinity of a matched series of mineralocorticoid receptor (MR) antagonists. Antagonists of MR are used for treatment of chronic heart failure and hypertension. Marketed MR antagonists, spironolactone and eplerenone, are also believed to be highly efficacious in treatment of chronic kidney disease in diabetes patients, but is contra-indicated due to the increased risk for hyperkalemia. These findings and a significant unmet medical need among patients with chronic kidney disease continues to stimulate efforts in the discovery of new MR antagonist with maintained efficacy but low or no risk for hyperkalemia. Applied on a matched series of MR antagonists the quantum mechanical based method gave an R2 = 0.76 for the experimental lipophilic ligand efficiency versus relative predicted binding affinity calculated with the M06-2X functional in gas phase and an R2 = 0.64 for experimental binding affinity versus relative predicted binding affinity calculated with the M06-2X functional including an implicit solvation model. The quantum mechanical approach using core fragments was compared to free energy perturbation calculations using the full sized compound structures.

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding.

PubMed

Cash, Jennifer N; Angerman, Elizabeth B; Kattamuri, Chandramohan; Nolan, Kristof; Zhao, Huaying; Sidis, Yisrael; Keutmann, Henry T; Thompson, Thomas B

2012-01-06

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.

Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

PubMed

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor*

PubMed Central

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722

Use of Chimeras, Point Mutants, and Molecular Modeling to Map the Antagonist-binding Site of 4,4′,4″,4‴-(Carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic Acid (NF449) at P2X1 Receptors for ATP*

PubMed Central

Farmer, Louise K.; Schmid, Ralf; Evans, Richard J.

2015-01-01

P2X receptor subtype-selective antagonists are promising candidates for treatment of a range of pathophysiological conditions. However, in contrast to high resolution structural understanding of agonist action in the receptors, comparatively little is known about the molecular basis of antagonist binding. We have generated chimeras and point mutations in the extracellular ligand-binding loop of the human P2X1 receptor, which is inhibited by NF449, suramin, and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate, with residues from the rat P2X4 receptor, which is insensitive to these antagonists. There was little or no effect on sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate in chimeric P2X1/4 receptors, indicating that a significant number of residues required for binding of these antagonists are present in the P2X4 receptor. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ∼60- and ∼135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding. These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were then discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin. PMID:25425641

Use of chimeras, point mutants, and molecular modeling to map the antagonist-binding site of 4,4',4″,4‴-(carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) at P2X1 receptors for ATP.

PubMed

Farmer, Louise K; Schmid, Ralf; Evans, Richard J

2015-01-16

P2X receptor subtype-selective antagonists are promising candidates for treatment of a range of pathophysiological conditions. However, in contrast to high resolution structural understanding of agonist action in the receptors, comparatively little is known about the molecular basis of antagonist binding. We have generated chimeras and point mutations in the extracellular ligand-binding loop of the human P2X1 receptor, which is inhibited by NF449, suramin, and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate, with residues from the rat P2X4 receptor, which is insensitive to these antagonists. There was little or no effect on sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate in chimeric P2X1/4 receptors, indicating that a significant number of residues required for binding of these antagonists are present in the P2X4 receptor. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ∼60- and ∼135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding. These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were then discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

PubMed

Tabassum, Nargis; Ma, Qianyun; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

2017-09-01

Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators.

PubMed

Han, Changho; Chatterjee, Arindam; Noetzel, Meredith J; Panarese, Joseph D; Smith, Emery; Chase, Peter; Hodder, Peter; Niswender, Colleen; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

2015-01-15

Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure. Copyright © 2014 Elsevier Ltd. All rights reserved.

Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model.

PubMed

Kai, Zhen-Peng; Zhu, Jing-Jing; Deng, Xi-Le; Yang, Xin-Ling; Chen, Shan-Shan

2018-04-03

Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT) antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C -terminal amidated octapeptide Manse-AT (6-13). We identified three residues essential for bioactivity (Thr⁴, Arg6 and Phe⁸) by assaying alanine-replacement analogs of Manse-AT (6-13). Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR) was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10-13), we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10-13) validated our hypothesis. The IC 50 value of antagonist Manse-AT (10-13) is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10-13) was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR*

PubMed Central

Li, Hao; Redinbo, Matthew R.; Venkatesh, Madhukumar; Ekins, Sean; Chaudhry, Anik; Bloch, Nicolin; Negassa, Abdissa; Mukherjee, Paromita; Kalpana, Ganjam; Mani, Sridhar

2013-01-01

The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications. PMID:23525103

Characterization of a novel non-peptide vasopressin V1 receptor antagonist (OPC-21268) in the rat.

PubMed

Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I

1993-08-01

A non-peptide, orally effective, vasopressin (AVP) V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl)-3,4-dihydro-2(1H)-quinolinone (OPC-21268) has recently been described. This paper reports the in-vitro and in-vivo characterization of OPC-21268 binding to vasopressin receptors in rat liver and kidney. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, 125I-labelled [d(CH2)5,sarcosine7]AVP to V1 receptors in both rat liver and kidney medulla membranes. The concentration of OPC-21268 that displaced 50% of specific AVP binding (IC50) was 40 +/- 3 nmol/l for liver V1 and 15 +/- 2 nmol/l for kidney V1 receptors (mean +/- S.E.M.; n = 3). OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)]d(CH2)5,D-Ile2,Ile4] AVP binding to V2 receptors in renal medulla membranes (IC50 > 0.1 mmol/l). After oral administration to rats, OPC-21268 was an effective V1 antagonist in a time- and dose-dependent manner. Binding kinetic studies showed that OPC-21268 acted as a competitive antagonist at the liver V1 receptor in vitro and in vivo, in addition to its in-vitro competitive effects at the renal V1 receptor. OPC-21268 shows promise as an orally active V1 antagonist.

Fas Binding to Calmodulin Regulates Apoptosis in Osteoclasts*

PubMed Central

Wu, Xiaojun; Ahn, Eun-Young; McKenna, Margaret A.; Yeo, Hyeonju; McDonald, Jay M.

2005-01-01

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca2+ receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536–4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 μm, and trifluoperazine, 10 μm, induce 7.3 ± 1.8-fold and 5.3 ± 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661–5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 μm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr−cg mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr−cg mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr−cg mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 ± 0.2-fold in Lpr−cg-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis. PMID:15965236

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

PubMed

Tahara, A; Tsukada, J; Tomura, Y; Wada, K i; Kusayama, T; Ishii, N; Yatsu, T; Uchida, W; Tanaka, A

2000-01-01

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139

Modulation of the binding of basic fibroblast growth factor and heparanase activity by purified λ-carrageenan oligosaccharides.

PubMed

Niu, Ting-Ting; Zhang, Dong-Sheng; Chen, Hai-Min; Yan, Xiao-Jun

2015-07-10

Inhibitors of angiogenesis and tumor metastasis are increasingly emerging as promising agents for cancer therapy. Here, we report λ-carrageenan oligosaccharides (λ-COs), highly-sulfated oligosaccharides acting as a basic fibroblast growth factor (bFGF) antagonist and heparanase inhibitor. λ-COs with degree of polymerization (DP) from 2 to 8 degraded by λ-carrageenase were separated and purified. The structures were identified by mass spectrometry. The activities of λ-COs are closely related with DP. λ-COs showed no cytotoxicity, but inactivated bFGF-induced cell proliferation; among them, λ-carraheptaose showed highest capability. Only λ-carraheptaose can effectively bind to bFGF. Binding kinetics showed that λ-carraheptaose and suramin had different binding modes, i.e., suramin displayed a fast association and fast dissociation, but λ-carraheptaose exhibited a slow association and slow dissociation. In addition, λ-COs showed the highest heparanase inhibitory ability and abolished the endothelial cell invasion. Thus, λ-COs may provide a tool to develop of new carbohydrate-based therapeutics against cancer and angiogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Similarity- and Substructure-Based Development of β2-Adrenergic Receptor Ligands Based on Unusual Scaffolds

PubMed Central

2017-01-01

The β2-adrenergic receptor (β2AR) is a G protein-coupled receptor (GPCR) and a well-explored target. Here, we report the discovery of 13 ligands, ten of which are novel, of this particular GPCR. They have been identified by similarity- and substructure-based searches using multiple ligands, which were described in an earlier study, as starting points. Of note, two of the molecules used as queries here distinguish themselves from other β2AR antagonists by their unique scaffold. The molecules described in this work allow us to explore the ligand space around the previously reported molecules in greater detail, leading to insights into their structure–activity relationship. We also report experimental binding and selectivity data and putative binding modes for the novel molecules. PMID:28523097

Pharmacological lineage analysis revealed the binding affinity of broad-spectrum substance P antagonists to receptors for gonadotropin-releasing peptide.

PubMed

Arai, Kazune; Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

2015-02-15

A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor

PubMed Central

Judson, Richard S.; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M.; Filer, Dayne L.; Houck, Keith A.; Martin, Matthew T.; Sipes, Nisha; Richard, Ann M.; Mansouri, Kamel; Setzer, R. Woodrow; Knudsen, Thomas B.; Crofton, Kevin M.; Thomas, Russell S.

2015-01-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. PMID:26272952

Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

PubMed

Lundstrom, K; Hawcock, A B; Vargas, A; Ward, P; Thomas, P; Naylor, A

1997-10-15

Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

Exploration of the Ca2+ interaction modes of the nifedipine calcium channel antagonist.

PubMed

Liu, Huichun; Zhang, Liang; Li, Ping; Cukier, Robert I; Bu, Yuxiang

2007-02-02

A comprehensive study is carried out using quantum chemical computation and molecular dynamics (MD) simulations to gain insight into the interaction between Ca(2+) ions and the most important class of calcium channel antagonists--nifedipine. First, the chelating structures and energetic characters of nifedipine-Ca(2+) in the gas phase are explored, and 25 isomers are found. The most favorable chelating mode is a tridentate one, that is, Ca(2+) binds to two carbonyl O atoms and one nitryl O atom, where Ca(2+) is above the plane of the three O atoms to form a pyramidal structure. Accurate geometric structures, relative stabilities, vertical and adiabatic binding energies, and charge distributions are discussed. The differences in the geometries and energies among these isomers are analyzed from the contributions of chelating sites, electrostatics and polarizations, steric repulsions, and charge distributions. The interconversions among isomers with similar geometries and energies are also investigated because of the importance of the geometric transformation in the biological system. Furthermore, certain numbers of water molecules are added to the nifedipine-Ca(2+) system to probe the effect of water. A detailed study is performed on the hydrated geometries on the basis of the most stable isomer 1. Stepwise hydration can weaken the nifedipine-Ca(2+) interaction, and the chelating sites of nifedipine are gradually replaced by the added water molecules. Hexacoordination is found to be the most favorable geometry no matter how many water molecules were added, which can be verified by the MD simulations. The transfer of water molecules from the inner shell to the outer shell is also supported by MD simulations of the hexahydrated complexes.

A DFT approach to discriminate the antagonist and partial agonist activity of ligands binding to the NMDA receptor

NASA Astrophysics Data System (ADS)

Haslak, Zeynep Pinar; Bozkurt, Esra; Dutagaci, Bercem; De Proft, Frank; Aviyente, Viktorya; De Vleeschouwer, Freija

2018-02-01

The activation of N-methyl-D-aspartate receptors is found to be intimately associated with neurodegenerative diseases which make them promising therapeutic targets. Despite the significantly increasing multidisciplinary interests centred on this ionotropic channel, design of new ligands with intended functional activity remains a great challenge. In this article, a computational study based on density functional theory is presented to understand the structural factors of ligands determining their function as antagonists and partial agonists. With this aim, the GluN1 subunit is chosen as being one of the essential components in the activation mechanism, and quantum chemical calculations are implemented for 30 antagonists and 30 partial agonists known to bind to this subunit with different binding affinities. Several quantum chemical descriptors are investigated which might unlock the difference between antagonists and partial agonists.

Identification and characterization of amino-piperidinequinolones and quinazolinones as MCHr1 antagonists.

PubMed

Blackburn, Christopher; LaMarche, Matthew J; Brown, James; Che, Jennifer Lee; Cullis, Courtney A; Lai, Sujen; Maguire, Martin; Marsilje, Thomas; Geddes, Bradley; Govek, Elizabeth; Kadambi, Vivek; Doherty, Colleen; Dayton, Brian; Brodjian, Sevan; Marsh, Kennan C; Collins, Christine A; Kym, Philip R

2006-05-15

Several potent, functionally active MCHr1 antagonists derived from quinolin-2(1H)-ones and quinazoline-2(1H)-ones have been synthesized and evaluated. Pyridylmethyl substitution at the quinolone 1-position results in derivatives with low-nM binding potency and good selectivity with respect to hERG binding.

Multiple opioid receptors in endotoxic shock: evidence for delta involvement and mu-delta interactions in vivo.

PubMed Central

D'Amato, R; Holaday, J W

1984-01-01

The use of selective delta and mu opioid antagonists has provided evidence that delta opioid receptors within the brain mediate the endogenous opioid component of endotoxic shock hypotension. The selectivity of these delta and mu antagonists was demonstrated by their differing effects upon morphine analgesia and endotoxic hypotension. The mu antagonist beta-funaltrexamine, at doses that antagonized morphine analgesia, failed to alter shock, whereas the delta antagonist M 154,129: [N,N-bisallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH] (ICI) reversed shock at doses that failed to block morphine analgesia. Therefore, selective delta antagonists may have therapeutic value in reversing circulatory shock without altering the analgesic actions of endogenous or exogenous opioids. Additional data revealed that prior occupancy of mu binding sites by irreversible opioid antagonists may allosterically attenuate the actions of antagonists with selectivity for delta binding sites. For endogenous opioid systems, this observation provides an opportunity to link in vivo physiological responses with receptor-level biochemical interactions. PMID:6326151

Quantitative autoradiographic analysis of muscarinic receptor subtypes and their role in representational memory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Messer, W.S.

1986-01-01

Autoradiographic techniques were used to examine the distribution of muscarinic receptors in rat brain slices. Agonist and selective antagonist binding were examined by measuring the ability for unlabeled ligands to inhibit (/sup 3/H)-1-QNB labeling of muscarinic receptors. The distribution of high affinity pirenzepine binding sites (M/sub 1/ subtype) was distinct from the distribution of high affinity carbamylcholine sites, which corresponded to the M/sub 2/ subtype. In a separate assay, the binding profile for pirenzepine was shown to differ from the profile for scopolamine, a classical muscarinic antagonist. Muscarinic antagonists, when injected into the Hippocampus, impaired performance of a representational memorymore » task. Pirenzepine, the M/sub 1/ selective antagonist, produced representational memory deficits. Scopolamine, a less selective muscarinic antagonist, caused increases in running times in some animals which prevented a definitive interpretation of the nature of the impairment. Pirenzepine displayed a higher affinity for the hippocampus and was more effective in producing a selective impairment of representational memory than scopolamine. The data indicated that cholinergic activity in the hippocampus was necessary for representation memory function.« less

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

PubMed

Müller, C E; Maurinsh, J; Sauer, R

2000-01-01

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

Molecular Gymnastics: Mechanisms of HIV-1 Resistance to CCR5 Antagonists and Impact on Virus Phenotypes.

PubMed

Roche, Michael; Borm, Katharina; Flynn, Jacqueline K; Lewin, Sharon R; Churchill, Melissa J; Gorry, Paul R

2016-01-01

Human immunodeficiency virus type 1 (HIV-1) enters host cells through the binding of its envelope glycoproteins (Env) to the host cell receptor CD4 and then subsequent binding to a chemokine coreceptor, either CCR5 or CXCR4. CCR5 antagonists are a relatively recent class addition to the armamentarium of anti-HIV-1 drugs. These compounds act by binding to a hydrophobic pocket formed by the transmembrane helices of CCR5 and altering the conformation of the extracellular domains, such that they are no longer recognized by Env. Maraviroc is the first drug within this class to be licenced for use in HIV-1 therapy regimens. HIV resistance to CCR5 antagonists occurs either through outgrowth of pre-existing CXCR4-using viruses, or through acquisition of the ability of CCR5-using HIV-1 to use the antagonist bound form of CCR5. In the latter scenario, the mechanism underlying resistance is through complex alterations in the way that resistant Envs engage CCR5. These significant changes are unlikely to occur without consequence to the viral entry phenotype and may also open up new avenues to target CCR5 antagonist resistant viruses. This review discusses the mechanism of action of CCR5 antagonists, how HIV resistance to CCR5 antagonists occurs, and the subsequent effects on Env function.

Molecular dynamic simulation of mGluR5 amino terminal domain: essential dynamics analysis captures the agonist or antagonist behaviour of ligands.

PubMed

Casoni, Alessandro; Clerici, Francesca; Contini, Alessandro

2013-04-01

We describe the application of molecular dynamics followed by principal component analysis to study the inter-domain movements of the ligand binding domain (LBD) of mGluR5 in response to the binding of selected agonists or antagonists. Our results suggest that the method is an attractive alternative to current approaches to predict the agonist-induced or antagonist-blocked LBD responses. The ratio between the eigenvalues of the first and second eigenvectors (R1,2) is also proposed as a numerical descriptor for discriminating the ligand behavior as a mGluR5 agonist or antagonist. Copyright © 2013 Elsevier Inc. All rights reserved.

Combined 3D-QSAR modeling and molecular docking study on azacycles CCR5 antagonists

NASA Astrophysics Data System (ADS)

Ji, Yongjun; Shu, Mao; Lin, Yong; Wang, Yuanqiang; Wang, Rui; Hu, Yong; Lin, Zhihua

2013-08-01

The beta chemokine receptor 5 (CCR5) is an attractive target for pharmaceutical industry in the HIV-1, inflammation and cancer therapeutic areas. In this study, we have developed quantitative structure activity relationship (QSAR) models for a series of 41 azacycles CCR5 antagonists using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and Topomer CoMFA methods. The cross-validated coefficient q2 values of 3D-QASR (CoMFA, CoMSIA, and Topomer CoMFA) methods were 0.630, 0.758, and 0.852, respectively, the non-cross-validated R2 values were 0.979, 0.978, and 0.990, respectively. Docking studies were also employed to determine the most probable binding mode. 3D contour maps and docking results suggested that bulky groups and electron-withdrawing groups on the core part would decrease antiviral activity. Furthermore, docking results indicated that H-bonds and π bonds were favorable for antiviral activities. Finally, a set of novel derivatives with predicted activities were designed.

Effect of SR-49059, a vasopressin V1a antagonist, on human vascular smooth muscle cells.

PubMed

Serradeil-Le Gal, C; Herbert, J M; Delisee, C; Schaeffer, P; Raufaste, D; Garcia, C; Dol, F; Marty, E; Maffrand, J P; Le Fur, G

1995-01-01

The effects of SR-49059, a new nonpeptide and selective arginine vasopressin (AVP) V1a antagonist, were investigated in binding and functional studies on cultured human aortic vascular smooth muscle cells (VSMC). Characterization of human vascular V1a receptors, using a specific V1a radioiodinated ligand, showed that [125I]-linear AVP antagonist binding to human VSMC membranes was time dependent, reversible, and saturable. A single population of high-affinity binding sites (apparent equilibrium dissociation constant = 15 +/- 6 pM; maximum binding density = 36 +/- 5 fmol/mg protein, i.e., approximately 3,000 sites/cell) with the expected V1a profile was identified. Exposure of these cells to AVP dose-dependently produced cytosolic free [Ca2+] increase [AVP concentration required to obtain a half-maximal response (EC50) = 23 +/- 9 nM] and proliferation (EC50 = 3.2 +/- 0.5 nM). SR-49059 strongly and stereospecifically inhibited [125I]-linear AVP antagonist binding to VSMC V1a receptors [inhibition constant (Ki) = 1.4 +/- 0.3 nM], AVP-evoked Ca2+ increase [concentration of inhibitor required to obtain 50% inhibition of specific binding (IC50) = 0.41 +/- 0.06 nM], and the mitogenic effects induced by 100 nM AVP (IC50 = 0.83 +/- 0.04 nM). OPC-21268, another nonpeptide V1a antagonist, was more than two orders of magnitude less potent than SR-49059 in these models. However, the consistent affinity (Ki = 138 +/- 21 nM) and activity found with OPC-21268 on human VSMC in comparison with the inactivity already observed for other human V1a receptors (liver, platelets, adrenals, and uterus) strongly suggested the existence of human AVP V1a-receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

NASA Astrophysics Data System (ADS)

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-01

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1.

PubMed

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-28

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr(6.63) forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr(6.63) to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr356(6.63) allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kozuka, M.; Ito, T.; Hirose, S.

1989-02-28

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using /sup 125/I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction thatmore » was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.« less

The Molecular Determinants of Small-Molecule Ligand Binding at P2X Receptors

PubMed Central

Pasqualetto, Gaia; Brancale, Andrea; Young, Mark T.

2018-01-01

P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP—their physiological ligand—is released as a neurotransmitter and in conditions of cell damage such as inflammation, and substantial evidence implicates P2X receptors in diseases including neuropathic pain, cancer, and arthritis. In 2009, the first P2X crystal structure, Danio rerio P2X4 in the apo- state, was published, and this was followed in 2012 by the ATP-bound structure. These structures transformed our understanding of the conformational changes induced by ATP binding and the mechanism of ligand specificity, and enabled homology modeling of mammalian P2X receptors for ligand docking and rational design of receptor modulators. P2X receptors are attractive drug targets, and a wide array of potent, subtype-selective modulators (mostly antagonists) have been developed. In 2016, crystal structures of human P2X3 in complex with the competitive antagonists TNP-ATP and A-317491, and Ailuropoda melanoleuca P2X7 in complex with a series of allosteric antagonists were published, giving fascinating insights into the mechanism of channel antagonism. In this article we not only summarize current understanding of small-molecule modulator binding at P2X receptors, but also use this information in combination with previously published structure-function data and molecular docking experiments, to hypothesize a role for the dorsal fin loop region in differential ATP potency, and describe novel, testable binding conformations for both the semi-selective synthetic P2X7 agonist 2′-(3′)-O-(4-benzoyl)benzoyl ATP (BzATP), and the P2X4-selective positive allosteric modulator ivermectin. We find that the distal benzoyl group of BzATP lies in close proximity to Lys-127, a residue previously implicated in BzATP binding to P2X7, potentially explaining the increased potency of BzATP at rat P2X7 receptors. We also present molecular docking of ivermectin to rat P2X4 receptors, illustrating a plausible binding conformation between the first and second transmembrane domains which not only tallies with previous mutagenesis studies, but would also likely have the effect of stabilizing the open channel structure, consistent with the mode of action of this positive allosteric modulator. From our docking simulations and analysis of sequence homology we propose a series of mutations likely to confer ivermectin sensitivity to human P2X1. PMID:29456508

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon

PubMed Central

Warner, Fiona J; Comis, Alfio; Miller, Robert C; Burcher, Elizabeth

1999-01-01

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (KD 0.47±0.05 nM), of high capacity (Bmax 2.1±0.1 fmol mg−1 wet weight tissue) and reversible (kinetically derived KD 0.36±0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide γ (NPγ)≥NKA≥[Lys5,MeLeu9,Nle10]NKA (4–10) (NK2 agonist)>>substance P (SP)>neurokinin B (NKB)≥[Pro9]SP (NK1 agonist)>>senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968≥MEN11420>GR94800≥MEN10627>MEN10376≥R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 μM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear. PMID:10455255

Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3.

PubMed

Leffler, Agnes; Ahlstedt, Ingela; Engberg, Susanna; Svensson, Arne; Billger, Martin; Oberg, Lisa; Bjursell, Magnus K; Lindström, Erik; von Mentzer, Bengt

2009-05-01

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK2R and NK3R) and dog (NK1R, NK2R and NK3R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs. The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca2+. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs. Data collected using the cloned dog NK1R confirmed that the dog NK1R displays similar pharmacology as the human and the gerbil NK1R, but differs greatly from the mouse and the rat NK1R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK2R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK3R likely influence the NK3R antagonist potency, explaining the 3-10-fold decrease in potency observed for the rat NK3R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK1R and NK3R.

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor.

PubMed

Judson, Richard S; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M; Filer, Dayne L; Houck, Keith A; Martin, Matthew T; Sipes, Nisha; Richard, Ann M; Mansouri, Kamel; Setzer, R Woodrow; Knudsen, Thomas B; Crofton, Kevin M; Thomas, Russell S

2015-11-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

Computational study of the binding mechanism between farnesoid X receptor α and antagonist N-benzyl-N-(3-(tertbutyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide.

PubMed

Du, Juan; Qiu, Miaoxue; Guo, Lizhong; Yao, Xiaojun

2018-05-02

Farnesoid X receptor α (FXRα) is a bile acid-activated transcription factor, which plays important roles in the regulation of multiple metabolic processes. Development of FXR antagonist has revealed great potential for the treatment of metabolic disorders. The compound N-Benzyl-N-(3-(tertbutyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino). Benzamide (NDB) was recently determined as a selective antagonist of FXRα, while the detailed interaction mechanism is not well understood. In this study, the combined computational methods including molecular dynamics simulations, binding free energy calculation, and principal component analysis were utilized to investigate the effect of NDB on the dynamics behaviors and dimerization of FXRα The binding free energy calculation indicated that the protein dimerization increases NDB affinity and the binding of NDB also stabilizes the interaction between two subunits of FXRα. Further decomposition of the overall binding free energies into individual residues identifies several residues significant for NDB binding, including Leu291, Met294, Ala295, His298, Met332, Ser336, Ala452, and Leu455. It also suggests that the interactions of L289(A)-W458(B), W458(A)-L289(B), R459(A)-N461(B), and N461(A)-R459(B) are important for the dimer stabilization. This study provides a molecular basis for the understanding of binding mechanism between antagonist NDB and FXRα and valuable information for the novel FXR modulators design for the treatment of metabolic syndrome.

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375

PubMed Central

Douglas, Stephen A; Behm, David J; Aiyar, Nambi V; Naselsky, Diane; Disa, Jyoti; Brooks, David P; Ohlstein, Eliot H; Gleason, John G; Sarau, Henry M; Foley, James J; Buckley, Peter T; Schmidt, Dulcie B; Wixted, William E; Widdowson, Katherine; Riley, Graham; Jin, Jian; Gallagher, Timothy F; Schmidt, Stanley J; Ridgers, Lance; Christmann, Lisa T; Keenan, Richard M; Knight, Steven D; Dhanak, Dashyant

2005-01-01

SB-706375 potently inhibited [125I]hU-II binding to both mammalian recombinant and ‘native' UT receptors (Ki 4.7±1.5 to 20.7±3.6 nM at rodent, feline and primate recombinant UT receptors and Ki 5.4±0.4 nM at the endogenous UT receptor in SJRH30 cells). Prior exposure to SB-706375 (1 μM, 30 min) did not alter [125I]hU-II binding affinity or density in recombinant cells (KD 3.1±0.4 vs 5.8±0.9 nM and Bmax 3.1±1.0 vs 2.8±0.8 pmol mg−1) consistent with a reversible mode of action. The novel, nonpeptidic radioligand [3H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (KD 2.6±0.4 nM, Bmax 0.86±0.12 pmol mg−1) in a manner that was inhibited by both U-II isopeptides and SB-706375 (Ki 4.6±1.4 to 17.6±5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. SB-706375 was a potent, competitive hU-II antagonist across species with pKb 7.29–8.00 in HEK293-UT receptor cells (inhibition of [Ca2+]i-mobilization) and pKb 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (Kapp∼20 nM). SB-706375 was a selective U-II antagonist with ⩾100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (Ki/IC50>1 μM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 μM). In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals. PMID:15852036

Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.

PubMed

Huang, Xin; Chen, Hao; Michelsen, Klaus; Schneider, Stephen; Shaffer, Paul L

2015-10-08

Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.

Discovery of Tertiary Sulfonamides as Potent Liver X Receptor Antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zuercher, William J.; Buckholz†, Richard G.; Campobasso, Nino

2010-08-12

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

PubMed

Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

2010-04-22

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Binding of [3H] SR 49059, a potent nonpeptide vasopressin V1a antagonist, to rat and human liver membranes.

PubMed

Serradeil-Le Gal, C; Raufaste, D; Marty, E; Garcia, C; Maffrand, J P; Le Fur, G

1994-02-28

The new potent and selective nonpeptide vasopressin V1a antagonist, SR 49059, was tritiated and used for the characterization of rat and human liver AVP V1a receptors. Binding of [3H] SR 49059 was time-dependent, reversible and saturable. A single class of high affinity binding sites was identified with Kd values of 0.63 +/- 0.13 and 2.95 +/- 0.64 nM, in rat and human liver membranes, respectively. The maximal binding capacity (Bmax) was about 7 times higher in rat than in human liver preparations. The relative potencies of several AVP/oxytocin agonists or antagonists to inhibit [3H] SR 49059 binding confirmed that this ligand labeled a homogeneous population of sites with the expected AVP V1a profile. Furthermore, [3H] SR 49059 or unlabeled SR 49059 displayed only slight species differences between rat and human V1a receptors, whereas OPC-21268, another nonpeptide V1a antagonist, exhibited a high species-related potency with more than 500 fold higher affinity for rat than for human liver V1a receptors. Thus, [3H] SR 49059 is the first nonpeptide AVP V1a ligand reported having highly specific activity, stability, specificity and affinity. This makes it a suitable probe for labeling AVP V1a receptors in rat and also in human tissues.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74.

PubMed

Miller, Philip S; Barwell, James; Poyner, David R; Wigglesworth, Mark J; Garland, Stephen L; Donnelly, Dan

2010-01-01

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1. Copyright 2009 Elsevier Inc. All rights reserved.

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

PubMed Central

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-01

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R. PMID:25628267

Divergence in Ubiquitin Interaction and Catalysis among the Ubiquitin-Specific Protease Family Deubiquitinating Enzymes.

PubMed

Tencer, Adam H; Liang, Qin; Zhuang, Zhihao

2016-08-23

Deubiquitinating enzymes (DUBs) are responsible for reversing mono- and polyubiquitination of proteins and play essential roles in numerous cellular processes. Close to 100 human DUBs have been identified and are classified into five families, with the ubiquitin-specific protease (USP) family being the largest (>50 members). The binding of ubiquitin (Ub) to USP is strikingly different from that observed for the DUBs in the ubiquitin C-terminal hydrolase (UCH) and ovarian tumor domain protease (OTU) families. We generated a panel of mutant ubiquitins and used them to probe the ubiquitin's interaction with a number of USPs. Our results revealed a remarkable divergence of USP-Ub interactions among the USP catalytic domains. Our double-mutant cycle analysis targeting the ubiquitin residues located in the tip, the central body, and the tail of ubiquitin also demonstrated different crosstalk among the USP-Ub interactions. This work uncovered intriguing divergence in the ubiquitin-binding mode in the USP family DUBs and raised the possibility of targeting the ubiquitin-binding hot spots on USPs for selective inhibition of USPs by small molecule antagonists.

Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

DOE PAGES

Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; ...

2015-05-07

We report that angiotensin II type 1 receptor (AT 1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT 1R blockers (ARBs), the structural basis for AT 1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT 1R in complex with its selective antagonist ZD7155 atmore » 2.9 Å resolution. The AT 1R-ZD7155 complex structure revealed key structural features ofAT 1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT 1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT 1R structure-function relationship and structure-based drug design.« less

Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius

We report that angiotensin II type 1 receptor (AT 1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT 1R blockers (ARBs), the structural basis for AT 1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT 1R in complex with its selective antagonist ZD7155 atmore » 2.9 Å resolution. The AT 1R-ZD7155 complex structure revealed key structural features ofAT 1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT 1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT 1R structure-function relationship and structure-based drug design.« less

PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats.

PubMed

Horswill, J G; Bali, U; Shaaban, S; Keily, J F; Jeevaratnam, P; Babbs, A J; Reynet, C; Wong Kai In, P

2007-11-01

Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo. A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo. In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight. PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.

PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats

PubMed Central

Horswill, J G; Bali, U; Shaaban, S; Keily, J F; Jeevaratnam, P; Babbs, A J; Reynet, C; Wong Kai In, P

2007-01-01

Background and purpose: Rimonabant (AcompliaTM, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo. Experimental approach: A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPγS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo. Key results: In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPγS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight. Conclusions and implications: PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist. PMID:17592509

Identification of key residues involved in adrenomedullin binding to the AM1 receptor

PubMed Central

Watkins, HA; Au, M; Bobby, R; Archbold, JK; Abdul-Manan, N; Moore, JM; Middleditch, MJ; Williams, GM; Brimble, MA; Dingley, AJ; Hay, DL

2013-01-01

Background and Purpose Adrenomedullin (AM) is a peptide hormone whose receptors are members of the class B GPCR family. They comprise a heteromer between the GPCR, the calcitonin receptor-like receptor and one of the receptor activity-modifying proteins 1–3. AM plays a significant role in angiogenesis and its antagonist fragment AM22–52 can inhibit blood vessel and tumour growth. The mechanism by which AM interacts with its receptors is unknown. Experimental Approach We determined the AM22–52 binding epitope for the AM1 receptor extracellular domain using biophysical techniques, heteronuclear magnetic resonance spectroscopy and alanine scanning. Key Results Chemical shift perturbation experiments located the main binding epitope for AM22–52 at the AM1 receptor to the C-terminal 8 amino acids. Isothermal titration calorimetry of AM22–52 alanine-substituted peptides indicated that Y52, G51 and I47 are essential for AM1 receptor binding and that K46 and P49 and R44 have a smaller role to play. Characterization of these peptides at the full-length AM receptors was assessed in Cos7 cells by cAMP assay. This confirmed the essential role of Y52, G51 and I47 in binding to the AM1 receptor, with their substitution resulting in ≥100-fold reduction in antagonist potency compared with AM22–52. R44A, K46A, S48A and P49A AM22–52 decreased antagonist potency by approximately 10-fold. Conclusions and Implications This study localizes the main binding epitope of AM22–52 to its C-terminal amino acids and distinguishes essential residues involved in this binding. This will inform the development of improved AM receptor antagonists. PMID:23351143

Cholecystokinin receptors on gallbladder muscle and pancreatic acinar cells: a comparative study

DOE Office of Scientific and Technical Information (OSTI.GOV)

von Schrenck, T.; Moran, T.H.; Heinz-Erian, P.

1988-10-01

To compare receptors for cholecystokinin (CCK) in pancreas and gallbladder, we measured binding of 125I-Bolton-Hunter-labeled CCK-8 (125I-BH-CCK-8) to tissue sections from guinea pig gallbladder and pancreas under identical conditions. In both tissues, binding had similar time-, temperature-, and pH dependence, was reversible, saturable and inhibited only by CCK related peptides or CCK receptor antagonists. Autoradiography localized 125I-BH-CCK-8 binding to the smooth muscle layer in the gallbladder. Binding of 125I-BH-CCK-8 to gallbladder sections was inhibited by various agonists with the following potencies (IC50):CCK-8 (0.4 nM) greater than des(SO3)CCK-8 (0.07 microM) greater than gastrin-17-I (1.7 +/- 0.3 microM) and by various receptormore » antagonists with the following potencies: L364,718 (1.5 nM) greater than CR 1409 (0.19 microM) greater than asperlicin = CBZ-CCK-(27-32)-NH2 (1 microM) greater than Bt2cGMP (120 microM). Similar potencies were found for the agonists and antagonists for pancreas sections. Inhibition of binding of 125I-BH-CCK-8 by 11 different analogues of proglumide gave similar potencies for both pancreas and gallbladder. The potencies of agonists in stimulating and antagonists in inhibiting CCK-stimulated contraction or amylase release correlated closely with their abilities to inhibit 125I-BH-CCK-8 binding to gallbladder or pancreas sections or acini, respectively. The present results demonstrate and characterize a method that can be used to compare the CCK receptors in guinea pig gallbladder and pancreas under identical conditions. Moreover, this study demonstrates that gallbladder and pancreatic CCK receptors have similar affinities for the various agonists and antagonists tested and, therefore, provides no evidence that they represent different subtypes of CCK receptors that can be distinguished pharmacologically.« less

Key Structures and Interactions for Binding of Mycobacterium tuberculosis Protein Kinase B Inhibitors from Molecular Dynamics Simulation.

PubMed

Punkvang, Auradee; Kamsri, Pharit; Saparpakorn, Patchreenart; Hannongbua, Supa; Wolschann, Peter; Irle, Stephan; Pungpo, Pornpan

2015-07-01

Substituted aminopyrimidine inhibitors have recently been introduced as antituberculosis agents. These inhibitors show impressive activity against protein kinase B, a Ser/Thr protein kinase that is essential for cell growth of M. tuberculosis. However, up to now, X-ray structures of the protein kinase B enzyme complexes with the substituted aminopyrimidine inhibitors are currently unavailable. Consequently, structural details of their binding modes are questionable, prohibiting the structural-based design of more potent protein kinase B inhibitors in the future. Here, molecular dynamics simulations, in conjunction with molecular mechanics/Poisson-Boltzmann surface area binding free-energy analysis, were employed to gain insight into the complex structures of the protein kinase B inhibitors and their binding energetics. The complex structures obtained by the molecular dynamics simulations show binding free energies in good agreement with experiment. The detailed analysis of molecular dynamics results shows that Glu93, Val95, and Leu17 are key residues responsible to the binding of the protein kinase B inhibitors. The aminopyrazole group and the pyrimidine core are the crucial moieties of substituted aminopyrimidine inhibitors for interaction with the key residues. Our results provide a structural concept that can be used as a guide for the future design of protein kinase B inhibitors with highly increased antagonistic activity. © 2014 John Wiley & Sons A/S.

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function.

PubMed

Xie, Y; Cohen, J B

2001-01-26

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.

5-HT1A receptor antagonists reduce food intake and body weight by reducing total meals with no conditioned taste aversion.

PubMed

Dill, M Joelle; Shaw, Janice; Cramer, Jeff; Sindelar, Dana K

2013-11-01

Serotonin acts through receptors controlling several physiological functions, including energy homeostasis regulation and food intake. Recent experiments demonstrated that 5-HT1A receptor antagonists reduce food intake. We sought to examine the microstructure of feeding with 5-HT1A receptor antagonists using a food intake monitoring system. We also examined the relationship between food intake, inhibition of binding and pharmacokinetic (PK) profiles of the antagonists. Ex vivo binding revealed that, at doses used in this study to reduce food intake, inhibition of binding of a 5-HT1A agonist by ~40% was reached in diet-induced obese (DIO) mice with a trend for higher binding in DIO vs. lean animals. Additionally, PK analysis detected levels from 2 to 24h post-compound administration. Male DIO mice were administered 5-HT1A receptor antagonists LY439934 (10 or 30 mg/kg, p.o.), WAY100635 (3 or 10mg/kg, s.c.), SRA-333 (10 or 30 mg/kg, p.o.), or NAD-299 (3 or 10mg/kg, s.c.) for 3 days and meal patterns were measured. Analyses revealed that for each antagonist, 24-h food intake was reduced through a specific decrease in the total number of meals. Compared to controls, meal number was decreased 14-35% in the high dose. Average meal size was not changed by any of the compounds. The reduction in food intake reduced body weight 1-4% compared to Vehicle controls. Subsequently, a conditioned taste aversion (CTA) assay was used to determine whether the feeding decrease might be an indicator of aversion, nausea, or visceral illness caused by the antagonists. Using a two bottle preference test, it was found that none of the compounds produced a CTA. The decrease in food intake does not appear to be a response to nausea or malaise. These results indicate that 5-HT1A receptor antagonist suppresses feeding, specifically by decreasing the number of meals, and induce weight loss without an aversive side effect. © 2013 Elsevier Inc. All rights reserved.

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

PubMed

Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

2003-01-01

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

Synthesis and serotonergic activity of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives: novel antagonists for the vascular 5-HT(1B)-like receptor.

PubMed

Moloney, G P; Martin, G R; Mathews, N; Milne, A; Hobbs, H; Dodsworth, S; Sang, P Y; Knight, C; Williams, M; Maxwell, M; Glen, R C

1999-07-15

The synthesis and vascular 5-HT(1B)-like receptor activity of a novel series of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives are described. Modifications to the 5-ethylene-linked heterocycle and to substituents on the 2-benzylamide side chain have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B)-like receptor of pK(B) > 7.0, up to 100-fold selectivity over alpha(1)-adrenoceptor affinity and 5-HT(2A) receptor affinity, and which exhibited a favorable pharmacokinetic profile. N-Benzyl-3-[2-(dimethylamino)ethyl]-5-[2-(4,4-dimethyl-2, 5-dioxo-1-imidazolidinyl)ethyl]-1H-indole-2-carboxamide (23) was identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B)-like receptor-mediated agonist activity in the rabbit femoral artery), and competitive vascular 5-HT(1B)-like receptor antagonist with a plasma elimination half-life of approximately 4 h in dog plasma and with good oral bioavailability. The selectivity of compounds from this series for the vascular 5-HT(1B)-like receptors over other receptor subtypes is discussed as well as a proposed mode of binding to the receptor pharmacophore. It has been proposed that the aromatic ring of the 2, N-benzylcarboxamide group can occupy an aromatic binding site rather than the indole ring. The resulting conformation allows an amine-binding site to be occupied by the ethylamine nitrogen and a hydrogen-bonding site to be occupied by one of the hydantoin carbonyls. The electronic nature of the 2,N-benzylcarboxamide aromatic group as well as the size of substituents on this aromatic group is crucial for producing potent and selective antagonists. The structural requirement on the 3-ethylamine side chain incorporating the protonatable nitrogen is achieved by the bulky 2, N-benzylcarboxamide group and its close proximity to the 3-side chain.

Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh,S.; Yamashita, A.; Gouaux, E.

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibitionmore » exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.« less

Unbinding Pathways of an Agonist and an Antagonist from the 5-HT3 Receptor

PubMed Central

Thompson, A. J.; Chau, P.-L.; Chan, S. L.; Lummis, S. C. R.

2006-01-01

The binding sites of 5-HT3 and other Cys-loop receptors have been extensively studied, but there are no data on the entry and exit routes of ligands for these sites. Here we have used molecular dynamics simulations to predict the pathway for agonists and antagonists exiting from the 5-HT3 receptor binding site. The data suggest that the unbinding pathway follows a tunnel at the interface of two subunits, which is ∼8 Å long and terminates ∼20 Å above the membrane. The exit routes for an agonist (5-HT) and an antagonist (granisetron) were similar, with trajectories toward the membrane and outward from the ligand binding site. 5-HT appears to form many hydrogen bonds with residues in the unbinding pathway, and experiments show that mutating these residues significantly affects function. The location of the pathway is also supported by docking studies of granisetron, which show a potential binding site for granisetron on the unbinding route. We propose that leaving the binding pocket along this tunnel places the ligands close to the membrane and prevents their immediate reentry into the binding pocket. We anticipate similar exit pathways for other members of the Cys-loop receptor family. PMID:16387779

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

DOE Office of Scientific and Technical Information (OSTI.GOV)

Middleton, R.E.; Cohen, J.B.

1991-07-16

The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Xiao-Min, E-mail: rxm200318@gmail.com; Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn; Gao, Yu, E-mail: francesscototti@gmail.com

2013-05-01

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions in experimental animals, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to TH receptors (TRs). However, previous data on TH receptor binding and TH activity of OH-PBDEs were very limited and sometimes inconsistent. In the present paper, we examined the binding potency of ten OH-PBDEs with different degrees of bromination to TR using a fluorescence competitive binding assay. The results showed that the ten OH-PBDEs bound to TR with potency that correlated to their bromination level. We further examined their effectmore » on TR using a coactivator binding assay and GH3 cell proliferation assay. Different TR activities of OH-PBDEs were observed depending on their degree of bromination. Four low-brominated OH-PBDEs (2′-OH-BDE-28, 3′-OH-BDE-28, 5-OH-BDE-47, 6-OH-BDE-47) were found to be TR agonists, which recruited the coactivator peptide and enhanced GH3 cell proliferation. However, three high-brominated OH-PBDEs (3-OH-BDE-100, 3′-OH-BDE-154, 4-OH-BDE-188) were tested to be antagonists. Molecular docking was employed to simulate the interactions of OH-PBDEs with TR and identify the structural determinants for TR binding and activity. According to the docking results, low-brominated OH-PBDEs, which are weak binders but TR agonists, bind with TR at the inner side of its binding pocket, whereas high-brominated compounds, which are potent binders but TR antagonists, reside at the outer region. These results indicate that OH-PBDEs have different activities on TR (agonistic or antagonistic), possibly due to their different binding geometries with the receptor. - Highlights: ► Thyroid hormone (TH) activity of OH-PBDEs with different Br number was evaluated. ► Four different experimental approaches were employed to investigate the mechanism. ► Low-brominated OH-PBDEs were agonists, but high-brominated ones were antagonists. ► Low-brominated OH-PBDEs bind to TH receptor differently than high-brominated ones.« less

Virtual High-Throughput Screening To Identify Novel Activin Antagonists

PubMed Central

Zhu, Jie; Mishra, Rama K.; Schiltz, Gary E.; Makanji, Yogeshwar; Scheidt, Karl A.; Mazar, Andrew P.; Woodruff, Teresa K.

2015-01-01

Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex’s binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases. PMID:26098096

Functional antagonistic properties of clozapine at the 5-HT3 receptor.

PubMed

Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

1996-08-23

The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

Stereochemical requirements for central and peripheral muscarinic and antimuscarinic activity of some acetylenic compounds related to oxotremorine.

PubMed Central

Dahlbom, R.; Jenden, D. J.; Resul, B.; Ringdahl, B.

1982-01-01

1 The enantiomers of some analogues of the central muscarinic agent, oxotremorine, were prepared and investigated for tremorogenic and tremorolytic activity in intact mice and for muscarinic and antimuscarinic activity on the isolated ileum of the guinea-pig. 2 The R-isomers were more potent than the S-isomers both in vivo and in vitro regardless of whether the compounds are agonists, partial agonists or competitive antagonists. 3 It is suggested that in the oxotremorine series, agonists and antagonists interact with a common receptor site, in contrast to classical muscarinic antagonists which are believed to bind also to accessory receptor areas, located close to the agonist binding site. PMID:7093587

Interleukin 1 receptor antagonist is a member of the interleukin 1 gene family: evolution of a cytokine control mechanism.

PubMed Central

Eisenberg, S P; Brewer, M T; Verderber, E; Heimdal, P; Brandhuber, B J; Thompson, R C

1991-01-01

Interleukin 1 receptor antagonist (IL-1ra) is a protein that binds to the IL-1 receptor and blocks the binding of both IL-1 alpha and -beta without inducing a signal of its own. Human IL-1ra has some sequence identity to human IL-1 beta, but the evolutionary relationship between these proteins has been unclear. We show that the genes for human, mouse, and rat IL-1ra are similar to the genes for IL-1 alpha and IL-1 beta in intron-exon organization, indicating that gene duplication events were important in the creation of this gene family. Furthermore, an analysis of sequence comparisons and mutation rates for IL-1 alpha, IL-1 beta, and IL-1ra suggests that the duplication giving rise to the IL-1ra gene was an early event in the evolution of the gene family. Comparisons between the mature sequences for IL-1ra, IL-1 alpha, and IL-1 beta suggest that IL-1ra has a beta-stranded structure like to IL-1 alpha and IL-1 beta, consistent with the three proteins being related. The N-terminal sequences of IL-1ra appear to be derived from a region of the genome different than those of IL-1 alpha and IL-1 beta, thus explaining their different modes of biosynthesis and suggesting an explanation for their different biological activities. Images PMID:1828896

Identification of two H3-histamine receptor subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

West, R.E. Jr.; Zweig, A.; Shih, N.Y.

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealedmore » two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.« less

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

PubMed

Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

2016-03-24

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

PubMed

Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

2008-10-01

GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

Opiate antagonist prevents μ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth.

PubMed

Sarkar, Dipak K; Sengupta, Amitabha; Zhang, Changqing; Boyadjieva, Nadka; Murugan, Sengottuvelan

2012-05-11

In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

The pure estrogen receptor antagonist ICI 182,780 promotes a novel interaction of estrogen receptor-alpha with the 3',5'-cyclic adenosine monophosphate response element-binding protein-binding protein/p300 coactivators.

PubMed

Jaber, Basem M; Gao, Tong; Huang, Luping; Karmakar, Sudipan; Smith, Carolyn L

2006-11-01

Estrogen receptor-alpha (ERalpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ERalpha agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ERalpha promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ERalpha ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ERalpha. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ERalpha targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ERalpha is insufficient to promote transcription of ERalpha target genes.

Inhibition of /sup 3/H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aharony, D.; Falcone, R.C.; Krell, R.D.

1987-12-01

The specific binding of (/sup 3/H)5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ((/sup 3/H)LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. (/sup 3/H)LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound (/sup 3/H)LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2more » less than 5 min) and full dissociation. Equilibrium saturation analysis of (/sup 3/H)LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of (/sup 3/H)LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.« less

Dopamine Transporter-Dependent and -Independent Striatal Binding of the Benztropine Analog JHW 007, a Cocaine Antagonist with Low Abuse Liability

PubMed Central

Kopajtic, Theresa A.; Liu, Yi; Surratt, Christopher K.; Donovan, David M.; Newman, Amy H.; Katz, Jonathan L.

2010-01-01

The benztropine analog N-(n-butyl)-3α-[bis(4′-fluorophenyl)methoxy]-tropane (JHW 007) displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks the effects of cocaine, including its self-administration. To determine sites responsible for the cocaine antagonist effects of JHW 007, its in vitro binding was compared with that of methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (WIN 35428) in rats, mice, and human DAT (hDAT)-transfected cells. A one-site model, with Kd values of 4.21 (rat) and 8.99 nM (mouse) best fit the [3H]WIN 35428 data. [3H]JHW 007 binding best fit a two-site model (rat, 7.40/4400 nM; mouse, 8.18/2750 nM), although a one-site fit was observed with hDAT membranes (43.7 nM). Drugs selective for the norepinephrine and serotonin transporters had relatively low affinity in competition with [3H]JHW 007 binding, as did drugs selective for other sites identified previously as potential JHW 007 binding sites. The association of [3H]WIN 35428 best fit a one-phase model, whereas the association of [3H]JHW 007 best fit a two-phase model in all tissues. Because cocaine antagonist effects of JHW 007 have been observed previously soon after injection, its rapid association observed here may contribute to those effects. Multiple [3H]JHW 007 binding sites were obtained in tissue from mice lacking the DAT, suggesting these as yet unidentified sites as potential contributors to the cocaine antagonist effects of JHW 007. Unlike WIN 35428, the binding of JHW 007 was Na+-independent. This feature of JHW 007 has been linked to the conformational status of the DAT, which in turn may contribute to the antagonism of cocaine. PMID:20855444

Abscisic Acid Acts as a Blocker of the Bitter Taste G Protein-Coupled Receptor T2R4.

PubMed

Pydi, Sai P; Jaggupilli, Appalaraju; Nelson, Ken M; Abrams, Suzanne R; Bhullar, Rajinder P; Loewen, Michele C; Chelikani, Prashen

2015-04-28

Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. In humans, 25 T2Rs mediate bitter taste sensation. In addition to the oral cavity, T2Rs are expressed in many extraoral tissues, including the central nervous system, respiratory system, and reproductive system. To understand the mechanistic roles of the T2Rs in oral and extraoral tissues, novel blockers or antagonists are urgently needed. Recently, we elucidated the binding pocket of T2R4 for its agonist quinine, and an antagonist and inhibitory neurotransmitter, γ-aminobutyric acid. This structure-function information about T2R4 led us to screen the plant hormone abscisic acid (ABA), its precursor (xanthoxin), and catabolite phaseic acid for their ability to bind and activate or inhibit T2R4. Molecular docking studies followed by functional assays involving calcium imaging confirmed that ABA is an antagonist with an IC50 value of 34.4 ± 1.1 μM. However, ABA precursor xanthoxin acts as an agonist on T2R4. Interestingly, molecular model-guided site-directed mutagenesis suggests that the T2R4 residues involved in quinine binding are also predominantly involved in binding to the novel antagonist, ABA. The antagonist ability of ABA was tested using another T2R4 agonist, yohimbine. Our results suggest that ABA does not inhibit yohimbine-induced T2R4 activity. The discovery of natural bitter blockers has immense nutraceutical and physiological significance and will help in dissecting the T2R molecular pathways in various tissues.

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

PubMed

Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

1998-11-06

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

An efficient synthesis of a rationally designed 1,5 disubstituted imidazole AT(1) angiotensin II receptor antagonist: reorientation of imidazole pharmacophore groups in losartan reserves high receptor affinity and confirms docking studies.

PubMed

Agelis, George; Roumelioti, Panagiota; Resvani, Amalia; Durdagi, Serdar; Androutsou, Maria-Eleni; Kelaidonis, Konstantinos; Vlahakos, Demetrios; Mavromoustakos, Thomas; Matsoukas, John

2010-09-01

A new 1,5 disubstituted imidazole AT(1) Angiotensin II (AII) receptor antagonist related to losartan with reversion of butyl and hydroxymethyl groups at the 2-, 5-positions of the imidazole ring was synthesized and evaluated for its antagonist activity (V8). In vitro results indicated that the reorientation of butyl and hydroxymethyl groups on the imidazole template of losartan retained high binding affinity to the AT(1) receptor concluding that the spacing of the substituents at the 2,5- positions is of primary importance. The docking studies are confirmed by binding assay results which clearly show a comparable binding score of the designed compound V8 with that of the prototype losartan. An efficient, regioselective and cost effective synthesis renders the new compound as an attractive candidate for advanced toxicological evaluation and a drug against hypertension.

Rational Design of Potent Antagonists to the Human Growth Hormone Receptor

NASA Astrophysics Data System (ADS)

Fuh, Germaine; Cunningham, Brian C.; Fukunaga, Rikiro; Nagata, Shigekazu; Goeddel, David V.; Wells, James A.

1992-06-01

A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially-a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

An efficient synthesis of a rationally designed 1,5 disubstituted imidazole AT1 Angiotensin II receptor antagonist: reorientation of imidazole pharmacophore groups in losartan reserves high receptor affinity and confirms docking studies

NASA Astrophysics Data System (ADS)

Agelis, George; Roumelioti, Panagiota; Resvani, Amalia; Durdagi, Serdar; Androutsou, Maria-Eleni; Kelaidonis, Konstantinos; Vlahakos, Demetrios; Mavromoustakos, Thomas; Matsoukas, John

2010-09-01

A new 1,5 disubstituted imidazole AT1 Angiotensin II (AII) receptor antagonist related to losartan with reversion of butyl and hydroxymethyl groups at the 2-, 5-positions of the imidazole ring was synthesized and evaluated for its antagonist activity ( V8). In vitro results indicated that the reorientation of butyl and hydroxymethyl groups on the imidazole template of losartan retained high binding affinity to the AT1 receptor concluding that the spacing of the substituents at the 2,5- positions is of primary importance. The docking studies are confirmed by binding assay results which clearly show a comparable binding score of the designed compound V8 with that of the prototype losartan. An efficient, regioselective and cost effective synthesis renders the new compound as an attractive candidate for advanced toxicological evaluation and a drug against hypertension.

Structural basis for Smoothened receptor modulation and chemoresistance to anti-cancer drugs

PubMed Central

Wang, Chong; Wu, Huixian; Evron, Tama; Vardy, Eyal; Han, Gye Won; Huang, Xi-Ping; Hufeisen, Sandy J.; Mangano, Thomas J.; Urban, Dan J.; Katritch, Vsevolod; Cherezov, Vadim; Caron, Marc G.; Roth, Bryan L.; Stevens, Raymond C.

2014-01-01

The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumors; however, mutations at SMO have been found to abolish their anti-tumor effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6–2.8Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D4736.55 elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules. PMID:25008467

Pharmacological characterization of CCKB receptors in human brain: no evidence for receptor heterogeneity.

PubMed

Kinze, S; Schöneberg, T; Meyer, R; Martin, H; Kaufmann, R

1996-10-11

In this paper, cholecystokinin (CCK) B-type binding sites were characterized with receptor binding studies in different human brain regions (various parts of cerebral cortex, basal ganglia, hippocampus, thalamus, cerebellar cortex) collected from 22 human postmortem brains. With the exception of the thalamus, where no specific CCK binding sites were found, a pharmacological characterization demonstrated a single class of high affinity CCK sites in all brain areas investigated. Receptor densities ranged from 0.5 fmol/mg protein (hippocampus) to 8.4 fmol/mg protein (nucleus caudatus). These CCK binding sites displayed a typical CCKA binding profile as shown in competition studies by using different CCK-related compounds and non peptide CCK antagonists discriminating between CCKA and CCKB sites. The rank order of agonist or antagonist potency in inhibiting specific sulphated [propionyl-3H]cholecystokinin octapeptide binding was similar and highly correlated for the brain regions investigated as demonstrated by a computer-assisted analysis. Therefore it is concluded that CCKB binding sites in human cerebral cortex, basal ganglia, cerebellar cortex share identical ligand binding characteristics.

Detection of angiotensin II binding to single adrenal zona glomerulosa cells by confocal Raman microspectroscopy

NASA Astrophysics Data System (ADS)

McCoy, Michael J.; Habermann, Timothy J.; Hanke, Craig J.; Adar, Fran; Campbell, William B.; Nithipatikom, Kasem

1999-04-01

We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.

Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination.

PubMed

Ren, Xiao-Min; Guo, Liang-Hong; Gao, Yu; Zhang, Bin-Tian; Wan, Bin

2013-05-01

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions in experimental animals, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to TH receptors (TRs). However, previous data on TH receptor binding and TH activity of OH-PBDEs were very limited and sometimes inconsistent. In the present paper, we examined the binding potency of ten OH-PBDEs with different degrees of bromination to TR using a fluorescence competitive binding assay. The results showed that the ten OH-PBDEs bound to TR with potency that correlated to their bromination level. We further examined their effect on TR using a coactivator binding assay and GH3 cell proliferation assay. Different TR activities of OH-PBDEs were observed depending on their degree of bromination. Four low-brominated OH-PBDEs (2'-OH-BDE-28, 3'-OH-BDE-28, 5-OH-BDE-47, 6-OH-BDE-47) were found to be TR agonists, which recruited the coactivator peptide and enhanced GH3 cell proliferation. However, three high-brominated OH-PBDEs (3-OH-BDE-100, 3'-OH-BDE-154, 4-OH-BDE-188) were tested to be antagonists. Molecular docking was employed to simulate the interactions of OH-PBDEs with TR and identify the structural determinants for TR binding and activity. According to the docking results, low-brominated OH-PBDEs, which are weak binders but TR agonists, bind with TR at the inner side of its binding pocket, whereas high-brominated compounds, which are potent binders but TR antagonists, reside at the outer region. These results indicate that OH-PBDEs have different activities on TR (agonistic or antagonistic), possibly due to their different binding geometries with the receptor. Copyright © 2013 Elsevier Inc. All rights reserved.

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

PubMed

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

2012-09-11

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

Meta-diamide insecticides acting on distinct sites of RDL GABA receptor from those for conventional noncompetitive antagonists.

PubMed

Nakao, Toshifumi; Banba, Shinich; Nomura, Michikazu; Hirase, Kangetsu

2013-04-01

The RDL GABA receptor is an attractive target of insecticides. Here we demonstrate that meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor antagonists showing high insecticidal activity against Spodoptera litura. We also suggest that the mode of action of the meta-diamides is distinct from that of conventional noncompetitive antagonists (NCAs), such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. Using a membrane potential assay, we examined the effects of the meta-diamide 3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7) and NCAs on mutant Drosophila RDL GABA receptors expressed in Drosophila Mel-2 cells. NCAs had little or no inhibitory activity against at least one of the three mutant receptors (A2'S, A2'G, and A2'N), which were reported to confer resistance to NCAs. In contrast, meta-diamide 7 inhibited all three A2' mutant receptors, at levels comparable to its activity with the wild-type receptor. Furthermore, the A2'S·T6'V mutation almost abolished the inhibitory effects of all NCAs. However, meta-diamide 7 inhibited the A2'S・T6'S mutant receptor at the same level as its activity with the wild-type receptor. In contrast, a G336M mutation in the third transmembrane domain of the RDL GABA receptor abolished the inhibitory activities of meta-diamide 7, although the G336M mutation had little effect on the inhibitory activities of conventional NCAs. Molecular modeling studies also suggested that the binding site of meta-diamides was different from those of NCAs. Meta-diamide insecticides are expected to be prominent insecticides effective against A2' mutant RDL GABA receptors with a different mode of action. Copyright © 2013 Elsevier Ltd. All rights reserved.

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes via Nonequilibrium Candidate Monte Carlo.

PubMed

Gill, Samuel C; Lim, Nathan M; Grinaway, Patrick B; Rustenburg, Ariën S; Fass, Josh; Ross, Gregory A; Chodera, John D; Mobley, David L

2018-05-31

Accurately predicting protein-ligand binding affinities and binding modes is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation time scales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes. In this technique, the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over 2 orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step toward applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding modes of ligands using enhanced sampling (BLUES) package which is freely available on GitHub.

Interaction of fenoterol stereoisomers with β2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

PubMed

Reinartz, Michael T; Kälble, Solveig; Wainer, Irving W; Seifert, Roland

2015-05-01

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the β2-adrenoceptor (β2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using β2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Macromolecular beta-adrenergic antagonists discriminating between receptor and antibody.

PubMed Central

Pitha, J; Zjawiony, J; Lefkowitz, R J; Caron, M G

1980-01-01

The beta-adrenergic antagonist, alprenolol, was attached in an irreversible manner to macromolecular dextran via side arms that differed in length. The ability of these macromolecules to bind to the beta-adrenergic receptor of frog erythrocytes and to catecholamine-binding antibodies raised against partially purified receptors was studied. Compared to the parent drug the potency of binding of macromolecular alprenolol to the receptor decreased about 1/10, 1/600, and 1/8000 when the length of the arm separating alprenolol from the dextran moiety was 13, 8, and 4 atoms, respectively. In contrast, the binding potencies of the parent drug and of all its macromolecular derivatives for the antibody were within the same order of magnitude. Thus, conversion of a drug to a macromolecular form may not only sustain its binding activity but may also lead in a higher selectivity. The macromolecular derivatives described here may be suitable probes for investigation of the location and of the molecular properties of the binding sites for beta-adrenergic drugs. PMID:6154947

Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure based modeling methods

PubMed Central

Politi, Regina; Rusyn, Ivan; Tropsha, Alexander

2016-01-01

The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure-Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R2=0.55 and CCR=0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R2=0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. PMID:25058446

Structural insights into selective agonist actions of tamoxifen on human estrogen receptor alpha.

PubMed

Chakraborty, Sandipan; Biswas, Pradip Kumar

2014-08-01

Tamoxifen-an anti-estrogenic ligand in breast tissues used as a first-line treatment in estrogen receptor (ER)-positive breast cancers-is associated with the development of resistance followed by resumption of tumor growth in about 30 % of cases. Whether tamoxifen assists in proliferation in such cases or whether any ligand-independent pathway to transcription exists is not fully understood; also, no ERα mutants have been detected so far that could lead to tamoxifen resistance. Using in silico conformational analysis of the ERα ligand binding domain (LBD), in the absence and presence of selective agonist (diethylstilbestrol; DES), antagonist (Faslodex; ICI), and selective estrogen receptor modulator (SERM; 4-hydroxy tamoxifen; 4-OHT) ligands, we have elucidated ligand-responsive structural modulations of the ERα-LBD dimer in its agonist and antagonist complexes to address the issue of "tamoxifen resistance". DES and ICI were found to stabilize the dimer in their agonist and antagonist conformations, respectively. The ERα-LBD dimer without the presence of any bound ligand also led to a stable structure in agonist conformation. However, binding of 4-OHT to the antagonist structure led to a flexible conformation allowing the protein to visit conformations populated by agonists as was evident from principal component analysis and radius of gyration plots. Further, the relaxed conformations of the 4-OHT bound protein exhibited a diminished size of the co-repressor binding pocket in the LBD, thus signaling a partial blockage of the co-repressor binding motif. Thus, the ability of 4-OHT-bound ERα-LBD to assume flexible conformations visited by agonists and reduced co-repressor binding surface at the LBD provide crucial structural insights into tamoxifen-resistance that complement our existing understanding.

Benzimidazoles as benzamide replacements within cyclohexane-based CC chemokine receptor 2 (CCR2) antagonists.

PubMed

Cherney, Robert J; Mo, Ruowei; Meyer, Dayton T; Pechulis, Anthony D; Guaciaro, Michael A; Lo, Yvonne C; Yang, Gengjie; Miller, Persymphonie B; Scherle, Peggy A; Zhao, Qihong; Cvijic, Mary Ellen; Barrish, Joel C; Decicco, Carl P; Carter, Percy H

2012-10-01

We describe the design, synthesis, and evaluation of benzimidazoles as benzamide replacements within a series of trisubstituted cyclohexane CCR2 antagonists. 7-Trifluoromethylbenzimidazoles displayed potent binding and functional antagonism of CCR2 while being selective over CCR3. These benzimidazoles were also incorporated into lactam-containing antagonists, thus completely eliminating the customary bis-amide. Copyright © 2012 Elsevier Ltd. All rights reserved.

High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

PubMed Central

Drisdel, Renaldo C.; Sharp, Douglas; Henderson, Tricia; Hales, Tim G.; Green, William N.

2008-01-01

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. PMID:17702741

The Conformational Variability of FimH: Which Conformation Represents the Therapeutic Target?

PubMed

Eris, Deniz; Preston, Roland C; Scharenberg, Meike; Hulliger, Fabian; Abgottspon, Daniela; Pang, Lijuan; Jiang, Xiaohua; Schwardt, Oliver; Ernst, Beat

2016-06-02

FimH is a bacterial lectin found at the tips of type 1 pili of uropathogenic Escherichia coli (UPEC). It mediates shear-enhanced adhesion to mannosylated surfaces. Binding of UPEC to urothelial cells initiates the infection cycle leading to urinary tract infections (UTIs). Antiadhesive glycomimetics based on α-d-mannopyranose offer an attractive alternative to the conventional antibiotic treatment because they do not induce a selection pressure and are therefore expected to have a reduced resistance potential. Genetic variation of the fimH gene in clinically isolated UPEC has been associated with distinct mannose binding phenotypes. For this reason, we investigated the mannose binding characteristics of four FimH variants with mannose-based ligands under static and hydrodynamic conditions. The selected FimH variants showed individually different binding behavior under both sets of conditions as a result of the conformational variability of FimH. Clinically relevant FimH variants typically exist in a dynamic conformational equilibrium. Additionally, we evaluated inhibitory potencies of four FimH antagonists representing different structural classes. Inhibitory potencies of three of the tested antagonists were dependent on the binding phenotype and hence on the conformational equilibrium of the FimH variant. However, the squarate derivative was the notable exception and inhibited FimH variants irrespective of their binding phenotype. Information on antagonist affinities towards various FimH variants has remained largely unconsidered despite being essential for successful antiadhesion therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

Binding of anticancer drug daunomycin to a TGGGGT G-quadruplex DNA probed by all-atom molecular dynamics simulations: additional pure groove binding mode and implications on designing more selective G-quadruplex ligands.

PubMed

Shen, Zhanhang; Mulholland, Kelly A; Zheng, Yujun; Wu, Chun

2017-09-01

DNA G-quadruplex structures are emerging cancer-specific targets for chemotherapeutics. Ligands that bind to and stabilize DNA G-quadruplexes have the potential to be anti-cancer drugs. Lack of binding selectivity to DNA G-quadruplex over DNA duplex remains a major challenge when attempting to develop G-quadruplex ligands into successful anti-cancer drugs. Thorough understanding of the binding nature of existing non-selective ligands that bind to both DNA quadruplex and DNA duplex will help to address this challenge. Daunomycin and doxorubicin, two commonly used anticancer drugs, are examples of non-selective DNA ligands. In this study, we extended our early all-atom binding simulation studies between doxorubicin and a DNA duplex (d(CGATCG) 2 ) to probe the binding between daunomycin and a parallel DNA quadruplex (d(TGGGGT) 4 ) and DNA duplex. In addition to the end stacking mode, which mimics the mode in the crystal structure, a pure groove binding mode was observed in our free binding simulations. The dynamic and energetic properties of these two binding modes are thoroughly examined, and a detailed comparison is made between DNA quadruplex binding modes and DNA duplex binding modes. Implications on the design of more selective DNA quadruplex ligands are also discussed. Graphical abstract Top stacking and groov binding modes from the MD simulations.

Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

PubMed Central

Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

2011-01-01

Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

Estrogenicity of halogenated bisphenol A: in vitro and in silico investigations.

PubMed

Zhang, Jie; Li, Tiezhu; Wang, Tuoyi; Yuan, Cuiping; Zhong, Shuning; Guan, Tianzhu; Li, Zhuolin; Wang, Yongzhi; Yu, Hansong; Luo, Quan; Wang, Yongjun; Zhang, Tiehua

2018-03-01

The binding interactions of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to human estrogen receptor α ligand binding domain (hERα-LBD) was investigated using a combined in vitro and in silico approach. First, the recombinant hERα-LBD was prepared as a soluble protein in Escherichia coli BL21(DE3)pLysS. A native fluorescent phytoestrogen, coumestrol, was employed as tracer for the fluorescence polarization assay. The results of the in vitro binding assay showed that bisphenol compounds could bind to hERα-LBD as the affinity ligands. All the tested halogenated BPAs exhibited weaker receptor binding than BPA, which might be explained by the steric effect of substituents. Molecular docking studies elucidated that the halogenated BPAs adopted different conformations in the flexible hydrophobic ligand binding pocket (LBP), which is mainly dependent on their distinct halogenation patterns. The compounds with halogen substituents on the phenolic rings and on the bridging alkyl moiety acted as agonists and antagonists for hERα, respectively. Interestingly, all the compounds in the agonist conformation of hERα formed a hydrogen bond with His524, while the compounds in the antagonist conformation formed a hydrogen bond with Thr347. These docking results suggested a pivotal role of His524/Thr347 in maintaining the hERα structure in the biologically active agonist/antagonist conformation. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation, which might be applicable for the structure-based design of novel bisphenol compounds with reduced toxicities and for environmental risk assessment. In addition, based on hERα-LBD as a recognition element, the proposed fluorescence polarization assay may offer an alternative to chromatographic techniques for the multi-residue determination of bisphenol compounds.

Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

PubMed

Schiffer, Lina; Müller, Anne-Rose; Hobler, Anna; Brixius-Anderko, Simone; Zapp, Josef; Hannemann, Frank; Bernhardt, Rita

2016-10-01

Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

PubMed

Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I; Lanciego, José L; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

2017-01-01

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB 2 receptors (CB 2 Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB 2 R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB 2 R. Using membrane preparations from CB 2 R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB 2 R where the synthetic cannabinoid, [ 3 H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB 2 R-selective compound, CM-157. The effect on binding to CB 2 R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the K D . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB 2 R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors

PubMed Central

Martínez-Pinilla, Eva; Varani, Katia; Reyes-Resina, Irene; Angelats, Edgar; Vincenzi, Fabrizio; Ferreiro-Vera, Carlos; Oyarzabal, Julen; Canela, Enric I.; Lanciego, José L.; Nadal, Xavier; Navarro, Gemma; Borea, Pier Andrea; Franco, Rafael

2017-01-01

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities. PMID:29109685

Guanosine-5'-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition.

PubMed

Molz, Simone; Dal-Cim, Tharine; Tasca, Carla I

2009-12-01

Guanine derivatives modulate the glutamatergic system through displacement of binding of glutamate to its receptors acting as antagonist of glutamate receptors in moderate to high micromolar concentrations. Guanosine-5'-monophosphate (GMP) is shown to be neuroprotective against glutamate- or oxygen/glucose deprivation-induced neurotoxicity and also against NMDA-induced apoptosis in hippocampal slices. However, in this study we are showing that high extracellular GMP concentrations (5mM) reduced cell viability in hippocampal brain slices. The toxic effect of GMP was not blocked by dipyridamole, a nucleoside transport inhibitor, nor mimicked by guanosine, suggesting an extracellular mode of action to GMP which does not involve its hydrolysis to guanosine. GMP-dependent cell damage was not blocked by P1 purinergic receptor antagonists, neither altered by adenosine A(1) or A(2A) receptor agonists. The blockage of the ionotropic glutamate receptors AMPA or NMDA, but not KA or metabotropic glutamate receptors, reversed the toxicity induced by GMP. GMP (5mM) induced a decrease in glutamate uptake into hippocampal slices, which was reversed by dl-TBOA. Therefore, GMP-induced hippocampal cell damage involves activation of ionotropic glutamate receptors and inhibition of glutamate transporters activity.

Superbinder SH2 domains act as antagonists of cell signaling.

PubMed

Kaneko, Tomonori; Huang, Haiming; Cao, Xuan; Li, Xing; Li, Chengjun; Voss, Courtney; Sidhu, Sachdev S; Li, Shawn S C

2012-09-25

Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes.

Characterization of a novel aquaretic agent, OPC-31260, as an orally effective, nonpeptide vasopressin V2 receptor antagonist.

PubMed

Yamamura, Y; Ogawa, H; Yamashita, H; Chihara, T; Miyamoto, H; Nakamura, S; Onogawa, T; Yamashita, T; Hosokawa, T; Mori, T

1992-04-01

1. OPC-31260, a benzazepine derivative, has been studied for its ability to antagonize the binding of arginine vasopressin (AVP) to receptors in rat liver (V1) and kidney (V2) plasma membranes, for antagonism of the antidiuretic action of AVP in alcohol-anaesthetized rats and for diuretic action in conscious normal rats. 2. OPC-31260 caused a competitive displacement of [3H]-AVP binding to both V1 and V2 receptors with IC50 values of 1.2 +/- 0.2 x 10(-6) M and 1.4 +/- 0.2 x 10(-8) M, respectively. 3. OPC-31260 at doses of 10 to 100 micrograms kg-1, i.v., inhibited the antidiuretic action of exogenously administered AVP in water-loaded, alcohol-anaesthetized rats in a dose-dependent manner. OPC-31260 did not exert an antidiuretic activity suggesting that it is not a partial V2 receptor agonist. 4. After oral administration at doses of 1 to 30 mg kg-1 in normal conscious rats, OPC-31260 dose-dependently increased urine flow and decreased urine osmolality. The diuretic action of OPC-31260 was characterized as aquaresis, the mode of diuretic action being different from previously known diuretic agents such as furosemide, hydrochlorothiazide and spironolactone. 5. The results indicate that OPC-31260 is a selective V2 receptor antagonist and behaves as an aquaretic agent. OPC-31260 will be a useful tool in studying the physiological role of AVP and in the treatment of various conditions characterized by water retention.

Characterization of a novel aquaretic agent, OPC-31260, as an orally effective, nonpeptide vasopressin V2 receptor antagonist

PubMed Central

Yamamura, Yoshitaka; Ogawa, Hidenori; Yamashita, Hiroshi; Chihara, Tomihiko; Miyamoto, Hisashi; Nakamura, Shigeki; Onogawa, Toshiyuki; Yamashita, Tatsuya; Hosokawa, Tetsumi; Mori, Toyoki; Tominaga, Michiaki; Yabuuchi, Youichi

1992-01-01

1 OPC-31260, a benzazepine derivative, has been studied for its ability to antagonize the binding of arginine vasopressin (AVP) to receptors in rat liver (V1) and kidney (V2) plasma membranes, for antagonism of the antidiuretic action of AVP in alcohol-anaesthetized rats and for diuretic action in conscious normal rats. 2 OPC-31260 caused a competitive displacement of [3H]-AVP binding to both V1 and V2 receptors with IC50 values of 1.2 ± 0.2 × 10-6 M and 1.4 ± 0.2 × 10-8 M, respectively. 3 OPC-31260 at doses of 10 to 100 μg kg-1, i.v., inhibited the antidiuretic action of exogenously administered AVP in water-loaded, alcohol-anaesthetized rats in a dose-dependent manner. OPC-31260 did not exert an antidiuretic activity suggesting that it is not a partial V2 receptor agonist. 4 After oral administration at doses of 1 to 30 mg kg-1 in normal conscious rats, OPC-31260 dose-dependently increased urine flow and decreased urine osmolality. The diuretic action of OPC-31260 was characterized as aquaresis, the mode of diuretic action being different from previously known diuretic agents such as furosemide, hydrochlorothiazide and spironolactone. 5 The results indicate that OPC-31260 is a selective V2 receptor antagonist and behaves as an aquaretic agent. OPC-31260 will be a useful tool in studying the physiological role of AVP and in the treatment of various conditions characterized by water retention. PMID:1387020

Binding of Capsaicin to the TRPV1 Ion Channel.

PubMed

Darré, Leonardo; Domene, Carmen

2015-12-07

Transient receptor potential (TRP) ion channels constitute a notable family of cation channels involved in the ability of an organisms to detect noxious mechanical, thermal, and chemical stimuli that give rise to the perception of pain, taste, and changes in temperature. One of the most experimentally studied agonist of TRP channels is capsaicin, which is responsible for the burning sensation produced when chili pepper is in contact with organic tissues. Thus, understanding how this molecule interacts and regulates TRP channels is essential to high impact pharmacological applications, particularly those related to pain treatment. The recent publication of a three-dimensional structure of the vanilloid receptor 1 (TRPV1) in the absence and presence of capsaicin from single particle electron cryomicroscopy experiments provides the opportunity to explore these questions at the atomic level. In the present work, molecular docking and unbiased and biased molecular dynamics simulations were employed to generate a structural model of the capsaicin-channel complex. In addition, the standard free energy of binding was estimated using alchemical transformations coupled with conformational, translational, and orientational restraints on the ligand. Key binding modes consistent with previous experimental data are identified, and subtle but essential dynamical features of the binding site are characterized. These observations shed some light into how TRPV1 interacts with capsaicin, and may help to refine design parameters for new TRPV1 antagonists, and potentially guide further developments of TRP channel modulators.

Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Politi, Regina; Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599; Rusyn, Ivan, E-mail: iir@unc.edu

2014-10-01

The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R{sup 2} = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict bindingmore » affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R{sup 2} = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables prediction of both affinity and efficacy. • Models can be used to identify environmental chemicals interacting with THRβ. • Models can be used to eliminate the THRβ-mediated mechanism of action.« less

Synthesis and biological evaluation of cyclopropyl analogues of 2-amino-5-phosphonopentanoic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dappen, M.S.; Pellicciari, R.; Natalini, B.

1991-01-01

A series of cyclopropyl analogues related to 2-amino-5-phosphonopentanoic acid (AP5) were synthesized and their biological activity was assessed as competitive antagonists for the N-methyl-D-aspartate (NMDA) receptor. In vitro receptor binding using (3H)-L-glutamate as the radioligand provided affinity data, while modulation of (3H)MK-801 binding was used as a functional assay. The analogues were also evaluated in (3H)kainate binding to assess selectivity over non-NMDA glutamate receptors. Of the compounds tested, 4,5-methano-AP5 analogue 26 was the most potent selective NMDA antagonist; however, potency was lower than that for (((+/-)-2-carboxypiperidin-4-yl)methyl)phosphonic acid (CGS 19755, 5).

[125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

PubMed Central

Dumont, Yvan; Quirion, Rémi

2000-01-01

GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]-GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]-GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [125I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]-GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]-GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. PMID:10694200

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

PubMed Central

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

2012-01-01

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF165 to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form ofVEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å2 in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2. PMID:22927390

Classification and virtual screening of androgen receptor antagonists.

PubMed

Li, Jiazhong; Gramatica, Paola

2010-05-24

Computational tools, such as quantitative structure-activity relationship (QSAR), are highly useful as screening support for prioritization of substances of very high concern (SVHC). From the practical point of view, QSAR models should be effective to pick out more active rather than inactive compounds, expressed as sensitivity in classification works. This research investigates the classification of a big data set of endocrine-disrupting chemicals (EDCs)-androgen receptor (AR) antagonists, mainly aiming to improve the external sensitivity and to screen for potential AR binders. The kNN, lazy IB1, and ADTree methods and the consensus approach were used to build different models, which improve the sensitivity on external chemicals from 57.1% (literature) to 76.4%. Additionally, the models' predictive abilities were further validated on a blind collected data set (sensitivity: 85.7%). Then the proposed classifiers were used: (i) to distinguish a set of AR binders into antagonists and agonists; (ii) to screen a combined estrogen receptor binder database to find out possible chemicals that can bind to both AR and ER; and (iii) to virtually screen our in-house environmental chemical database. The in silico screening results suggest: (i) that some compounds can affect the normal endocrine system through a complex mechanism binding both to ER and AR; (ii) new EDCs, which are nonER binders, but can in silico bind to AR, are recognized; and (iii) about 20% of compounds in a big data set of environmental chemicals are predicted as new AR antagonists. The priority should be given to them to experimentally test the binding activities with AR.

Understanding the stereospecific interactions of 3-deoxyphosphatidylinositol derivatives with the PTEN phosphatase domain

PubMed Central

Wang, Qin; Wei, Yang; Mottamal, Madhusoodanan; Roberts, Mary F.; Krilov, Goran

2011-01-01

PTEN is an important control element of PI3K/AKT signaling involved in controlling the processes of embryonic development, cell migration and apoptosis. While its dysfunction is implicated in a large fraction of cancers, PTEN activity in the same pathway may also contribute to metabolic syndromes such as diabetes. In those cases, selective inhibitors of PTEN may be useful. A new class of chiral PTEN inhibitors based on the 3-deoxy-phosphatidylinositol derivatives was recently identified [Wang et al. (2008) J. Am. Chem. Soc. 130, 7746]. However, lack of detailed understanding of protein-ligand interactions has hampered efforts to develop effective agonists or antagonists of PTEN. Here, we use computational modeling to characterize the interactions of the diverse 3-deoxyphosphatidylinositol inhibitors with the PTEN protein. We show that, while each of the compounds binds with the inositol headgroup inserting into the proposed active site of the PTEN phosphatase domain, hydrogen bonding restrictions lead to distinct binding geometries for ligand pairs of opposite chirality. We furthermore demonstrate that the binding modes differ primarily in the orientation of acyl tails of the ligands and that the activity of the compounds is primarily controlled by the effectiveness of tail-protein contacts. These findings are confirmed by binding affinity calculations which are in good agreement with experiment. Finally, we show that while more potent D-series ligands bind in a manner similar to that of the native substrate, an alternate hydrophobic pocket suitable for binding the opposite chirality L-series inhibitors exists, offering the possibility of designing highly selective PTEN- targeting compounds. PMID:20538496

Small Molecule Ligands of Methyl-Lysine Binding Proteins

PubMed Central

Herold, J. Martin; Wigle, Tim J.; Norris, Jacqueline L.; Lam, Robert; Korboukh, Victoria K.; Gao, Cen; Ingerman, Lindsey A.; Kireev, Dmitri B.; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J.; Arrowsmith, Cheryl H.; Jin, Jian; Janzen, William P.; Frye, Stephen V.

2011-01-01

Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design. PMID:21417280

Structural basis for subtype-specific inhibition of the P2X7 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karasawa, Akira; Kawate, Toshimitsu

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of themore » drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.« less

Probing interaction of a fluorescent ligand with HIV TAR RNA

NASA Astrophysics Data System (ADS)

Qi, Liang; Zhang, Jing; He, Tian; Huo, Yuan; Zhang, Zhi-Qi

2017-02-01

Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaakola, Veli-Pekka; Griffith, Mark T.; Hanson, Michael A.

2009-01-15

The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A{sub 2A} adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extendedmore » conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.« less

Identification of isosilybin a from milk thistle seeds as an agonist of peroxisome proliferator-activated receptor gamma.

PubMed

Pferschy-Wenzig, Eva-Maria; Atanasov, Atanas G; Malainer, Clemens; Noha, Stefan M; Kunert, Olaf; Schuster, Daniela; Heiss, Elke H; Oberlies, Nicholas H; Wagner, Hildebert; Bauer, Rudolf; Dirsch, Verena M

2014-04-25

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism. Agonists of this nuclear receptor are used in the treatment of type 2 diabetes and are also studied as a potential treatment of other metabolic diseases, including nonalcoholic fatty liver disease. Silymarin, a concentrated phenolic mixture from milk thistle (Silybum marianum) seeds, is used widely as a supportive agent in the treatment of a variety of liver diseases. In this study, the PPARγ activation potential of silymarin and its main constituents was investigated. Isosilybin A (3) caused transactivation of a PPARγ-dependent luciferase reporter in a concentration-dependent manner. This effect could be reversed upon co-treatment with the PPARγ antagonist T0070907. In silico docking studies suggested a binding mode for 3 distinct from that of the inactive silymarin constituents, with one additional hydrogen bond to Ser342 in the entrance region of the ligand-binding domain of the receptor. Hence, isosilybin A (3) has been identified as the first flavonolignan PPARγ agonist, suggesting its further investigation as a modulator of this nuclear receptor.

[(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

PubMed

Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

2002-02-11

This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

Crystal structure of the adenosine A 2A receptor bound to an antagonist reveals a potential allosteric pocket

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phasemore » diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.« less

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

A Novel Chemical Inhibitor of ABA Signaling Targets All ABA Receptors.

PubMed

Ye, Yajin; Zhou, Lijuan; Liu, Xue; Liu, Hao; Li, Deqiang; Cao, Minjie; Chen, Haifeng; Xu, Lin; Zhu, Jian-Kang; Zhao, Yang

2017-04-01

Abscisic acid (ABA), the most important stress-induced phytohormone, regulates seed dormancy, germination, plant senescence, and the abiotic stress response. ABA signaling is repressed by group A type 2C protein phosphatases (PP2Cs), and then ABA binds to its receptor of the ACTIN RESISTANCE1 (PYR1), PYR1-LIKE (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family, which, in turn, inhibits PP2Cs and activates downstream ABA signaling. The agonist/antagonist of ABA receptors have the potential to reveal the ABA signaling machinery and to become lead compounds for agrochemicals; however, until now, no broad-spectrum antagonists of ABA receptors blocking all PYR/PYL-PP2C interactions have been identified. Here, using chemical genetics screenings, we identified ABA ANTAGONIST1 (AA1), the first broad-spectrum antagonist of ABA receptors in Arabidopsis ( Arabidopsis thaliana ). Physiological analyses revealed that AA1 is sufficiently active to block ABA signaling. AA1 interfered with all the PYR/PYL-HAB1 interactions, and the diminished PYR/PYL-HAB1 interactions, in turn, restored the activity of HAB1. AA1 binds to all 13 members. Molecular dockings, the non-AA1-bound PYL2 variant, and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2. Using AA1, we tested the genetic relationships of ABA receptors with other core components of ABA signaling, demonstrating that AA1 is a powerful tool with which to sidestep this genetic redundancy of PYR/PYLs. In addition, the application of AA1 delays leaf senescence. Thus, our study developed an efficient broad-spectrum antagonist of ABA receptors and demonstrated that plant senescence can be chemically controlled through AA1, with a simple and easy-to-synthesize structure, allowing its availability and utility as a chemical probe synthesized in large quantities, indicating its potential application in agriculture. © 2017 American Society of Plant Biologists. All Rights Reserved.

A Novel Chemical Inhibitor of ABA Signaling Targets All ABA Receptors1

PubMed Central

Ye, Yajin; Liu, Xue; Liu, Hao; Li, Deqiang; Cao, Minjie; Chen, Haifeng; Zhu, Jian-kang

2017-01-01

Abscisic acid (ABA), the most important stress-induced phytohormone, regulates seed dormancy, germination, plant senescence, and the abiotic stress response. ABA signaling is repressed by group A type 2C protein phosphatases (PP2Cs), and then ABA binds to its receptor of the ACTIN RESISTANCE1 (PYR1), PYR1-LIKE (PYL), and REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family, which, in turn, inhibits PP2Cs and activates downstream ABA signaling. The agonist/antagonist of ABA receptors have the potential to reveal the ABA signaling machinery and to become lead compounds for agrochemicals; however, until now, no broad-spectrum antagonists of ABA receptors blocking all PYR/PYL-PP2C interactions have been identified. Here, using chemical genetics screenings, we identified ABA ANTAGONIST1 (AA1), the first broad-spectrum antagonist of ABA receptors in Arabidopsis (Arabidopsis thaliana). Physiological analyses revealed that AA1 is sufficiently active to block ABA signaling. AA1 interfered with all the PYR/PYL-HAB1 interactions, and the diminished PYR/PYL-HAB1 interactions, in turn, restored the activity of HAB1. AA1 binds to all 13 members. Molecular dockings, the non-AA1-bound PYL2 variant, and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2. Using AA1, we tested the genetic relationships of ABA receptors with other core components of ABA signaling, demonstrating that AA1 is a powerful tool with which to sidestep this genetic redundancy of PYR/PYLs. In addition, the application of AA1 delays leaf senescence. Thus, our study developed an efficient broad-spectrum antagonist of ABA receptors and demonstrated that plant senescence can be chemically controlled through AA1, with a simple and easy-to-synthesize structure, allowing its availability and utility as a chemical probe synthesized in large quantities, indicating its potential application in agriculture. PMID:28193765

A Blocking Group Scan Using a Spherical Organometallic Complex Identifies an Unprecedented Binding Mode with Potent Activity In Vitro and In Vivo for the Opioid Peptide Dermorphin.

PubMed

Strack, Martin; Bedini, Andrea; Yip, King T; Lombardi, Sara; Siegmund, Daniel; Stoll, Raphael; Spampinato, Santi M; Metzler-Nolte, Nils

2016-10-04

Herein, the selective enforcement of one particular receptor-ligand interaction between specific domains of the μ-selective opioid peptide dermorphin and the μ opioid receptor is presented. For this, a blocking group scan is described which exploits the steric demand of a bis(quinolinylmethyl)amine rhenium(I) tricarbonyl complex conjugated to a number of different, strategically chosen positions of dermorphin. The prepared peptide conjugates lead to the discovery of two different binding modes: An expected N-terminal binding mode corresponds to the established view of opioid peptide binding, whereas an unexpected C-terminal binding mode is newly discovered. Surprisingly, both binding modes provide high affinity and agonistic activity at the μ opioid receptor in vitro. Furthermore, the unprecedented C-terminal binding mode shows potent dose-dependent antinociception in vivo. Finally, in silico docking studies support receptor activation by both dermorphin binding modes and suggest a biological relevance for dermorphin itself. Relevant ligand-protein interactions are similar for both binding modes, which is in line with previous protein mutation studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Noribogaine is a G-protein biased κ-opioid receptor agonist.

PubMed

Maillet, Emeline L; Milon, Nicolas; Heghinian, Mari D; Fishback, James; Schürer, Stephan C; Garamszegi, Nandor; Mash, Deborah C

2015-12-01

Noribogaine is the long-lived human metabolite of the anti-addictive substance ibogaine. Noribogaine efficaciously reaches the brain with concentrations up to 20 μM after acute therapeutic dose of 40 mg/kg ibogaine in animals. Noribogaine displays atypical opioid-like components in vivo, anti-addictive effects and potent modulatory properties of the tolerance to opiates for which the mode of action remained uncharacterized thus far. Our binding experiments and computational simulations indicate that noribogaine may bind to the orthosteric morphinan binding site of the opioid receptors. Functional activities of noribogaine at G-protein and non G-protein pathways of the mu and kappa opioid receptors were characterized. Noribogaine was a weak mu antagonist with a functional inhibition constants (Ke) of 20 μM at the G-protein and β-arrestin signaling pathways. Conversely, noribogaine was a G-protein biased kappa agonist 75% as efficacious as dynorphin A at stimulating GDP-GTP exchange (EC50=9 μM) but only 12% as efficacious at recruiting β-arrestin, which could contribute to the lack of dysphoric effects of noribogaine. In turn, noribogaine functionally inhibited dynorphin-induced kappa β-arrestin recruitment and was more potent than its G-protein agonistic activity with an IC50 of 1 μM. This biased agonist/antagonist pharmacology is unique to noribogaine in comparison to various other ligands including ibogaine, 18-MC, nalmefene, and 6'-GNTI. We predict noribogaine to promote certain analgesic effects as well as anti-addictive effects at effective concentrations>1 μM in the brain. Because elevated levels of dynorphins are commonly observed and correlated with anxiety, dysphoric effects, and decreased dopaminergic tone, a therapeutically relevant functional inhibition bias to endogenously released dynorphins by noribogaine might be worthy of consideration for treating anxiety and substance related disorders. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

The Binding Mode Prediction and Similar Ligand Potency in the Active Site of Vitamin D Receptor with QM/MM Interaction, MESP, and MD Simulation.

PubMed

Selvaraman, Nagamani; Selvam, Saravana Kumar; Muthusamy, Karthikeyan

2016-08-01

Non-secosteroidal ligands are well-known vitamin D receptor (VDR) agonists. In this study, we described a combined QM/MM to define the protein-ligand interaction energy a strong positive correlation in both QM-MM interaction energy and binding free energy against the biological activity. The molecular dynamics simulation study was performed, and specific interactions were extensively studied. The molecular docking results and surface analysis shed light on steric and electrostatic complementarities of these non-secosteroidal ligands to VDR. Finally, the drug likeness properties were also calculated and found within the acceptable range. The results show that bulky group substitutions in side chain decrease the VDR activity, whereas a small substitution increased it. Functional analyses of H393A and H301A mutations substantiate their roles in the VDR agonistic and antagonistic activities. Apart from the His393 and His301, two other amino acids in the hinge region viz. Ser233 and Arg270 acted as an electron donor/acceptor specific to the agonist in the distinct ligand potency. The results from this study disclose the binding mechanism of VDR agonists and structural modifications required to improve the selectivity. © 2016 John Wiley & Sons A/S.

Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

PubMed

Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

2012-03-14

An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

Characterization of bradykinin receptors in human lung fibroblasts using the binding of 3[H][Des-Arg10,Leu9]kallidin and [3H]NPC17731.

PubMed

Zhang, S P; Codd, E E

1998-01-01

Bradykinin (BK) receptors are involved in pain and inflammation. Two BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. Both B1 and B2 receptors are G-protein coupled membrane receptors. B1 receptors are present in smooth muscle tissue, whereas B2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg10,Leu9]kallidin (DALKD) is a selective B1 receptor antagonist, and NPC17731 is a selective B2 receptor antagonist. To develop binding assays for the two known BK receptor subtypes, [3H]DALKD and [3H]NPC17731 were used as selective ligands for B1 and B2 receptors respectively. Both ligands bound to the CCD-16 human lung fibroblast membranes reaching equilibrium at 25 degrees C within 30 min. Binding was stable for at least 60 min. The Kd of [3H]DALKD was 0.33 nM and Bmax was 52 fmol/mg membrane protein. The Kd of [3H]NPC17731 was 0.39 nM and Bmax was 700 fmol/mg membrane protein. Competition for [3H]DALKD binding with BK receptor agonists was in the order: [des-Arg10]KD (DAKD) > KD > [des-Arg9]BK (DABK) > BK, and competition for [3H]DALKD binding with BK receptor antagonists was in the order: DALKD > [des-Arg10]Hoe 140 (DAHoe 140) > [des-Arg9,Leu8]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [3H]DALKD bound selectively to B1 receptors. By contrast, competition for [3H]NPC17731 binding by BK agonists was in the order: BK > KD > DAKD > DABK, and competition for [3H]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 > DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [3H]NPC17731 labeled B2 receptors selectively. These results demonstrate that [3H]DALKD and [3H]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous evaluation of B1 and B2 BK receptor subtypes.

Design of fluorinated 5-HT(4)R antagonists: influence of the basicity and lipophilicity toward the 5-HT(4)R binding affinities.

PubMed

Fontenelle, Clement Q; Wang, Zhong; Fossey, Christine; Cailly, Thomas; Linclau, Bruno; Fabis, Frederic

2013-12-01

Analogues of potent 5-HT(4)R antagonists possessing a fluorinated N-alkyl chain have been synthesized in order to investigate the effect of the resulting change in basicity and lipophilicity on the affinity and selectivity profile. We demonstrate that for this series, the affinity is decreased with decreased basicity of the piperidine's nitrogen atom. In contrast, the resulting increase in lipophilicity has minimal impact on binding affinity and selectivity. 3,3,3-Trifluoropropyl and 4,4,4-trifluorobutyl derivatives 6d and 6e have shown to bind to the 5-HT(4)R while maintaining their pharmacological profile and selectivity toward other 5-HT receptors. Copyright © 2013 Elsevier Ltd. All rights reserved.

Selective regulation of beta 1- and beta 2-adrenoceptors in the human heart by chronic beta-adrenoceptor antagonist treatment.

PubMed Central

Michel, M. C.; Pingsmann, A.; Beckeringh, J. J.; Zerkowski, H. R.; Doetsch, N.; Brodde, O. E.

1988-01-01

1. In 44 patients undergoing coronary artery bypass grafting, the effect of chronic administration of the beta-adrenoceptor antagonists sotalol, propranolol, pindolol, metoprolol and atenolol on beta-adrenoceptor density in right atria (containing 70% beta 1- and 30% beta 2-adrenoceptors) and in lymphocytes (having only beta 2-adrenoceptors) was studied. 2. beta-Adrenoceptor density in right atrial membranes and in intact lymphocytes was assessed by (-)-[125I]-iodocyanopindolol (ICYP) binding; the relative amount of right atrial beta 1- and beta 2-adrenoceptors was determined by inhibition of ICYP binding by the selective beta 2-adrenoceptor antagonist ICI 118,551 and analysis of the resulting competition curves by the iterative curve fitting programme LIGAND. 3. With the exception of pindolol, all beta-adrenoceptor antagonists increased right atrial beta-adrenoceptor density compared to that observed in atria from patients not treated with beta-adrenoceptor antagonists. 4. All beta-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor density; on the other hand, only sotalol and propranolol also increased right atrial beta 2-adrenoceptor density, whereas metoprolol and atenolol did not affect it and pindolol decreased it. 5. Similarly, in corresponding lymphocytes, only sotalol or propranolol increased beta 2-adrenoceptor density, while metoprolol and atenolol did not affect it and pindolol decreased it. 6. It is concluded that beta-adrenoceptor antagonists subtype-selectively regulate cardiac and lymphocyte beta-adrenoceptor subtypes. The selective increase in cardiac beta 1-adrenoceptor density evoked by metoprolol and atenolol may be one of the reasons for the beneficial effects observed in patients with end-stage congestive cardiomyopathy following intermittent treatment with low doses of selective beta 1-adrenoceptor antagonists. PMID:2902891

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

2012-03-15

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structuremore » of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.« less

Identifying the binding mode of a molecular scaffold

NASA Astrophysics Data System (ADS)

Chema, Doron; Eren, Doron; Yayon, Avner; Goldblum, Amiram; Zaliani, Andrea

2004-01-01

We describe a method for docking of a scaffold-based series and present its advantages over docking of individual ligands, for determining the binding mode of a molecular scaffold in a binding site. The method has been applied to eight different scaffolds of protein kinase inhibitors (PKI). A single analog of each of these eight scaffolds was previously crystallized with different protein kinases. We have used FlexX to dock a set of molecules that share the same scaffold, rather than docking a single molecule. The main mode of binding is determined by the mode of binding of the largest cluster among the docked molecules that share a scaffold. Clustering is based on our `nearest single neighbor' method [J. Chem. Inf. Comput. Sci., 43 (2003) 208-217]. Additional criteria are applied in those cases in which more than one significant binding mode is found. Using the proposed method, most of the crystallographic binding modes of these scaffolds were reconstructed. Alternative modes, that have not been detected yet by experiments, could also be identified. The method was applied to predict the binding mode of an additional molecular scaffold that was not yet reported and the predicted binding mode has been found to be very similar to experimental results for a closely related scaffold. We suggest that this approach be used as a virtual screening tool for scaffold-based design processes.

Design, Synthesis, and Evaluation of Nonretinoid Retinol Binding Protein 4 Antagonists for the Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

PubMed Central

2015-01-01

Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4–/– mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%. PMID:25210858

Design, synthesis, and evaluation of nonretinoid retinol binding protein 4 antagonists for the potential treatment of atrophic age-related macular degeneration and Stargardt disease.

PubMed

Cioffi, Christopher L; Dobri, Nicoleta; Freeman, Emily E; Conlon, Michael P; Chen, Ping; Stafford, Douglas G; Schwarz, Daniel M C; Golden, Kathy C; Zhu, Lei; Kitchen, Douglas B; Barnes, Keith D; Racz, Boglarka; Qin, Qiong; Michelotti, Enrique; Cywin, Charles L; Martin, William H; Pearson, Paul G; Johnson, Graham; Petrukhin, Konstantin

2014-09-25

Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4(-/-) mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%.

Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.

PubMed Central

Halmos, G; Schally, A V; Pinski, J; Vadillo-Buenfil, M; Groot, K

1996-01-01

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites. PMID:8637885

Tacr1 gene variation and neurokinin 1 receptor expression is associated with antagonist efficacy in genetically selected alcohol-preferring rats.

PubMed

Schank, Jesse R; Tapocik, Jenica D; Barbier, Estelle; Damadzic, Ruslan; Eskay, Robert L; Sun, Hui; Rowe, Kelly E; King, Courtney E; Yao, Mengdi; Flanigan, Meghan E; Solomon, Matthew G; Karlsson, Camilla; Cheng, Kejun; Rice, Kenner C; Heilig, Markus

2013-04-15

Genetic deletion or antagonism of the neurokinin 1 receptor (NK1R) decreases alcohol intake, alcohol reward, and stress-induced alcohol relapse in rodents, while TACR1 variation is associated with alcoholism in humans. We used L822429, a specific antagonist with high affinity for the rat NK1R, and examined whether sensitivity to NK1R blockade is altered in alcohol-preferring (P) rats. Operant alcohol self-administration and progressive ratio responding were analyzed in P-rats and their founder Wistar line. We also analyzed Tacr1 expression and binding and sequenced the Tacr1 promoter from both lines. Systemic L822429 decreased alcohol self-administration in P-rats but did not affect the lower rates of alcohol self-administration in Wistar rats. Tacr1 expression was elevated in the prefrontal cortex and the amygdala of P-rats. In central amygdala, elevated Tacr1 expression was accompanied by elevated NK1R binding. Central amygdala (but not prefrontal cortex) infusion of L822429 replicated the systemic antagonist effects on alcohol self-administration in P-rats. All P-rats, but only 18% of their founder Wistar population, were CC homozygous for a-1372G/C single nucleotide polymorphism. In silico analysis indicated that the Tacr1-1372 genotype could modulate binding of the transcription factors GATA-2 and E2F-1. Electromobility shift and luciferase reporter assays suggested that the-1372C allele confers increased transcription factor binding and transcription. Genetic variation at the Tacr1 locus may contribute to elevated rates of alcohol self-administration, while at the same time increasing sensitivity to NK1R antagonist treatment. Published by Elsevier Inc.

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists

PubMed Central

Hiranita, Takato; Kopajtic, Theresa A.; Rice, Kenner C.; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H.; McCurdy, Christopher R.

2016-01-01

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

The opioid receptor pharmacology of GSK1521498 compared to other ligands with differential effects on compulsive reward-related behaviours.

PubMed

Kelly, Eamonn; Mundell, Stuart J; Sava, Anna; Roth, Adelheid L; Felici, Antonio; Maltby, Kay; Nathan, Pradeep J; Bullmore, Edward T; Henderson, Graeme

2015-01-01

The novel opioid receptor antagonist, GSK1421498, has been shown to attenuate reward-driven compulsive behaviours, such as stimulant drug seeking or binge eating, in animals and humans. Here, we report new data on the receptor pharmacology of GSK121498, in comparison to naltrexone, naloxone, 6-β-naltrexol and nalmefene. To determine whether the novel opioid antagonist, GSK1521498, is an orthosteric or allosteric antagonist at the μ opioid receptor (MOPr) and whether it has neutral antagonist or inverse agonist properties. A combination of radioligand binding assays and [(35)S]GTPγS binding assays was employed. GSK1521498 completely displaced [(3)H]naloxone binding to MOPr and did not alter the rate of [(3)H]naloxone dissociation from MOPr observations compatible with it binding to the orthosteric site on MOPr. GSK1521498 exhibited inverse agonism when MOPr was overexpressed but not when the level of MOPr expression was low. In parallel studies under conditions of high receptor expression density, naloxone, naltrexone, 6-β-naltrexol and nalmefene exhibited partial agonism, not inverse agonism as has been reported previously for naloxone and naltrexone. In brain tissue from mice receiving a prolonged morphine pre-treatment, GSK1521498 exhibited slight inverse agonism. Differences between GSK1521498 and naltrexone in their effects on compulsive reward seeking are arguably linked to the more selective and complete MOPr antagonism of GSK1521498 versus the partial MOPr agonism of naltrexone. GSK1521498 is also pharmacologically differentiated by its inverse agonist efficacy at high levels of MOPr expression, but this may be less likely to contribute to behavioural differentiation at patho-physiological levels of expression.

Small Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy

PubMed Central

Han, Bingshe; Park, Dongkyoo; Li, Rui; Xie, Maohua; Owonikoko, Taofeek K.; Zhang, Guojing; Sica, Gabriel L.; Ding, Chunyong; Zhou, Jia; Magis, Andrew T.; Chen, Zhuo G.; Shin, Dong M.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Curran, Walter J.; Deng, Xingming

2015-01-01

SUMMARY The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a small molecule Bcl2-BH4 domain-antagonist (BDA-366) that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival to a cell death inducer. BDA-366 suppresses growth of lung cancer xenografts derived from cell lines and patient without significant normal tissue toxicity at effective doses. mTOR inhibition up-regulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a strategy to improve lung cancer outcome. PMID:26004684

Discovery of potent and novel smoothened antagonists via structure-based virtual screening and biological assays.

PubMed

Lu, Wenfeng; Zhang, Dihua; Ma, Haikuo; Tian, Sheng; Zheng, Jiyue; Wang, Qin; Luo, Lusong; Zhang, Xiaohu

2018-05-23

The Hedgehog (Hh) signaling pathway plays a critical role in controlling patterning, growth and cell migration during embryonic development. Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. As a key member of the Hh pathway, the Smoothened (Smo) receptor, a member of the G protein-coupled receptor (GPCR) family, has emerged as an attractive therapeutic target for the treatment and prevention of human cancers. The recent determination of several crystal structures of Smo in complex with different antagonists offers the possibility to perform structure-based virtual screening for discovering potent Smo antagonists with distinct chemical scaffolds. In this study, based on the two Smo crystal complexes with the best capacity to distinguish the known Smo antagonists from decoys, the molecular docking-based virtual screening was conducted to identify promising Smo antagonists from ChemDiv library. A total of 21 structurally novel and diverse compounds were selected for experimental testing, and six of them exhibited significant inhibitory activity against the Hh pathway activation (IC 50  < 10 μM) in a GRE (Gli-responsive element) reporter gene assay. Specifically, the most potent compound (compound 20: 47 nM) showed comparable Hh signaling inhibition to vismodegib (46 nM). Compound 20 was further confirmed to be a potent Smo antagonist in a fluorescence based competitive binding assay. Optimization using substructure searching method led to the discovery of 12 analogues of compound 20 with decent Hh pathway inhibition activity, including four compounds with IC 50 lower than 1 μM. The important residues uncovered by binding free energy calculation (MM/GBSA) and binding free energy decomposition were highlighted and discussed. These findings suggest that the novel scaffold afforded by compound 20 can be used as a good starting point for further modification/optimization and the clarified interaction patterns may also guide us to find more potent Smo antagonists. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

CC12, A High Affinity Ligand for 3H-Cimetidine Binding, is an Improgan Antagonist

PubMed Central

Hough, Lindsay B.; Nalwalk, Julia W.; Phillips, James G.; Kern, Brian; Shan, Zhixing; Wentland, Mark P.; de Esch, Iwan J.P.; Janssen, Elwin; Barr, Travis; Stadel, Rebecca

2007-01-01

Summary Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of 3H-cimetidine (3HCIM) in brain fractions was used to discover 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole, which behaved in vivo as the first improgan antagonist. The synthesis and pharmacological properties of this drug (named CC12) are described herein. In rats, CC12 (50 – 500 nmol, icv) produced dose-dependent inhibition of improgan (200 – 400 nmol) antinociception on the tail flick and hot plate tests. When given alone to rats, CC12 had no effects on nociceptive latencies, or on other observable behavioral or motor functions. Maximal inhibitory effects of CC12 (500 nmol) were fully surmounted with a large icv dose of improgan (800 nmol), demonstrating competitive antagonism. In mice, CC12 (200-400 nmol, icv) behaved as a partial agonist, producing incomplete improgan antagonism, but also limited antinociception when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's antagonist properties are not explained by activity at 25 sites relevant to analgesia, including known receptors for cannabinoids, opioids or histamine. The use of CC12 as an improgan antagonist will facilitate the characterization of improgan analgesia. Furthermore, because CC12 was also found presently to inhibit opioid and cannabinoid antinociception, it is suggested that this drug modifies a biochemical mechanism shared by several classes of analgesics. Elucidation of this mechanism will enhance understanding of the biochemistry of pain relief. PMID:17336343

Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: A combined synthesis, binding and docking study

PubMed Central

McCullough, Christopher; Neumann, Terrence S.; Gone, Jayapal Reddy; He, Zhengjie; Herrild, Christian; Wondergem, Julie; Pandey, Rajesh K.; Donaldson, William A.; Sem, Daniel S.

2014-01-01

Various estrogen analogs were synthesized and tested for binding to human ERα using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg394/Glu353 is present. Di-hydroxyl compounds sometimes bind in two orientations, which are flipped in terms of relative positioning of their hydroxyl groups. Di-hydroxyl compounds were predicted to bind with their aliphatic hydroxyl group interacting with His524 in ERα. One nonsteroid-based dihdroxyl compound was 1000-fold specific for ERβ over ERα, and was also 25-fold specific for agonist ERβ versus antagonist activity. Docking predictions suggest this specificity may be due to interaction of the aliphatic hydroxyl with His475 in the agonist form of ERβ, versus with Thr299 in the antagonist form. But, the presence of this aliphatic hydroxyl is not required in all compounds, since mono-hydroxyl (phenolic) compounds bind ERα with high affinity, via hydroxyl hydrogen bonding interactions with the ERα Arg394/Glu353/water triad, and van der Waals interactions with the rest of the molecule. PMID:24315190

Design, synthesis and biological activity of 6-substituted carbamoyl benzimidazoles as new nonpeptidic angiotensin II AT₁ receptor antagonists.

PubMed

Zhang, Jun; Wang, Jin-Liang; Zhou, Zhi-Ming; Li, Zhi-Huai; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

2012-07-15

A series of 6-substituted carbamoyl benzimidazoles were designed and synthesised as new nonpeptidic angiotensin II AT(1) receptor antagonists. The preliminary pharmacological evaluation revealed a nanomolar AT(1) receptor binding affinity for all compounds in the series, and a potent antagonistic activity in an isolated rabbit aortic strip functional assay for compounds 6f, 6g, 6h and 6k was also demonstrated. Furthermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that compound 6g is an orally active AT(1) receptor antagonist with low toxicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antiestrogenic activity of flavnoid phytochemicals mediated via c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha

USDA-ARS?s Scientific Manuscript database

Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling in...

Dopamine transporter-dependent and -independent striatal binding of the benztropine analog JHW 007, a cocaine antagonist with low abuse liability

USDA-ARS?s Scientific Manuscript database

The benztropine analog JHW 007 displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks effects of cocaine,including its self-administration. To determine sites responsible for the cocaine-antagonist effects of JHW 007, ...

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

PubMed Central

FAULHAMMER, DIRK; ESCHGFÄLLER, BERND; STARK, SANDRA; BURGSTALLER, PETRA; ENGLBERGER, WERNER; ERFURTH, JEANNETTE; KLEINJUNG, FRANK; RUPP, JOHANNA; VULCU, SEBASTIAN DAN; SCHRÖDER, WERNER; VONHOFF, STEFAN; NAWRATH, HERMANN; GILLEN, CLEMENS; KLUSSMANN, SVEN

2004-01-01

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are l-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to d-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC50 values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPγS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K+ channels. PMID:14970396

A computational model of the nicotinic acetylcholine binding site

NASA Astrophysics Data System (ADS)

Gálvez-ruano, Enrique; Iriepa-Canalda, Isabel; Morreale, Antonio; Lipkowitz, Kenny B.

1999-01-01

We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro-β-erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).

Serotonergic and dopaminergic distinctions in the behavioral pharmacology of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD).

PubMed

Schindler, Emmanuelle A D; Dave, Kuldip D; Smolock, Elaine M; Aloyo, Vincent J; Harvey, John A

2012-03-01

After decades of social stigma, hallucinogens have reappeared in the clinical literature demonstrating unique benefits in medicine. The precise behavioral pharmacology of these compounds remains unclear, however. Two commonly studied hallucinogens, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD), were investigated both in vivo and in vitro to determine the pharmacology of their behavioral effects in an animal model. Rabbits were administered DOI or LSD and observed for head bob behavior after chronic drug treatment or after pretreatment with antagonist ligands. The receptor binding characteristics of DOI and LSD were studied in vitro in frontocortical homogenates from naïve rabbits or ex vivo in animals receiving an acute drug injection. Both DOI- and LSD-elicited head bobs required serotonin(2A) (5-HT(2A)) and dopamine(1) (D(1)) receptor activation. Serotonin(2B/2C) receptors were not implicated in these behaviors. In vitro studies demonstrated that LSD and the 5-HT(2A/2C) receptor antagonist, ritanserin, bound frontocortical 5-HT(2A) receptors in a pseudo-irreversible manner. In contrast, DOI and the 5-HT(2A/2C) receptor antagonist, ketanserin, bound reversibly. These binding properties were reflected in ex vivo binding studies. The two hallucinogens also differed in that LSD showed modest D(1) receptor binding affinity whereas DOI had negligible binding affinity at this receptor. Although DOI and LSD differed in their receptor binding properties, activation of 5-HT(2A) and D(1) receptors was a common mechanism for eliciting head bob behavior. These findings implicate these two receptors in the mechanism of action of hallucinogens. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

PubMed

Chiara, David C; Trinidad, Jonathan C; Wang, Dong; Ziebell, Michael R; Sullivan, Deirdre; Cohen, Jonathan B

2003-01-21

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

PubMed

Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

2005-01-01

This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

PubMed

Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

1998-04-10

The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

PubMed

Ovigne, J M; Vermot-Desroches, C; Lecron, J C; Portier, M; Lupker, J; Pecceu, F; Wijdenes, J

1998-06-01

The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography.

PubMed Central

Gupta, N; McAllister, R; Drance, S M; Rootman, J; Cynader, M S

1994-01-01

Muscarinic cholinergic agents are used to lower intraocular pressure in the medical management of glaucoma and subtypes of muscarinic receptors have now been recognised in many tissues including the eye. To localise muscarinic receptors and their M1 and M2 subtypes in the human eye, in vitro ligand binding and autoradiographic techniques with densitometric quantitation on postmortem eye sections were used. As ligands, [3H] quinuclydinyl benzylate (QNB) (non-subtype specific muscarinic antagonist), [3H]pirenzipine (M1 antagonist), [3H]oxotremorine (M2 muscarinic agonist), [3H]AFDX-116(11[(2[diethylaminomethyl]1-piperidinyl)acetyl]5 , 11dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one) (M2 antagonist) were studied. Specific binding sites for QNB, pirenzipine, and AFDX-116 were localised in the entire ciliary muscle, the iris, and ciliary epithelium. [3H]oxotremorine localised only in the longitudinal portion of the ciliary muscle, and additionally, was not localised in the iris or ciliary epithelium. These results suggest that oxotremorine, by binding selectively to receptors on the longitudinal ciliary muscle and inducing its contraction, may modulate outflow facility independently from accommodation and miosis. Images PMID:7918268

Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism.

PubMed

Nolan, Kristof; Kattamuri, Chandramohan; Rankin, Scott A; Read, Randy J; Zorn, Aaron M; Thompson, Thomas B

2016-08-23

The DAN family, including Gremlin-1 and Gremlin-2 (Grem1 and Grem2), represents a large family of secreted BMP (bone morphogenetic protein) antagonists. However, how DAN proteins specifically inhibit BMP signaling has remained elusive. Here, we report the structure of Grem2 bound to GDF5 at 2.9-Å resolution. The structure reveals two Grem2 dimers binding perpendicularly to each GDF5 monomer, resembling an H-like structure. Comparison to the unbound Grem2 structure reveals a dynamic N terminus that undergoes significant transition upon complex formation, leading to simultaneous interaction with the type I and type II receptor motifs on GDF5. Binding studies show that DAN-family members can interact with BMP-type I receptor complexes, whereas Noggin outcompetes the type I receptor for ligand binding. Interestingly, Grem2-GDF5 forms a stable aggregate-like structure in vitro that is not clearly observed for other antagonists, including Noggin and Follistatin. These findings exemplify the structural and functional diversity across the various BMP antagonist families. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila-pridinol, diphenidol and sila-diphenidol type.

PubMed Central

Waelbroeck, M.; Camus, J.; Tastenoy, M.; Lambrecht, G.; Mutschler, E.; Kropfgans, M.; Sperlich, J.; Wiesenberger, F.; Tacke, R.; Christophe, J.

1993-01-01

1. We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using [3H]-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide. 2. The affinity of the antagonists either increased or decreased with temperature. van't Hoff plots were linear in the 278-310 degrees K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to -29 kJ mol-1) to large positive values (up to +30 kJ mol-1). 3. (R)-configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed. 4. When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes. 5. Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change of drug binding. 6. Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects. 7. Taken together, these results suggest that hydrogen bond-forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and M2 muscarinic receptors. PMID:8102927

Actions of Agonists, Fipronil and Ivermectin on the Predominant In Vivo Splice and Edit Variant (RDLbd, I/V) of the Drosophila GABA Receptor Expressed in Xenopus laevis Oocytes

PubMed Central

Suwanmanee, Siros; Buckingham, Steven David; Biggin, Philip; Sattelle, David

2014-01-01

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides. PMID:24823815

Synthesis and serotonergic activity of variously substituted (3-amido)phenylpiperazine derivatives and benzothiophene-4-piperazine derivatives: novel antagonists for the vascular 5-HT1B receptor.

PubMed

Moloney, Gerard P; Garavelas, Agatha; Martin, Graeme R; Maxwell, Miles; Glen, Robert C

2004-04-01

The synthesis and vascular 5-HT(1B) receptor activity of a novel series of substituted 3-amido phenylpiperazine and 4-(4-methyl-1-piperazinyl)-1-benzo[b]thiophene derivatives is described. Modifications to the amido linked sidechains of the 3-amidophenyl-piperazine derivatives and to the 2-sidechain of the 1-benzo[b]thiophene derivatives have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B) receptor of pK(B) > 7.0. From the 3-amidophenyl-piperazine series, N-(4-(4-chlorophenyl)thiazol-2-yl-3-(4-methyl-1-piperazinyl)benzamide (30) and from the benzo[b]thiophene-4-piperazine series N-(2-ethylphenyl)-4-(4-methyl-1- piperazinyl)-1-benzo[b]thiophene-2-carboxamide (38) were identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B) receptor mediated agonist activity in the rabbit femoral artery) and competitive vascular 5-HT(1B) receptor antagonist. The affinity of compounds from these two series of compounds for the vascular 5-HT(1B) receptor is discussed as well as a proposed mode of binding to the receptor pharmacophore.

DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

PubMed Central

Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

2008-01-01

Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

Differential binding of /sup 3/H-imipramine and /sup 3/H-mianserin in rat cerebral cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dumbrille-Ross, A.; Tang, S.W.; Coscina, D.V.

1981-11-16

Drug competition profiles, effect of raphe lesion, and sodium dependency of the binding of two antidepressant drugs /sup 3/H-imipramine and /sup 3/H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common ''antidepressant receptor.'' Of the neurotransmitters tested, only serotonin displaced binding of both /sup 3/H-imipramine and /sup 3/H-mianserin. /sup 3/H-Mianserin binding was potently displaced by serotonin S/sub 2/ antagonists and exhibited a profile similar to that of /sup 3/H-spiperone binding. In the presence of the serotonin S/sub 2/ antagonist spiperone, antihistamines (H/sub 1/) potently displaced /sup 3/H-mianserin binding. /sup 3/H-Imipramine binding was displacedmore » potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing /sup 3/H-imipramine binding was not similar to their order in displacing /sup 3/H-spiperone or -3H-serotonin binding. Prior midbrain raphe lesions greatly decreased the binding of /sup 3/H-imipramine but did not alter binding of /sup 3/H-mianserin. Binding of /sup 3/H-imipramine but not /sup 3/H-mianserin was sodium dependent. These results show that /sup 3/H-imipramine and /sup 3/H-mianserin bind to different receptors. /sup 3/H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. /sup 3/H-Mianserin binds to postsynaptic receptors, possibly both serotonin S/sub 2/ and histamine H/sub 1/ receptors, the binding of which is sodium independent.« less

OPC-21268, an orally effective, nonpeptide vasopressin V1 receptor antagonist.

PubMed

Yamamura, Y; Ogawa, H; Chihara, T; Kondo, K; Onogawa, T; Nakamura, S; Mori, T; Tominaga, M; Yabuuchi, Y

1991-04-26

An orally effective, nonpeptide, vasopressin V1 receptor antagonist, OPC-21268, has been identified. This compound selectively antagonized binding to the V1 subtype of the vasopressin receptor in a competitive manner. In vivo, the compound acted as a specific antagonist of arginine vasopressin (AVP)-induced vasoconstriction. After oral administration in conscious rats, the compound also antagonized pressor responses to AVP. OPC-21268 can be used to study the physiological role of AVP and may be therapeutically useful in the treatment of hypertension and congestive heart failure.

In vitro histamine H/sub 2/-antagonist activity of the novel compound HUK 978

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coombes, J.D.; Norris, D.B.; Rising, T.J.

1985-11-04

Histamine stimulated adenylate cyclase from guinea-pig fundic mucosa and /sup 3/H-tiotidine binding in guinea-pig cerebral cortex were used to assess the in-vitro histamine H/sub 2/-activity of the novel H/sub 2/-antagonist HUK 978. The results showed that HUK 978 was a more potent H/sub 2/-antagonist than either cimetidine or ranitidine. HUK 978 was also shown to be devoid of activity at the histamine H-/sub 1/-receptor, the muscarinic receptor and the ..cap alpha.. and ..beta..-adrenergic receptors.

Discovery and Cocrystal Structure of Benzodiazepinedione HDM2 Antagonists that Activate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grasberger,B.; Lu, T.; Schubert, C.

2005-01-01

HDM2 binds to an {alpha}-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their {alpha}-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling

PubMed Central

Singhal, Hari; Greene, Marianne E.; Zarnke, Allison L.; Laine, Muriel; Al Abosy, Rose; Chang, Ya-Fang; Dembo, Anna G.; Schoenfelt, Kelly; Vadhi, Raga; Qiu, Xintao; Rao, Prakash; Santhamma, Bindu; Nair, Hareesh B.; Nickisch, Klaus J.; Long, Henry W.; Becker, Lev; Brown, Myles; Greene, Geoffrey L.

2018-01-01

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic. PMID:29435103

Stimuli of Sensory-Motor Nerves Terminate Arterial Contractile Effects of Endothelin-1 by CGRP and Dissociation of ET-1/ETA-Receptor Complexes

PubMed Central

Meens, Merlijn J. P. M. T.; Compeer, Matthijs G.; Hackeng, Tilman M.; van Zandvoort, Marc A.; Janssen, Ben J. A.; De Mey, Jo G. R.

2010-01-01

Background Endothelin-1 (ET-1), a long-acting paracrine mediator, is implicated in cardiovascular diseases but clinical trials with ET-receptor antagonists were not successful in some areas. We tested whether the quasi-irreversible receptor-binding of ET-1 (i) limits reversing effects of the antagonists and (ii) can be selectively dissociated by an endogenous counterbalancing mechanism. Methodology/Principal findings In isolated rat mesenteric resistance arteries, ETA-antagonists, endothelium-derived relaxing factors and synthetic vasodilators transiently reduced contractile effects of ET-1 but did not prevent persistent effects of the peptide. Stimuli of peri-vascular vasodilator sensory-motor nerves such as capsaicin not only reduced but also terminated long-lasting effects of ET-1. This was prevented by CGRP-receptor antagonists and was mimicked by exogenous calcitonin gene-related peptide (CGRP). Using 2-photon laser scanning microscopy in vital intact arteries, capsaicin and CGRP, but not ETA-antagonism, were observed to promote dissociation of pre-existing ET-1/ETA-receptor complexes. Conclusions Irreversible binding and activation of ETA-receptors by ET-1 (i) occur at an antagonist-insensitive site of the receptor and (ii) are selectively terminated by endogenously released CGRP. Hence, natural stimuli of sensory-motor nerves that stimulate release of endogenous CGRP can be considered for therapy of diseases involving ET-1. PMID:20532232

Pharmacological and pharmacokinetic characterization of 2-piperazine-alpha-isopropyl benzylamine derivatives as melanocortin-4 receptor antagonists.

PubMed

Chen, Chen; Tucci, Fabio C; Jiang, Wanlong; Tran, Joe A; Fleck, Beth A; Hoare, Sam R; Wen, Jenny; Chen, Takung; Johns, Michael; Markison, Stacy; Foster, Alan C; Marinkovic, Dragan; Chen, Caroline W; Arellano, Melissa; Harman, John; Saunders, John; Bozigian, Haig; Marks, Daniel

2008-05-15

A series of 2-piperazine-alpha-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 receptor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting compounds 8-12 bound selectively to MC4R over other melanocortin receptor subtypes and behaved as functional antagonists. These compounds were also studied for their permeability using Caco-2 cell monolayers and metabolic stability in human liver microsomes. Most compounds exhibited low permeability and high efflux ratio possibly due to their high molecular weights. They also showed moderate metabolic stability which might be associated with their moderate to high lipophilicity. Pharmacokinetic properties of these MC4R antagonists, including brain penetration, were studied in mice after oral and intravenous administrations. Two compounds identified to possess high binding affinity and selectivity, 10d and 11d, were studied in a murine cachexia model. After intraperitoneal (ip) administration of 1mg/kg dose, mice treated with 10d had significantly more food intake and weight gain than the control animals, demonstrating efficacy by blocking the MC4 receptor. Similar in vivo effects were also observed when 11d was dosed orally at 20mg/kg. These results provide further evidence that a potent and selective MC4R antagonist has potential in the treatment of cancer cachexia.

Stereoselective L-(3H)quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.

The muscarinic antagonist L-(/sup 3/H)quinuclidinyl benzilate (L-(/sup 3/H)QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-(/sup 3/H)QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-(/sup 3/H)QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-(/sup 3/H)QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably lessmore » effective. These pharmacological characteristics of the L-(/sup 3/H)QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-(/sup 3/H)QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-(/sup 3/H)QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-(/sup 3/H)QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.« less

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

PubMed Central

Izzo, Nicholas J.; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F.; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M.

2014-01-01

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics. PMID:25390368

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

PubMed

Izzo, Nicholas J; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M

2014-01-01

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module.

PubMed

Wojciechowski, Michał; Różycki, Bartosz; Huy, Pham Dinh Quoc; Li, Mai Suan; Bayer, Edward A; Cieplak, Marek

2018-03-22

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

PubMed

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

Structure-based drug design enables conversion of a DFG-in binding CSF-1R kinase inhibitor to a DFG-out binding mode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyers, Marvin J.; Pelc, Matthew; Kamtekar, Satwik

2010-08-11

The work described herein demonstrates the utility of structure-based drug design (SBDD) in shifting the binding mode of an HTS hit from a DFG-in to a DFG-out binding mode resulting in a class of novel potent CSF-1R kinase inhibitors suitable for lead development.

Accuracy of binding mode prediction with a cascadic stochastic tunneling method.

PubMed

Fischer, Bernhard; Basili, Serena; Merlitz, Holger; Wenzel, Wolfgang

2007-07-01

We investigate the accuracy of the binding modes predicted for 83 complexes of the high-resolution subset of the ASTEX/CCDC receptor-ligand database using the atomistic FlexScreen approach with a simple forcefield-based scoring function. The median RMS deviation between experimental and predicted binding mode was just 0.83 A. Over 80% of the ligands dock within 2 A of the experimental binding mode, for 60 complexes the docking protocol locates the correct binding mode in all of ten independent simulations. Most docking failures arise because (a) the experimental structure clashed in our forcefield and is thus unattainable in the docking process or (b) because the ligand is stabilized by crystal water. 2007 Wiley-Liss, Inc.

Avoiding false positives and optimizing identification of true negatives in estrogen receptor binding and agonist/antagonist assays

EPA Science Inventory

The potential for chemicals to affect endocrine signaling is commonly evaluated via in vitro receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented fr...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang

Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differsmore » between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.« less

Aptamer antagonists of myelin-derived inhibitors promote axon growth.

PubMed

Wang, Yuxuan; Khaing, Zin Z; Li, Na; Hall, Brad; Schmidt, Christine E; Ellington, Andrew D

2010-03-16

Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators.

Aptamer Antagonists of Myelin-Derived Inhibitors Promote Axon Growth

PubMed Central

Wang, Yuxuan; Khaing, Zin Z.; Li, Na; Hall, Brad; Schmidt, Christine E.; Ellington, Andrew D.

2010-01-01

Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators. PMID:20300533

An assessment of the effects of serotonin 6 (5-HT6) receptor antagonists in rodent models of learning.

PubMed

Lindner, Mark D; Hodges, Donald B; Hogan, John B; Orie, Anitra F; Corsa, Jason A; Barten, Donna M; Polson, Craig; Robertson, Barbara J; Guss, Valerie L; Gillman, Kevin W; Starrett, John E; Gribkoff, Valentin K

2003-11-01

Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.

Vasopressin and a nonpeptide antidiuretic hormone receptor antagonist (OPC-31260).

PubMed

Burrell, L M; Phillips, P A; Stephenson, J M; Risvanis, J; Johnston, C I

1994-03-01

The development of nonpeptide orally active AVP analogues has provided a new tool with which to assess the physiological and pathophysiological role of vasopressin (AVP). We have previously characterised the nonpeptide vasopressin V1 receptor antagonist OPC-21268, and now report the in vitro characterisation of the nonpeptide V2 receptor antagonist OPC-31260 in the rat. OPC-31260 caused a concentration-dependent displacement of the selective AVP V2 receptor antagonist radioligand, [3H]desGly-NH2(9)[d(CH2)5, D-Ile2,Ile4]AVP from V2 receptors in rat kidney medulla membranes. The concentration of OPC-31260 that displaced 50% of specific AVP binding (IC50) was 20 +/- 2 nmol/l for renal V2 receptors. OPC-31260 also caused a concentration-dependent displacement of the selective AVP V1 receptor antagonist radioligand, [125I]-[d(CH2)5,sarcosine7]AVP from V1 receptors in both rat liver and kidney medulla membranes. The IC50 was 500 +/- 30 nmol/l for both renal and liver V1 receptors. After oral administration to rats, OPC-31260 was an effective inhibitor of AVP at renal V2 and liver V1 receptors in a time-dependent manner. In vitro binding kinetic studies showed that OPC-31260 was a competitive antagonist at both the renal V2 receptor and the hepatic V1 receptor. OPC-31260 is a nonpeptide, orally effective competitive inhibitor of AVP with a V2:V1 receptor selectivity ratio of 25:1 indicating relative V2 receptor selectivity.

Novel long‐acting antagonists of muscarinic ACh receptors

PubMed Central

Randáková, Alena; Rudajev, Vladimír; Doležal, Vladimír; Boulos, John

2018-01-01

Background and Purpose The aim of this study was to develop potent and long‐acting antagonists of muscarinic ACh receptors. The 4‐hexyloxy and 4‐butyloxy derivatives of 1‐[2‐(4‐oxidobenzoyloxy)ethyl]‐1,2,3,6‐tetrahydropyridin‐1‐ium were synthesized and tested for biological activity. Antagonists with long‐residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long‐acting effects allow for reduced daily doses and adverse effects. Experimental Approach The binding and antagonism of functional responses to the agonist carbachol mediated by 4‐hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4‐butyloxy analogues. Key Results The 4‐hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M1 to 4 μM at M3 receptors. Under washing conditions to reverse antagonism, the half‐life of their antagonistic action ranged from 1.7 h at M2 to 5 h at M5 receptors. Conclusions and Implications The 4‐hexyloxy derivatives were found to be potent long‐acting M1‐preferring antagonists. In view of current literature, M1‐selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits. PMID:29498041

Antibodies to B7.1 define the GFCC'C" face of the N-terminal domain as critical for co-stimulatory interactions.

PubMed

Wang, Suyue; Veldman, Geertruida M; Stahl, Mark; Xing, Yuzhe; Tobin, James F; Erbe, David V

2002-09-02

Antagonists of the B7 family of co-stimulatory molecules have the potential for altering immune responses therapeutically. To better define the requirements for such inhibitors, we have mapped the binding of an entire panel of blocking antibodies specific for human B7.1. By mutagenesis, each of the residues critical for blocking antibody binding appeared to fall entirely within the N-terminal V-set domain of B7.1. Thus, although antibody-antigen interacting surfaces can be quite large, these results indicate that a relatively small portion of the GFCC'C" face of this domain is crucial for further antagonist development.

Platelet-activating factor (PAF) receptor-binding antagonist activity of Malaysian medicinal plants.

PubMed

Jantan, I; Rafi, I A A; Jalil, J

2005-01-01

Forty-nine methanol extracts of 37 species of Malaysian medicinal plants were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets, using 3H-PAF as a ligand. Among them, the extracts of six Zingiberaceae species (Alpinia galanga Swartz., Boesenbergia pandurata Roxb., Curcuma ochorrhiza Val., C. aeruginosa Roxb., Zingiber officinale Rosc. and Z. zerumbet Koenig.), two Cinnamomum species (C. altissimum Kosterm. and C. pubescens Kochummen.), Goniothalamus malayanus Hook. f. Momordica charantia Linn. and Piper aduncum L. are potential sources of new PAF antagonists, as they showed significant inhibitory effects with IC50 values ranging from 1.2 to 18.4 microg ml(-1).

Antagonism of Human Formyl Peptide Receptor 1 (FPR1) by Chromones and Related Isoflavones

PubMed Central

Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Cheng, Ni; Ye, Richard D.; Quinn, Mark T.

2014-01-01

Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. Because FPRs play an important role in the regulation of inflammatory reactions implicated in disease pathogenesis, FPR antagonists may represent novel therapeutics for modulating innate immunity. Previously, 4H-chromones were reported to be potent and competitive FPR1 antagonists. In the present studies, 96 additional chromone analogs, including related synthetic and natural isoflavones were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited fMLF-induced intracellular Ca2+ mobilization in FPR1-HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-HL60 and FPR1-RBL cells. Compound 10 (6-hexyl-2-methyl-3-(1-methyl-1H-benzimidazol-2-yl)-4-oxo-4H-chromen-7-yl acetate) was found to be the most potent FPR1-specific antagonist, with binding affinity Ki~100 nM. These chromones inhibited Ca2+ flux and chemotaxis in human neutrophils with nanomolar-micromolar IC50 values. In addition, the most potent novel FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells. These antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, RBL cells transfected with murine Fpr1, or interleukin 8-induced Ca2+ flux in human neutrophils and RBL cells transfected with CXC chemokine receptor 1 (CXCR1). Moreover, pharmacophore modeling showed that the active chromones had a significantly higher degree of similarity with the pharmacophore template as compared to inactive analogs. Thus, the chromone/isoflavone scaffold represents a relevant backbone for development of novel FPR1 antagonists. PMID:25450672

Synergism of virtual screening and medicinal chemistry: identification and optimization of allosteric antagonists of metabotropic glutamate receptor 1.

PubMed

Noeske, Tobias; Trifanova, Dina; Kauss, Valerjans; Renner, Steffen; Parsons, Christopher G; Schneider, Gisbert; Weil, Tanja

2009-08-01

We report the identification of novel potent and selective metabotropic glutamate receptor 1 (mGluR1) antagonists by virtual screening and subsequent hit optimization. For ligand-based virtual screening, molecules were represented by a topological pharmacophore descriptor (CATS-2D) and clustered by a self-organizing map (SOM). The most promising compounds were tested in mGluR1 functional and binding assays. We identified a potent chemotype exhibiting selective antagonistic activity at mGluR1 (functional IC(50)=0.74+/-0.29 microM). Hit optimization yielded lead structure 16 with an affinity of K(i)=0.024+/-0.001 microM and greater than 1000-fold selectivity for mGluR1 versus mGluR5. Homology-based receptor modelling suggests a binding site compatible with previously reported mutation studies. Our study demonstrates the usefulness of ligand-based virtual screening for scaffold-hopping and rapid lead structure identification in early drug discovery projects.

Importance of the pharmacological profile of the bound ligand in enrichment on nuclear receptors: toward the use of experimentally validated decoy ligands.

PubMed

Lagarde, Nathalie; Zagury, Jean-François; Montes, Matthieu

2014-10-27

The evaluation of virtual ligand screening methods is of major importance to ensure their reliability. Taking into account the agonist/antagonist pharmacological profile should improve the quality of the benchmarking data sets since ligand binding can induce conformational changes in the nuclear receptor structure and such changes may vary according to the agonist/antagonist ligand profile. We indeed found that splitting the agonist and antagonist ligands into two separate data sets for a given nuclear receptor target significantly enhances the quality of the evaluation. The pharmacological profile of the ligand bound in the binding site of the target structure was also found to be an additional critical parameter. We also illustrate that active compound data sets for a given pharmacological activity can be used as a set of experimentally validated decoy ligands for another pharmacological activity to ensure a reliable and challenging evaluation of virtual screening methods.

Michelob_x is the missing inhibitor of apoptosis protein antagonist in mosquito genomes

PubMed Central

Zhou, Lei; Jiang, Guohua; Chan, Gina; Santos, Carl P; Severson, David W; Xiao, Lei

2005-01-01

Apoptosis is implicated in the life cycle of the malaria parasite in mosquitoes. The genome project for the primary malaria vector Anopheles gambiae showed a significant expansion of the inhibitor of apoptosis protein (IAP) and caspase gene families in comparison with Drosophila. However, because of extensive sequence divergence, no orthologue was identified for the reaper/grim-like IAP antagonist genes that have a pivotal role in cell death regulation in Drosophila. Using a customized searching strategy, we identified michelob_x(mx), a gene not predicted by the genome project, as the missing IAP antagonist in the An. gambiae and other mosquito genomes. Mx has a highly conserved amino-terminal IAP-binding motif. Expression of Mx induces rapid cell death in insect cell lines and is a potent tissue ablator in vivo. Its proapoptotic activity is totally dependent on the IAP-binding motif. Like reaper in Drosophila, mx is transcriptionally induced by ultraviolet irradiation to mediate cell death. PMID:16041319

High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

PubMed Central

Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

2010-01-01

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

PubMed

Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

1999-02-15

We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

Synthesis, biological assessment and molecular modeling of new dihydroquinoline-3-carboxamides and dihydroquinoline-3-carbohydrazide derivatives as cholinesterase inhibitors, and Ca channel antagonists.

PubMed

Tomassoli, Isabelle; Ismaili, Lhassane; Pudlo, Marc; de Los Ríos, Cristóbal; Soriano, Elena; Colmena, Inés; Gandía, Luis; Rivas, Luis; Samadi, Abdelouahid; Marco-Contelles, José; Refouvelet, Bernard

2011-01-01

The synthesis, biological evaluation, and molecular modeling of new 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides(4), 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (6), and some hexahydropyrimido[5,4-c]quinoline-2,5-diones (9) produced earlier by our laboratory, as AChE/BuChE inhibitors, is described. From these analyses compound 4c resulted equipotent regarding the inhibition of cholinesterases'; inhibitors 6k, 9a, 9b were selective for AChE, whereas product 4d proved selective for BuChE. Docking analysis has been carry out in order to identify the binding mode in the active site, and to explain the observed selectivities. Only compound 9a has been shown to decrease K(+)-induced calcium signals in bovine chromaffin cells. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Receptors for luteinizing hormone-releasing hormone (LHRH) in benign prostatic hyperplasia (BPH) as potential molecular targets for therapy with LHRH antagonist cetrorelix.

PubMed

Rozsa, Bernadett; Nadji, Mehrdad; Schally, Andrew V; Dezso, Balazs; Flasko, Tibor; Toth, Gyorgy; Mile, Melinda; Block, Norman L; Halmos, Gabor

2011-04-01

The majority of men will develop symptoms of benign prostatic hyperplasia (BPH) after 70 years of age. Various studies indicate that antagonists of LHRH, such as cetrorelix, exert direct inhibitory effects on BPH mediated by specific LHRH receptors. Our aim was to investigate the mRNA for LHRH and LHRH receptors and the expression of LHRH receptors in specimens of human BPH. The expression of mRNA for LHRH (n=35) and LHRH receptors (n=55) was investigated by RT-PCR in surgical specimens of BPH, using specific primers. The characteristics of binding sites for LHRH on 20 samples were determined by ligand competition assays. The LHRH receptor expression was also examined in 64 BPH specimens by immunohistochemistry. PCR products for LHRH were found in 18 of 35 (51%) BPH tissues and mRNA for LHRH receptors was detected in 39 of 55 (71%) BPH specimens. Eighteen of 20 (90%) samples showed a single class of high affinity binding sites for [D-Trp(6) ]LHRH with a mean K(d) of 4.04 nM and a mean B(max) of 527.6 fmol/mg membrane protein. LHRH antagonist cetrorelix showed high affinity binding to LHRH receptors in BPH. Positive immunohistochemical reaction for LHRH receptors was present in 42 of 64 (67%) BPH specimens. A high incidence of LHRH receptors in BPH supports the use of LHRH antagonists such as cetrorelix, for treatment of patients with lower urinary tract symptoms from BPH. Copyright © 2010 Wiley-Liss, Inc.

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

PubMed Central

2012-01-01

Introduction The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Methods Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. Results MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Conclusions Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist. PMID:22251451

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells.

PubMed

Lappano, Rosamaria; Santolla, Maria Francesca; Pupo, Marco; Sinicropi, Maria Stefania; Caruso, Anna; Rosano, Camillo; Maggiolini, Marcello

2012-01-17

The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist.

[11C]AZ10419096 - a full antagonist PET radioligand for imaging brain 5-HT1B receptors.

PubMed

Lindberg, Anton; Nag, Sangram; Schou, Magnus; Takano, Akihiro; Matsumoto, Junya; Amini, Nahid; Elmore, Charles S; Farde, Lars; Pike, Victor W; Halldin, Christer

2017-11-01

The serotonergic system is widely present in all regions of the central nervous system (CNS) and plays a key modulatory role in many of its functions. Positron emission tomography (PET) is used to study several serotonin receptors in CNS in vivo. The G-protein coupled receptor 5-HT 1B is mostly present in the occipital cortex and in midbrain and is linked to several psychiatric disorders. There is evidence that agonist PET radioligands for neuroreceptors are more sensitive to endogenous neurotransmitters than antagonists. Our previously developed 5-HT 1B receptor PET radioligand, [ 11 C]AZ10419369, is now considered a partial agonist. In this work we are aiming to develop a full antagonist PET radioligand for imaging brain 5-HT 1B receptors, and evaluate its sensitivity to increased endogenous serotonin concentration. [ 11 C]AZ10419096 was synthesized by rapid methylation of the prepared corresponding N-desmethyl precursor with [ 11 C]methyl triflate. Five PET measurements were performed in cynomolgus monkeys, consisting of two at baseline, one after treatment of a monkey with a 5-HT 1B antagonist, AR-A000002, and two in which fenfluramine was administered during scanning to induce endogenous serotonin release. [ 11 C]AZ10419096 was synthesized in high yield and purity within 30 min, including purification, formulation and sterile filtration. The baseline PET measurements demonstrated [ 11 C]AZ10419096 to have favorable radioligand characteristics, including high specific binding in brain regions that have high 5-HT 1B density, such as occipital cortex and globus pallidus, as well as subsequent rapid elimination from brain and a minor abundance of lipophilic radiometabolites in plasma. AR-A00002 completely blocked radioligand receptor-specific binding. Fenfluramine produced a distinct displacement of radioligand consistent with an expected increase of synaptic endogenous serotonin concentration. [ 11 C]AZ10419096, a full 5-HT 1B antagonist PET radioligand, demonstrates high specific binding in monkey brain that is sensitive to competition from a known 5-HT 1B antagonist as well as to putatively increased endogenous serotonin levels. Published by Elsevier Inc.

Mixed-mode sorption of hydroxylated atrazine degradation products to sell: A mechanism for bound residue

USGS Publications Warehouse

Lerch, R.N.; Thurman, E.M.; Kruger, E.L.

1997-01-01

This study tested the hypothesis that sorption of hydroxylated atrazine degradation products (HADPs: hydroxyatrazine, HA; deethylhydroxyatrazine, DEHA; and deisopropylhydroxyatrazine, DIHA) to soils occurs by mixed-mode binding resulting from two simultaneous mechanisms: (1) cation exchange and (2) hydrophobic interaction. The objective was to use liquid chromatography and soil extraction experiments to show that mixed-mode binding is the mechanism controlling HADP sorption to soils and is also a mechanism for bound residue. Overall, HADP binding to solid-phase extraction (SPE) sorbents occurred in the order: cation exchange >> octadecyl (C18) >> cyanopropyl. Binding to cation exchange SPE and to a high-performance liquid chromatograph octyl (C8) column showed evidence for mixed-mode binding. Comparison of soil extracted by 0.5 M KH2P04, pH 7.5, or 25% aqueous CH3CN showed that, for HA and DIHA, cation exchange was a more important binding mechanism to soils than hydrophobic interaction. Based on differences between several extractants, the extent of HADP mixed-mode binding to soil occurred in the following order: HA > DIHA > DEHA. Mixed-mode extraction recovered 42.8% of bound atrazine residues from aged soil, and 88% of this fraction was identified as HADPs. Thus, a significant portion of bound atrazine residues in soils is sorbed by the mixed-mode binding mechanisms.

Binding modes and pathway of RHPS4 to human telomeric G-quadruplex and duplex DNA probed by all-atom molecular dynamics simulations with explicit solvent.

PubMed

Mulholland, Kelly; Siddiquei, Farzana; Wu, Chun

2017-07-19

RHPS4, a potent binder to human telomeric DNA G-quadruplex, shows high efficacy in tumor cell growth inhibition. However, it's preferential binding to DNA G-quadruplex over DNA duplex (about 10 fold) remains to be improved toward its clinical application. A high resolution structure of the single-stranded telomeric DNA G-quadruplexes, or B-DNA duplex, in complex with RHPS4 is not available yet, and the binding nature of this ligand to these DNA forms remains to be elusive. In this study, we carried out 40 μs molecular dynamics binding simulations with a free ligand to decipher the binding pathway of RHPS4 to a DNA duplex and three G-quadruplex folders (parallel, antiparallel and hybrid) of the human telomeric DNA sequence. The most stable binding mode identified for the duplex, parallel, antiparallel and hybrid G-quadruplexes is an intercalation, bottom stacking, top intercalation and bottom intercalation mode, respectively. The intercalation mode with similar binding strength to both the duplex and the G-quadruplexes, explains the lack of binding selectivity of RHPS4 to the G-quadruplex form. Therefore, a ligand modification that destabilizes the duplex intercalation mode but stabilizes the G-quadruplex intercalation mode will improve the binding selectivity toward G-quadruplex. The intercalation mode of RHPS4 to both the duplex and the antiparallel and the hybrid G-quadruplex follows a base flipping-insertion mechanism rather than an open-insertion mechanism. The groove binding, the side binding and the intercalation with flipping out of base were observed to be intermediate states before the full intercalation state with paired bases.

Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS | Center for Cancer Research

Cancer.gov

A Better Fit. An improved anticoagulant drug called RUC-2 (ball and stick structure) fits snugly into its binding pocket on integrin (blue), a protein found on the surface of platelets. RUC-2 binds both subunits of integrin, inhibiting the excessive blood coagulation that can lead to strokes and heart attacks. Unlike similar drugs that alter integrin's structure when they bind

Interaction of a vasopressin antagonist with vasopressin receptors in the septum of the rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dorsa, D.M.; Brot, M.D.; Shewey, L.M.

1988-01-01

The ability of d(CH2)5-Tyr(Me)-arginine-8-vasopressin, an antagonist of peripheral pressoric (V1-type) vasopressin receptors, to label vasopressin binding sites in the septum of the rat brain was evaluated. Using crude membrane preparations from the septum, /sup 3/H-arginine-8-vasopressin (AVP) specifically labels a single class of binding sites with a Kd of 2.9 nM and maximum binding site concentration of 19.8 fmole/mg protein. /sup 3/H-Antag also labels a single class of membrane sites but with higher affinity (Kd = 0.47 nM) and lower capacity (10.1 fmole/mg protein) than /sup 3/H-AVP. The rank order of potency of various competitor peptides for /sup 3/H-AVP and /supmore » 3/H-Antag binding was similar. Oxytocin was 100-1,000 fold less potent than AVP in competing for binding with both ligands. /sup 3/H-AVP and /sup 3/H-Antag showed similar labeling patterns when incubated with septal tissue slices. Unlabeled Antag also effectively antagonized vasopressin-stimulated phosphatidylinositol hydrolysis in septal tissue slices.« less

Complex Actions of Thyroid Hormone Receptor Antagonist NH-3 on Gene Promoters in Different Cell Lines

PubMed Central

Shah, Vanya; Nguyen, Phuong; Nguyen, Ngoc-Ha; Togashi, Marie; Scanlan, Thomas S.; Baxter, John D.; Webb, Paul

2014-01-01

It is desirable to obtain new antagonists for thyroid hormone (TRs) and other nuclear receptors (NRs). We previously used X-ray structural models of TR ligand binding domains (LBDs) to design compounds, such as NH-3, that impair coactivator binding to activation function 2 (AF-2) and block thyroid hormone (triiodothyronine, T3) actions. However, TRs bind DNA and are transcriptionally active without ligand. Thus, NH-3 could modulate TR activity via effects on other coregulator interaction surfaces, such as activation function (AF-1) and corepressor binding sites. Here, we find that NH-3 blocks TR-LBD interactions with coactivators and corepressors and also inhibits activities of AF-1 and AF-2 in transfections. While NH-3 lacks detectable agonist activity at T3-activated genes in GC pituitary cells it nevertheless activates spot 14 (S14) in HTC liver cells with the latter effect accompanied by enhanced histone H4 acetylation and coactivator recruitment at the S14 promoter. Surprisingly, T3 promotes corepressor recruitment to target promoters. NH-3 effects vary; we observe transient recruitment of N-CoR to S14 in GC cells and dismissal and rebinding of N-CoR to the same promoter in HTC cells. We propose that NH-3 will generally behave as an antagonist by blocking AF-1 and AF-2 but that complex effects on coregulator recruitment may result in partial/mixed agonist effects that are independent of blockade of T3 binding in some contexts. These properties could ultimately be utilized in drug design and development of new selective TR modulators. PMID:18930112

Involvement of sigma-1 receptors in the antidepressant-like effects of dextromethorphan.

PubMed

Nguyen, Linda; Robson, Matthew J; Healy, Jason R; Scandinaro, Anna L; Matsumoto, Rae R

2014-01-01

Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1) receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047) were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP) 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [(3)H](+)-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors.

Involvement of Sigma-1 Receptors in the Antidepressant-like Effects of Dextromethorphan

PubMed Central

Nguyen, Linda; Robson, Matthew J.; Healy, Jason R.; Scandinaro, Anna L.; Matsumoto, Rae R.

2014-01-01

Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1) receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047) were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP) 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [3H](+)-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors. PMID:24587167

Unsurmountable antagonism of brain 5-hydroxytryptamine2 receptors by (+)-lysergic acid diethylamide and bromo-lysergic acid diethylamide.

PubMed

Burris, K D; Sanders-Bush, E

1992-11-01

Lysergic acid diethylamide (LSD) and its structural analogue 2-bromo-lysergic acid diethylamide (BOL) act as unsurmountable antagonists of serotonin-elicited contractions in smooth muscle preparations. Two different models, allosteric and kinetic, have been invoked to explain these findings. The present studies investigate the mechanism of antagonism of brain 5-hydroxytryptamine (5HT)2 receptors, utilizing cells transfected with 5HT2 receptor cDNA cloned from rat brain. A proximal cellular response, phosphoinositide hydrolysis, was examined in order to minimize possible postreceptor effects. Even though LSD behaved as a partial agonist and BOL as a pure antagonist, both drugs blocked the effect of serotonin in an unsurmountable manner, i.e., increasing concentrations of serotonin could not overcome the blocking effect of LSD or BOL. Radioligand binding studies showed that preincubation of membranes with either LSD or BOL reduced the density of [3H]ketanserin binding sites, suggesting that the drugs bind tightly to the 5HT2 receptor and are not displaced during the binding assay. Two additional experiments supported this hypothesis. First, the off-rate of [3H] LSD was slow (20 min), relative to that of [3H]ketanserin (approximately 4 min). Second, when the length of incubation with [3H]ketanserin was increased to 60 min, the LSD-induced decrease in Bmax was essentially eliminated. The possibility that LSD and BOL decrease [3H]ketanserin binding by interacting with an allosteric site was rejected, because neither drug altered the rate of dissociation of [3H]ketanserin. The most parsimonious interpretation of these results is that unsurmountable antagonism reflects prolonged occupancy of the receptor by slowly reversible antagonists.

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

PubMed

Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

2002-07-01

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

Binding of indomethacin methyl ester to cyclooxygenase-2. A computational study.

PubMed

Sárosi, Menyhárt-Botond

2018-06-05

Inhibitors selective towards the second isoform of prostaglandin synthase (cyclooxygenase, COX-2) are promising nonsteroidal anti-inflammatory drugs and antitumor medications. Methylation of the carboxylate group in the relatively nonselective COX inhibitor indomethacin confers significant COX-2 selectivity. Several other modifications converting indomethacin into a COX-2 selective inhibitor have been reported. Earlier experimental and computational studies on neutral indomethacin derivatives suggest that the methyl ester derivative likely binds to COX-2 with a similar binding mode as that observed for the parent indomethacin. However, docking studies followed by molecular dynamics simulations revealed two possible binding modes in COX-2 for indomethacin methyl ester, which differs from the experimental binding mode found for indomethacin. Both alternative binding modes might explain the observed COX-2 selectivity of indomethacin methyl ester. Graphical abstract Binding of indomethacin methyl ester to cyclooxygenase-2.

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

PubMed

Katz, Jonathan L; Hiranita, Takato; Kopajtic, Theresa A; Rice, Kenner C; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H; McCurdy, Christopher R

2016-07-01

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. U.S. Government work not protected by U.S. copyright.

Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17β-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps

NASA Astrophysics Data System (ADS)

Negri, Matthias; Recanatini, Maurizio; Hartmann, Rolf W.

2011-09-01

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17β-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the βFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17β-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17β-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.

The effect of the sigma-1 receptor selective compound LS-1-137 on the DOI-induced head twitch response in mice.

PubMed

Malik, Maninder; Rangel-Barajas, Claudia; Mach, Robert H; Luedtke, Robert R

2016-09-01

Several receptor mediated pathways have been shown to modulate the murine head twitch response (HTR). However, the role of sigma receptors in the murine (±)-2,5-dimethoxy-4-iodoamphetamine (DOI)-induced HTR has not been previously investigated. We examined the ability of LS-1-137, a novel sigma-1 vs. sigma-2 receptor selective phenylacetamide, to modulate the DOI-induced HTR in DBA/2J mice. We also assessed the in vivo efficacy of reference sigma-1 receptor antagonists and agonists PRE-084 and PPCC. The effect of the sigma-2 receptor selective antagonist RHM-1-86 was also examined. Rotarod analysis was performed to monitor motor coordination after LS-1-137 administration. Radioligand binding techniques were used to determine the affinity of LS-1-137 at 5-HT2A and 5-HT2C receptors. LS-1-137 and the sigma-1 receptor antagonists haloperidol and BD 1047 were able to attenuate a DOI-induced HTR, indicating that LS-1-137 was acting in vivo as a sigma-1 receptor antagonist. LS-1-137 did not compromise rotarod performance within a dose range capable of attenuating the effects of DOI. Radioligand binding studies indicate that LS-1-137 exhibits low affinity binding at both 5-HT2A and 5-HT2C receptors. Based upon the results from these and our previous studies, LS-1-137 is a neuroprotective agent that attenuates the murine DOI-induced HTR independent of activity at 5-HT2 receptor subtypes, D2-like dopamine receptors, sigma-2 receptors and NMDA receptors. LS-1-137 appears to act as a sigma-1 receptor antagonist to inhibit the DOI-induced HTR. Therefore, the DOI-induced HTR can be used to assess the in vivo efficacy of sigma-1 receptor selective compounds. Copyright © 2016. Published by Elsevier Inc.

Trifluoperazine Regulation of Calmodulin Binding to Fas: A Computational Study

PubMed Central

Pan, Di; Yan, Qi; Chen, Yabing; McDonald, Jay M; Song, Yuhua

2011-01-01

Death-inducing signaling complex (DISC) formation is a critical step in Fas-mediated signaling for apoptosis. Previous experiments have demonstrated that the calmodulin (CaM) antagonist, trifluoperazine (TFP) regulates CaM-Fas binding and affects Fas-mediated DISC formation. In this study, we investigated the anti-cooperative characteristics of TFP binding to CaM and the effect of TFP on the CaM-Fas interaction from both structural and thermodynamic perspectives using combined molecular dynamics simulations and binding free energy analyses. We studied the interactions of different numbers of TFP molecules with CaM and explored the effects of the resulting conformational changes in CaM on CaM-Fas binding. Results from these analyses showed that the number of TFP molecules bound to CaM directly influenced α-helix formation and hydrogen bond occupancy within the α-helices of CaM, contributing to the conformational and motion changes in CaM. These changes affected CaM binding to Fas, resulting in secondary structural changes in Fas and conformational and motion changes of Fas in CaM-Fas complexes, potentially perturbing the recruitment of Fas-associated death domain (FADD) for DISC formation. The computational results from this study reveal the structural and molecular mechanisms that underlie the role of the CaM antagonist, TFP, in regulation of CaM-Fas binding and Fas-mediated DISC formation in a concentration-dependent manner. PMID:21656570

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

PubMed

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-10-31

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

PubMed Central

Chisamore, Michael J.; Cunningham, Michael E.; Flores, Osvaldo; Wilkinson, Hilary A.; Chen, J. Don

2009-01-01

Background The orphan nuclear receptor estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor α (ERα). An endogenous ligand has not been found. Novel ERRα antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRα have been recently reported. Research suggests that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERRα specific antagonist. Methodology/Principal Findings We demonstrate this ERRα ligand inhibits ERRα transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERα (ESR1) mRNA levels were not affected upon treatment with the ERRα antagonist, but other ERRα (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRα antagonist prevents the constitutive interaction between ERRα and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRα protein degradation via the ubiquitin proteasome pathway is increased by the ERRα-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRα protein is decreased when cells are treated with the ligand. Knocking-down ERRα (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRα antagonist. Conclusions/Significance We report the mechanism of action of a novel ERRα specific antagonist that inhibits transcriptional activity of ERRα, disrupts the constitutive interaction between ERRα and nuclear coactivators, and induces proteasome-dependent ERRα protein degradation. Additionally, we confirmed that knocking-down ERRα lead to similar genomic effects demonstrated in vitro when treated with the ERRα specific antagonist. PMID:19462000

RECOMBINANT ANDROGEN RECEPTOR (AR) BINDING ACROSS VERTEBRATE SPECIES: COMPARISON OF BINDING OF ENVIRONMENTAL COMPOUNDS TO HUMAN, RAINBOW TROUT AND FATHEAD MINNOW AR.

EPA Science Inventory

In vitro screening assays designed to identify androgen mimics or antagonists typically use mammalian (rat, human) androgen receptors (AR). Although the amino acid sequences of receptors from nonmammalian vertebrates are not identical to the mammalian receptors, it is uncertain ...

Novel Functional Properties of Drosophila CNS Glutamate Receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yan; Dharkar, Poorva; Han, Tae-Hee

Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation bymore » its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation.« less

Novel Functional Properties of Drosophila CNS Glutamate Receptors.

PubMed

Li, Yan; Dharkar, Poorva; Han, Tae-Hee; Serpe, Mihaela; Lee, Chi-Hon; Mayer, Mark L

2016-12-07

Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. VIDEO ABSTRACT. Published by Elsevier Inc.

Anti-androgenic effects of S-40542, a novel non-steroidal selective androgen receptor modulator (SARM) for the treatment of benign prostatic hyperplasia.

PubMed

Nejishima, Hiroaki; Yamamoto, Noriko; Suzuki, Mika; Furuya, Kazuyuki; Nagata, Naoya; Yamada, Shizuo

2012-10-01

Selective androgen receptor modulators (SARMs) would provide alternative therapeutic agent for androgen-related diseases. We identified a tetrahydroquinoline (THQ) derivative, 1-(8-nitro-3a, 4, 5, 9b-tetrahydro-3H-cyclopenta[c]quinolin-4-yl) ethane-1, 2-diol (S-40542) as a novel SARM antagonist. Affinity for nuclear receptors of S-40542 was evaluated in receptor-binding studies. Androgen receptor (AR) transcriptional activity of S-40542 was investigated by luciferase reporter assay in DU145AR cells. Normal and benign prostatic hyperplasia (BPH) model rats were repeatedly treated with S-40542 and flutamide. The tissue weights of prostate and levator ani muscle as well as blood levels of testosterone and luteinizing hormone were measured. S-40542 bound to the AR with high affinity. S-40542 at relatively high concentrations increased the transcriptional activity. This agent also showed a concentration-dependent AR antagonistic action in the presence of 1 nM 5α-dihydrotestosterone. Repeated treatment with S-40542 and flutamide decreased dose-dependently the weights of the prostate to a similar extent. In contrast, the tissue weight-reducing effect by S-40542 treatment on the levator ani muscle was much weaker than that of flutamide. S-40542 had little effect on the blood level of testosterone and luteinizing hormone, whereas flutamide increased the level of both hormones. Furthermore, S-40542 decreased dose-dependently prostate weight of BPH rats. The current results indicate that S-40542 possesses the prostate-selective SARM activity, suggestive of clinical benefit against benign prostate hyperplasia. THQ compounds may be useful for the research of mode of action of SARMs and for the development of safe SARM antagonists. Copyright © 2012 Wiley Periodicals, Inc.

Identification of a putative binding site critical for general anesthetic activation of TRPA1.

PubMed

Ton, Hoai T; Phan, Thieu X; Abramyan, Ara M; Shi, Lei; Ahern, Gerard P

2017-04-04

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABA A receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

Calcium antagonism: aldosterone and vascular responses to catecholamines and angiotensin II in man.

PubMed

Elliott, H L

1993-12-01

Effects of calcium antagonists on pressor mechanisms: A number of differences have been reported in the variable extent to which calcium antagonists interfere with various pressor mechanisms. In theory, high lipid solubility, membrane-binding characteristics and a prolonged duration of action appear to be requirements for a calcium antagonist to affect mechanisms such as vasodilation, endogenous vasoconstrictor responses, hormone release and natriuretic activity. Reduction in peripheral vascular resistance: A reduction in peripheral vascular resistance is fundamental to the antihypertensive effect not only of calcium antagonists but also of angiotensin converting enzyme inhibitors and alpha 1-adrenoceptor antagonists. However, only the calcium antagonists interfere directly with the pressor responses mediated by both the adrenergic nervous system and the renin-angiotensin system. Mechanism of lacidipine effects: Preliminary results with the new dihydropyridine calcium antagonist lacidipine indicate that it not only has vasodilator activity but that it also interferes with both adrenergic and non-adrenergic endogenous vasoconstrictor mechanisms. This may provide additional potentially beneficial cardiovascular effects, particularly in relation to left ventricular hypertrophy and dysfunction.

Concentration-dependent Cu(II) binding to prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

2008-03-01

The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs

PubMed Central

Cléry, Antoine; Allain, Frédéric H-T

2017-01-01

Abstract RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear. Therefore, we utilized microcalorimetry and semi-empirical analyses to comparatively analyze the cognate ssRNA binding thermodynamics of four different RRM domains, each with a different RNA binding mode. The different binding modes are: canonical binding to the β-sheet surface; canonical binding with involvement of N- and C-termini; binding to conserved loops; and binding to an α-helix. Our results identify enthalpy as the sole and general force driving association at physiological temperatures. Also, networks of weak interactions are a general feature regulating stability of the different RRM–ssRNA complexes. In agreement, non-polyelectrolyte effects contributed between ∼75 and 90% of the overall free energy of binding in the considered complexes. The various RNA binding modes also displayed enormous heat capacity differences, that upon dissection revealed large differential changes in hydration, conformations and dynamics upon binding RNA. Altogether, different modes employed by RRMs to bind cognate ssRNAs utilize various thermodynamics strategies during the association process. PMID:28334819

Exploiting Free-Energy Minima to Design Novel EphA2 Protein-Protein Antagonists: From Simulation to Experiment and Return.

PubMed

Russo, Simonetta; Callegari, Donatella; Incerti, Matteo; Pala, Daniele; Giorgio, Carmine; Brunetti, Jlenia; Bracci, Luisa; Vicini, Paola; Barocelli, Elisabetta; Capoferri, Luigi; Rivara, Silvia; Tognolini, Massimiliano; Mor, Marco; Lodola, Alessio

2016-06-06

The free-energy surface (FES) of protein-ligand binding contains information useful for drug design. Here we show how to exploit a free-energy minimum of a protein-ligand complex identified by metadynamics simulations to design a new EphA2 antagonist with improved inhibitory potency. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

PubMed Central

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A.; García-Pérez, Carlos A.; Guerrero-Rodríguez, Sandra L.; Ruiz, Angel J.; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M.; Velasco-Velázquez, Marco A.

2016-01-01

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists. PMID:27009862

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

PubMed Central

Moody, Terry W.; Tashakkori, Nicole; Mantey, Samuel A.; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T.

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists. PMID:28785244

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists.

PubMed

Moody, Terry W; Tashakkori, Nicole; Mantey, Samuel A; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB 2 R), neuromedin B receptor (BB 1 R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB 1 R, BB 2 R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB 1 R, BB 2 R, and BRS-3 with similar affinity ( K i = 1.4-10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca 2+ in human lung cancer cells transfected with BB 1 R, BB 2 R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

PubMed

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A; García-Pérez, Carlos A; Guerrero-Rodríguez, Sandra L; Ruiz-Moreno, Angel J; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

2016-04-26

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

Design and synthesis of N-(3,3-diphenylpropenyl)alkanamides as a novel class of high-affinity MT2-selective melatonin receptor ligands.

PubMed

Bedini, Annalida; Spadoni, Gilberto; Gatti, Giuseppe; Lucarini, Simone; Tarzia, Giorgio; Rivara, Silvia; Lorenzi, Simone; Lodola, Alessio; Mor, Marco; Lucini, Valeria; Pannacci, Marilou; Scaglione, Francesco

2006-12-14

A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated by previously described 3D-QSAR and receptor models. Compounds belonging to the classes of 2,3- and [3,3-diphenylprop(en)yl]alkanamides and of o- or [(m-benzyl)phenyl]ethyl-alkanamides were synthesized and tested on MT(1) and MT(2) receptors. The class of 3,3-diphenyl-propenyl-alkanamides was the most interesting one, with compounds having MT(2) receptor affinity similar to that of MLT, remarkable MT(2) selectivity, and partial agonist or antagonist behavior. In particular, the (E)-m-methoxy cyclobutanecarboxamido derivative 18f and the di-(m-methoxy) acetamido one, 18g, have sub-nM affinity for the MT(2) subtype, with more than 100-fold selectivity over MT(1), 18f being an antagonist and 18g a partial agonist on GTPgammaS test. Docking of 18g into a previously developed MT(2) receptor model showed a binding scheme consistent with that of other antagonists. The MT(2) expected binding affinities of the new compounds were calculated by a previously developed 3D-QSAR CoMFA model, giving satisfactory predictions.

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers II: Sigma-2/PGRMC1 Receptors Mediate Abeta 42 Oligomer Binding and Synaptotoxicity

PubMed Central

Izzo, Nicholas J.; Xu, Jinbin; Zeng, Chenbo; Kirk, Molly J.; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Cruchaga, Carlos; Goate, Alison; Cahill, Michael A.; Arancio, Ottavio; Mach, Robert H.; Craven, Rolf; Head, Elizabeth; LeVine, Harry; Spires-Jones, Tara L.; Catalano, Susan M.

2014-01-01

Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics. PMID:25390692

Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of macrophage migration inhibitory factor (MIF)

PubMed Central

Hare, Alissa A.; Leng, Lin; Gandavadi, Sunilkumar; Du, Xin; Cournia, Zoe; Bucala, Richard; Jorgensen, William L.

2010-01-01

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto-enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1-μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC50 values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF-CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts. PMID:20728358

Effect of SKI2670, a novel, orally active, non-peptide GnRH antagonist, on hypothalamic-pituitary-gonadal axis.

PubMed

Kim, Seon Mi; Yoo, Taekyung; Lee, So Young; Kim, Eun Jeong; Lee, Soo Min; Lee, Min Hee; Han, Min Young; Jung, Seung-Hyun; Choi, Jung-Hye; Ryu, Keun Ho; Kim, Hun-Taek

2015-10-15

Suppression of the hypothalamic-pituitary-gonadal axis has been widely utilized for the management of gonadal-hormone-dependent diseases such as endometriosis. Efforts to develop orally available gonadotropin-releasing hormone (GnRH) antagonists for the treatment of gonadal-hormone-dependent diseases led to the discovery of SKI2670, a novel non-peptide GnRH antagonist. The present study was undertaken to pharmacologically characterize SKI2670 in vitro and in vivo. We measured binding affinity and antagonistic activity of SKI2670 for the GnRH receptors. Immediate suppression of gonadotropins by single dosing of SKI2670 was examined in castrated monkeys. Subsequently, influence on gonadal hormones by prolonged administration of SKI2670 was assessed in naive female monkeys. To investigate in vivo efficacy of SKI2670, regression of ectopic implants by repeated administration of SKI2670 was examined in a rat endometriosis model. SKI2670 is a potent functional antagonist for the human GnRH receptor, with subnanomolar binding affinity. In castrated monkeys, single administration of SKI2670 lowered serum luteinizing hormone (LH) levels stronger with longer duration when compared to elagolix at equivalent doses. Moreover, repeated dosing of SKI2670 suppressed serum levels of gonadotropins and gonadal hormones in intact female monkeys while elagolix suppressed serum LH levels only. Finally, it exhibited regressive effects on ectopic implants in a rat endometriosis model without bone loss. Our findings demonstrate robust GnRH antagonistic efficacy of SKI2670 in animal models, suggesting that SKI2670-induced suppression of the hypothalamic-pituitary-gonadal axis may be beneficial for the treatment of gonadal-hormone-dependent diseases such as endometriosis in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

PubMed Central

Krayukhina, Elena; Noda, Masanori; Ishii, Kentaro; Maruno, Takahiro; Wakabayashi, Hirotsugu; Tada, Minoru; Suzuki, Takuo; Ishii-Watabe, Akiko; Kato, Masahiko; Uchiyama, Susumu

2017-01-01

ABSTRACT A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins. PMID:28387583

Critical review and evaluation of the uterotrophic bioassay for the identification of possible estrogen agonists and antagonists: in support of the validation of the OECD uterotrophic protocols for the laboratory rodent. Organisation for Economic Co-operation and Development.

PubMed

Owens, J William; Ashby, John

2002-01-01

A current issue for regulatory agencies is endocrine-related modes of action such as those mediated by the estrogen, androgen, and thyroid nuclear receptors. At the national and international levels, the consensus recommendation for the assessment of such modes of action is a tiered series of in vitro and in vivo protocols. The tiered framework begins with screens for structural alerts and then moves to rapid, mechanistic in vitro screening assays, and then to in vivo screening bioassays. The objective of these screens is to identify substances that may warrant testing for endocrine-mediated adverse effects. The final framework tier as needed is to test these substances in long-term bioassays for adverse endocrine-mediated reproductive and/or developmental effects. The subject of this review, the rodent uterotrophic bioassay, is intended to be a rapid in vivo screening bioassay for possible estrogen agonists and based on the response of the estrogen-sensitive uterus. The central metric of bioassay is a statistically significant increase in the weight of the uterus after 3 consecutive days of test substance administration. The extensive background literature is summarized in this review on the mode of action underlying the bioassay and the uterine response to estrogens. The review includes the bioassay's history of development and how its employment has changed and evolved over time. The review describes two major uterotrophic bioassay versions, the intact, immature female and the mature, ovariectomized female, and the protocol factors likely to influence relevance, reproducibility, and reliability of bioassay. The emphasis of the review is the ability of the uterotrophic bioassay to identify the substances of current interest: weak estrogen agonists with binding affinities relative to the natural 17beta-estradiol in the log 0 to log -3 range. Using selected model substances having RBAs in this target range, the bioassay's performance in a hierarchical, tiered approach is evaluated, including the predictive capability of the uterotrophic bioassay based on available reproductive and developmental testing data. The review concludes that the uterotrophic bioassay is reliable and can identify substances that may act via an estrogen-mode of action, supporting the validity of the uterotrophic bioassay and its regulatory use as an in vivo mechanistic screening bioassay for estrogen agonists and antagonists.

Detailed Analysis of the Binding Mode of Vanilloids to Transient Receptor Potential Vanilloid Type I (TRPV1) by a Mutational and Computational Study

PubMed Central

Mori, Yoshikazu; Ogawa, Kazuo; Warabi, Eiji; Yamamoto, Masahiro; Hirokawa, Takatsugu

2016-01-01

Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel and a multimodal sensor protein. Since the precise structure of TRPV1 was obtained by electron cryo-microscopy, the binding mode of representative agonists such as capsaicin and resiniferatoxin (RTX) has been extensively characterized; however, detailed information on the binding mode of other vanilloids remains lacking. In this study, mutational analysis of human TRPV1 was performed, and four agonists (capsaicin, RTX, [6]-shogaol and [6]-gingerol) were used to identify amino acid residues involved in ligand binding and/or modulation of proton sensitivity. The detailed binding mode of each ligand was then simulated by computational analysis. As a result, three amino acids (L518, F591 and L670) were newly identified as being involved in ligand binding and/or modulation of proton sensitivity. In addition, in silico docking simulation and a subsequent mutational study suggested that [6]-gingerol might bind to and activate TRPV1 in a unique manner. These results provide novel insights into the binding mode of various vanilloids to the channel and will be helpful in developing a TRPV1 modulator. PMID:27606946

Vinclozolin: a case study on the identification of endocrine active substances in the past and a future perspective.

PubMed

van Ravenzwaay, Bennard; Kolle, Susanne N; Ramirez, Tzutzuy; Kamp, Hennicke G

2013-12-16

In the late 1980s vinclozolin was tested for prenatal developmental toxicity in rats for registration purposes in USA. At 1000mg/kgbw, 95% of all fetuses were female upon visual inspection (ano-genital distance determination). Anti-androgenic effects (AA) were also noted in a subsequent 2-generation study. These findings triggered mechanistic investigations at BASF and at US-EPA. Results published by the latter were the starting point of the endocrine disruption (ED) discussion in the 1990s. AA effects of vinclozolin are mediated by two metabolites, which have an antagonistic effect on the androgen receptor. Currently, determination of ED has become a major end-point in toxicology testing and the US-EPA has set up an elaborated testing paradigm to fulfill this requirement. Future screening for ED can be improved making use of new technologies. ED modes of action can be determined by three alternative (3R) methods. Steroid synthesis in H295R cells (1), androgen-receptor binding in modified yeast (2) and metabolomics (3). Using vinclozolin as a case study, results indicate: (1) an effect on steroid synthesis in vitro, (2) an antagonistic effect on the androgen receptor and (3) that the metabolome profile of vinclozolin is similar to that of other receptor mediated anti-androgens (e.g. flutamide). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Small-Molecule “BRCA1-Mimetics” Are Antagonists of Estrogen Receptor-α

PubMed Central

Ma, Yongxian; Tomita, York; Preet, Anju; Clarke, Robert; Englund, Erikah; Grindrod, Scott; Nathan, Shyam; De Oliveira, Eliseu; Brown, Milton L.

2014-01-01

Context: Resistance to conventional antiestrogens is a major cause of treatment failure and, ultimately, death in breast cancer. Objective: The objective of the study was to identify small-molecule estrogen receptor (ER)-α antagonists that work differently from tamoxifen and other selective estrogen receptor modulators. Design: Based on in silico screening of a pharmacophore database using a computed model of the BRCA1-ER-α complex (with ER-α liganded to 17β-estradiol), we identified a candidate group of small-molecule compounds predicted to bind to a BRCA1-binding interface separate from the ligand-binding pocket and the coactivator binding site of ER-α. Among 40 candidate compounds, six inhibited estradiol-stimulated ER-α activity by at least 50% in breast carcinoma cells, with IC50 values ranging between 3 and 50 μM. These ER-α inhibitory compounds were further studied by molecular and cell biological techniques. Results: The compounds strongly inhibited ER-α activity at concentrations that yielded little or no nonspecific toxicity, but they produced only a modest inhibition of progesterone receptor activity. Importantly, the compounds blocked proliferation and inhibited ER-α activity about equally well in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Representative compounds disrupted the interaction of BRCA1 and ER-α in the cultured cells and blocked the interaction of ER-α with the estrogen response element. However, the compounds had no effect on the total cellular ER-α levels. Conclusions: These findings suggest that we have identified a new class of ER-α antagonists that work differently from conventional antiestrogens (eg, tamoxifen and fulvestrant). PMID:25264941

Structural Biology and Evolution of the TGF-β Family

PubMed Central

Hinck, Andrew P.; Mueller, Thomas D.; Springer, Timothy A.

2017-01-01

We review the evolution and structure of members of the transforming growth factor β (TGF-β) family, antagonistic or agonistic modulators, and receptors that regulate TGF-β signaling in extracellular environments. The growth factor (GF) domain common to all family members and many of their antagonists evolved from a common cystine knot growth factor (CKGF) domain. The CKGF superfamily comprises six distinct families in primitive metazoans, including the TGF-β and Dan families. Compared with Wnt/Frizzled and Notch/Delta families that also specify body axes, cell fate, tissues, and other families that contain CKGF domains that evolved in parallel, the TGF-β family was the most fruitful in evolution. Complexes between the prodomains and GFs of the TGF-β family suggest a new paradigm for regulating GF release by conversion from closed- to open-arm procomplex conformations. Ternary complexes of the final step in extracellular signaling show how TGF-β GF dimers bind type I and type II receptors on the cell surface, and enable understanding of much of the specificity and promiscuity in extracellular signaling. However, structures suggest that when GFs bind repulsive guidance molecule (RGM) family coreceptors, type I receptors do not bind until reaching an intracellular, membrane-enveloped compartment, blurring the line between extra- and intracellular signaling. Modulator protein structures show how structurally diverse antagonists including follistatins, noggin, and members of the chordin family bind GFs to regulate signaling; complexes with the Dan family remain elusive. Much work is needed to understand how these molecular components assemble to form signaling hubs in extracellular environments in vivo. PMID:27638177

Secondary metabolites of Cynodon dactylon as an antagonist to angiotensin II type1 receptor: Novel in silico drug targeting approach for diabetic retinopathy

PubMed Central

Jananie, R. K.; Priya, V.; Vijayalakshmi, K.

2012-01-01

Objectives: To study the ability of the secondary metabolites of Cynodon dactylon to serve as an antagonist to angiotensin II type 1 receptor (AT1); activation of this receptor plays a vital role in diabetic retinopathy (DR). Materials and Methods: In silico methods are mainly harnessed to reduce time, cost and risk associated with drug discovery. Twenty-four compounds were identified as the secondary metabolites of hydroalcoholic extract of C. dactylon using the GCMS technique. These were considered as the ligands or inhibitors that would serve as an antagonist to the AT1. The ACD/Chemsketch tool was used to generate 3D structures of the ligands. A molecular file format converter tool was used to convert the generated data to the PDB format (Protein Data Bank) and was used for docking studies. The AT1 structure was retrieved from the Swissprot data base and PDB and visualized using the Rasmol tool. Domain analysis was carried from the Pfam data base; following this, the active site of the target protein was identified using a Q-site finder tool. The ability of the ligands to bind with the active site of AT1 was studied using the Autodocking tool. The docking results were analyzed using the WebLab viewer tool. Results: Sixteen ligands showed effective binding with the target protein; diazoprogesteron, didodecyl phthalate, and 9,12-octadecadienoyl chloride (z,z) may be considered as compounds that could be used to bind with the active site sequence of AT1. Conclusions: The present study shows that the metabolites of C. dactylon could serve as a natural antagonist to AT1 that could be used to treat diabetic retinopathy. PMID:22368412

Molecular mechanism of R-bicalutamide switching from androgen receptor antagonist to agonist induced by amino acid mutations using molecular dynamics simulations and free energy calculation

NASA Astrophysics Data System (ADS)

Liu, Hongli; Han, Rui; Li, Jiazhong; Liu, Huanxiang; Zheng, Lifang

2016-12-01

R-bicalutamide, a first generation antiandrogen, was used to treat prostate cancer for decades. Although it is very effective at the beginning, resistance appears after 2-3 years of treatment. Mutation of androgen receptor (AR) is considered a main reason for drug resistance. It is reported that AR W741C, W741L, W741C_T877A, T877A, F876L, F876L_T877A and L701H mutations can convert R-bicalutamide from AR antagonist to agonist, but the switching mechanisms are not clear. In this study, molecular dynamics simulations and molecular mechanics generalized Born surface area (MM-GBSA) calculations were performed to analyze the interaction mechanisms between R-bicalutamide and wild type/mutant ARs. The results indicate that helix H12, which lies on the top of AR LBD like a cover, plays a vital role in R-bicalutamide binding. When interacting with AR, the B-ring of R-bicalutamide pushes H12 aside, distorting the coactivator binding site (AF2) resulting in the inactivation of transcription. Several residue mutations appear to enlarge the distance between the B-ring of R-bicalutamide and H12, reducing steric clash, which is conducive to a closed H12 conformation, leading to the formation of the coactivator binding site AF2 and increased transcription. Hydrogen bond and per-residue free energy decomposition analyses are also investigated to explore the interacting mechanisms, and M895 is found to be a key residue in the antagonist mechanism. The obtained molecular mechanisms will aid rational screening and design of novel AR antagonists, even to mutant AR.

Molecular mechanism of R-bicalutamide switching from androgen receptor antagonist to agonist induced by amino acid mutations using molecular dynamics simulations and free energy calculation.

PubMed

Liu, Hongli; Han, Rui; Li, Jiazhong; Liu, Huanxiang; Zheng, Lifang

2016-12-01

R-bicalutamide, a first generation antiandrogen, was used to treat prostate cancer for decades. Although it is very effective at the beginning, resistance appears after 2-3 years of treatment. Mutation of androgen receptor (AR) is considered a main reason for drug resistance. It is reported that AR W741C, W741L, W741C_T877A, T877A, F876L, F876L_T877A and L701H mutations can convert R-bicalutamide from AR antagonist to agonist, but the switching mechanisms are not clear. In this study, molecular dynamics simulations and molecular mechanics generalized Born surface area (MM-GBSA) calculations were performed to analyze the interaction mechanisms between R-bicalutamide and wild type/mutant ARs. The results indicate that helix H12, which lies on the top of AR LBD like a cover, plays a vital role in R-bicalutamide binding. When interacting with AR, the B-ring of R-bicalutamide pushes H12 aside, distorting the coactivator binding site (AF2) resulting in the inactivation of transcription. Several residue mutations appear to enlarge the distance between the B-ring of R-bicalutamide and H12, reducing steric clash, which is conducive to a closed H12 conformation, leading to the formation of the coactivator binding site AF2 and increased transcription. Hydrogen bond and per-residue free energy decomposition analyses are also investigated to explore the interacting mechanisms, and M895 is found to be a key residue in the antagonist mechanism. The obtained molecular mechanisms will aid rational screening and design of novel AR antagonists, even to mutant AR.

Neutralization of Staphylococcal Enterotoxin B by an Aptamer Antagonist

PubMed Central

Wang, Kaiyu; Gan, Longjie; Jiang, Li; Zhang, Xianhui; Yang, Xiangyue; Chen, Min

2015-01-01

Staphylococcal enterotoxin B (SEB) is a major virulence factor for staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T lymphocytic population, releasing proinflammatory cytokines. Blocking SEB-initiated toxicity may be an effective strategy for treating TSS. Using a process known as systematic evolution of ligands by exponential enrichment (SELEX), we identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd = 64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and cytokine secretion in human peripheral blood mononuclear cells. Moreover, a PEGylated aptamer antagonist significantly reduced mortality in a “double-hit” mouse model of SEB-induced TSS, established via sensitization with d-galactosamine followed by SEB challenge. Therefore, our novel aptamer antagonist may offer potential therapeutic efficacy against SEB-mediated TSS. PMID:25624325

Nonpeptidic angiotensin II AT₁ receptor antagonists derived from 6-substituted aminocarbonyl and acylamino benzimidazoles.

PubMed

Zhang, Jun; Wang, Jin-Liang; Yu, Wei-Fa; Zhou, Zhi-Ming; Tao, Wen-Chang; Wang, Yi-Cheng; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

2013-11-01

Both 6-substituted aminocarbonyl and acylamino benzimidazole derivatives were designed and synthesized as nonpeptidic angiotensin II AT₁ receptor antagonists. Compounds 6f, 6g, 11e, 11f, 11g, and 12 showed nanomolar AT₁ receptor binding affinity and high AT₁ receptor selectivity over AT₂ receptor in a preliminary pharmacological evaluation. Among them, the two most active compounds 6f (AT₁ IC₅₀ = 3 nM, AT₂ IC₅₀ > 10,000 nM, PA₂ = 8.51) and 11g (AT₁ IC₅₀ = 0.1 nM, AT₂ IC₅₀ = 149 nM, PA₂ = 8.43) exhibited good antagonistic activity in isolated rabbit aortic strip functional assay. In addition, they were orally active AT₁ receptor antagonists in spontaneous hypertensive rats. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy

PubMed Central

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-01-01

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicteddual binding modes across multiple bacterial species, our approach opens up newpossibilities for understanding assembly and catalytic properties of a broadrange of multi-enzyme complexes. DOI: http://dx.doi.org/10.7554/eLife.10319.001 PMID:26519733

A review of fungal antagonists of powdery mildews and their potential as biocontrol agents.

PubMed

Kiss, Levente

2003-04-01

There are approximately 40 fungal species that have so far been reported as natural antagonists of powdery mildews or have been tested as their potential biocontrol agents. This review summarizes the published data on their identification, taxonomy, ecology, modes of action and biocontrol efficacy. The results obtained with the two products already registered, AQ10 Biofungicide and Sporodex, are also discussed.

DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS

EPA Science Inventory

Screening for endocrine disrupting chemicals (EDCs) that act as estrogens or antiestrogens relies on the use of in vitro binding and gene expression assays coupled with short-term diagnostic in vivo assays. Although binding assays are useful to identify chemicals that are competi...

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

PubMed

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su; Hille, Bertil

2017-07-11

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M 1 muscarinic acetylcholine receptors (M 1 Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M 1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M 1 R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

PubMed Central

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su

2017-01-01

Binding of agonists to G-protein–coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane. PMID:28652372

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, B.T.

1989-06-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or themore » maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).« less

Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response.

PubMed

Paramo, Teresa; Tomasio, Susana M; Irvine, Kate L; Bryant, Clare E; Bond, Peter J

2015-12-09

Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the "membrane-like" nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics.

Energetics of Endotoxin Recognition in the Toll-Like Receptor 4 Innate Immune Response

PubMed Central

Paramo, Teresa; Tomasio, Susana M.; Irvine, Kate L.; Bryant, Clare E.; Bond, Peter J.

2015-01-01

Bacterial outer membrane lipopolysaccharide (LPS) potently stimulates the mammalian innate immune system, and can lead to sepsis, the primary cause of death from infections. LPS is sensed by Toll-like receptor 4 (TLR4) in complex with its lipid-binding coreceptor MD-2, but subtle structural variations in LPS can profoundly modulate the response. To better understand the mechanism of LPS-induced stimulation and bacterial evasion, we have calculated the binding affinity to MD-2 of agonistic and antagonistic LPS variants including lipid A, lipid IVa, and synthetic antagonist Eritoran, and provide evidence that the coreceptor is a molecular switch that undergoes ligand-induced conformational changes to appropriately activate or inhibit the receptor complex. The plasticity of the coreceptor binding cavity is shown to be essential for distinguishing between ligands, whilst similar calculations for a model bacterial LPS bilayer reveal the “membrane-like” nature of the protein cavity. The ability to predict the activity of LPS variants should facilitate the rational design of TLR4 therapeutics. PMID:26647780

Oxime Ethers of (E)-11-Isonitrosostrychnine as Highly Potent Glycine Receptor Antagonists.

PubMed

Mohsen, Amal M Y; Mandour, Yasmine M; Sarukhanyan, Edita; Breitinger, Ulrike; Villmann, Carmen; Banoub, Maha M; Breitinger, Hans-Georg; Dandekar, Thomas; Holzgrabe, Ulrike; Sotriffer, Christoph; Jensen, Anders A; Zlotos, Darius P

2016-12-23

A series of (E)-11-isonitrosostrychnine oxime ethers, 2-aminostrychnine, (strychnine-2-yl)propionamide, 18-oxostrychnine, and N-propylstrychnine bromide were synthesized and evaluated pharmacologically at human α1 and α1β glycine receptors in a functional fluorescence-based and a whole-cell patch-clamp assay and in [ 3 H]strychnine binding studies. 2-Aminostrychnine and the methyl, allyl, and propargyl oxime ethers were the most potent α1 and α1β antagonists in the series, displaying IC 50 values similar to those of strychnine at the two receptors. Docking experiments to the strychnine binding site of the crystal structure of the α3 glycine receptor indicated the same orientation of the strychnine core for all analogues. For the most potent oxime ethers, the ether substituent was accommodated in a lipophilic receptor binding pocket. The findings identify the oxime hydroxy group as a suitable attachment point for linking two strychnine pharmacophores by a polymethylene spacer and are, therefore, important for the design of bivalent ligands targeting glycine receptors.

Crystal structure of the μ-opioid receptor bound to a morphinan antagonist

PubMed Central

Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien

2012-01-01

Summary Opium is one of the world’s oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many of their undesirable side effects (sedation, apnea and dependence) by binding to and activating the G-protein-coupled μ-opioid receptor (μOR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the μOR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most GPCRs published to date, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the μOR crystallizes as a two-fold symmetric dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction. PMID:22437502

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

PubMed

Schrattenholz, A; Roth, U; Godovac-Zimmermann, J; Maelicke, A

1997-10-28

Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

Characterization of the slow calcium channel binding sites for ( sup 3 H)SR 33557 in rat heart sarcolemmal membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatelain, P.; Beaufort, P.; Meysmans, L.

1991-01-01

SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. ({sup 3}H)SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific ({sup 3}H)SR 33557 binding, with Cd2+ being themore » most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace ({sup 3}H)SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit ({sup 3}H)SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.« less

Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

PubMed

Atabey, N; Gao, Y; Yao, Z J; Breckenridge, D; Soon, L; Soriano, J V; Burke, T R; Bottaro, D P

2001-04-27

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI.

PubMed

Jarvis, Cassie; Han, Zhenfu; Kalas, Vasilios; Klein, Roger; Pinkner, Jerome S; Ford, Bradley; Binkley, Jana; Cusumano, Corinne K; Cusumano, Zachary; Mydock-McGrane, Laurel; Hultgren, Scott J; Janetka, James W

2016-02-17

Uropathogenic E. coli (UPEC) employ the mannose-binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half-life in vivo. In a rational design strategy, we obtained an X-ray structure of lead mannosides and then designed mannosides with improved drug-like properties. We show that cyclizing the carboxamide onto the biphenyl B-ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2-methyl-4-(1-oxo-1,2-dihydroisoquinolin-7-yl)phenyl α-d-mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N-Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22, as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria-mediated hemagglutination and prevent biofilm formation by UPEC with single-digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction of ligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides

PubMed Central

Blume, Arthur J.

1978-01-01

This study shows that nucleotides, as well as ions, regulate the opiate receptors of brain. GMP-P(NH)P and Na+ reduce the amount of steady-state specific [3H]dihydromorphine binding and increase the rate of dissociation of the ligand from the opiate receptor. In contrast, Mn2+ decreases the rate of ligand dissociation and antagonizes the ability of Na+ to increase dissociation. The effects of GMP-P(NH)P on steady-state binding and dissociation are not reversed by washing. Only GTP, GDP, ITP, and IMP-P(NH)P, in addition to GMP-P(NH)P, increase the rate of dihydromorphine dissociation. The site of nucleotide action appears to have high affinity: <1 μM GMP-P(NH)P produces half-maximal increases in ligand dissociation. GMP-P(NH)P- and Na+-directed increases in dissociation have also been found for the opiate agonists [3H]etorphine, [3H]Leu-enkephalin, and [3H]Met-enkephalin and the opiate antagonist [3H]naltrexone. Mn2+-directed decreases in dissociation have been found for the agonist [3H]-etorphine and the antagonist [3H]naltrexone. Although the plasma membrane receptors for a number of other neuro-transmitters and hormones are also regulated by guanine nucleotides, the opiate receptors appear unique because only they show nucleotide regulation of both agonist and antagonist binding. PMID:205867

Structure-activity relationships studies on weakly basic N-arylsulfonylindoles with an antagonistic profile in the 5-HT6 receptor

NASA Astrophysics Data System (ADS)

Mella, Jaime; Villegas, Francisco; Morales-Verdejo, César; Lagos, Carlos F.; Recabarren-Gajardo, Gonzalo

2017-07-01

We recently reported a series of 39 weakly basic N-arylsulfonylindoles as novel 5-HT6 antagonists. Eight of the compounds exhibited moderate to high binding affinities, with 2-(4-(2-Methoxyphenyl)piperazin-1-yl)-1-(1-tosyl-1H-indol-3-yl)ethanol 16 showing the highest binding affinity (pKi = 7.87). Given these encouraging results and as a continuation of our research, we performed an extensive step-by-step search for the best 3D-QSAR model that allows us to rationally propose novel molecules with improved 5-HT6 affinity based on our previously reported series. A comparative molecular similarity indices analysis (CoMSIA) model built on a docking-based alignment was developed, wherein steric, electrostatic, hydrophobic and hydrogen bond properties are correlated with biological activity. The model was validated internally and externally (q2 = 0.721; r2pred = 0.938), and identified the sulfonyl and hydroxyl groups and the piperazine ring among the main regions of the molecules that can be modified to create new 5-HT6 antagonists.

Ligand-guided optimization of CXCR4 homology models for virtual screening using a multiple chemotype approach

NASA Astrophysics Data System (ADS)

Neves, Marco A. C.; Simões, Sérgio; Sá e Melo, M. Luisa

2010-12-01

CXCR4 is a G-protein coupled receptor for CXCL12 that plays an important role in human immunodeficiency virus infection, cancer growth and metastasization, immune cell trafficking and WHIM syndrome. In the absence of an X-ray crystal structure, theoretical modeling of the CXCR4 receptor remains an important tool for structure-function analysis and to guide the discovery of new antagonists with potential clinical use. In this study, the combination of experimental data and molecular modeling approaches allowed the development of optimized ligand-receptor models useful for elucidation of the molecular determinants of small molecule binding and functional antagonism. The ligand-guided homology modeling approach used in this study explicitly re-shaped the CXCR4 binding pocket in order to improve discrimination between known CXCR4 antagonists and random decoys. Refinement based on multiple test-sets with small compounds from single chemotypes provided the best early enrichment performance. These results provide an important tool for structure-based drug design and virtual ligand screening of new CXCR4 antagonists.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rimmaudo, Laura Elizabeth; Torre, Eulalia de la; Sacerdote de Lustig, Eugenia

Angiogenesis is a process of new blood vessel development from pre-existing vasculature and it plays an essential role in tumor growth and metastases. Here, we investigate the expression of muscarinic acetylcholine receptors (mAchR) and their participation in tumor cell proliferation and angiogenesis ability. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate indicate that LMM3 cells derived from a murine mammary adenocarcinoma express a single class of functional mAchR. Competition binding assays with selective muscarinic antagonists indicate a predominance of M{sub 3} receptor subtype. The muscarinic agonist carbachol (CARB) stimulates LMM3 cell proliferation in a concentration dependent manner. Themore » maximal effect induced by 10{sup -9} M CARB was totally blunted by atropine and by the selective M{sub 3} and M{sub 1} antagonists, para-fluoro hexahydro sila-difenidol (pf-HHSiD) and pirenzepine, respectively. In addition, pf-HHSiD completely blocked in vivo CARB-induced neovascular formation and vascular endothelial growth factor-A in LMM3 tumor cells. We can conclude that mAchR expressed in LMM3 mammary tumor cells positively regulate proliferation and angiogenesis required for tumor progression.« less

Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors.

PubMed

Disingrini, Teresa; Muth, Mathias; Dallanoce, Clelia; Barocelli, Elisabetta; Bertoni, Simona; Kellershohn, Kerstin; Mohr, Klaus; De Amici, Marco; Holzgrabe, Ulrike

2006-01-12

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

PubMed

Murase, Akio; Nakao, Kazunari; Takada, Junji

2008-01-01

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor

PubMed Central

Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W.

2010-01-01

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')- , (R,S')-, and (S,R')-fenoterol, to the β2-adrenergic receptor (β2-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human β2-AR. Competitive displacement studies using [3H]CGP-12177 as the marker ligand were conducted at 4°, 15°, 25°, 30° and 37°C, the binding affinities calculated and the standard enthalpic (ΔH°) and standard entropic (ΔS°) contribution to the standard free energy change (ΔG°) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the β2-AR, and, therefore, the results of this study are inconsistent with the previously described “thermodynamic agonist-antagonist discrimination”, in which the binding of an agonist to the β-AR is entropy-driven and the binding of an antagonist is enthalpy driven. In addition, the data demonstrate that the chirality of the carbon atom containing the β-hydroxyl group of the fenoterol molecule (the β-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the β-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process. PMID:20144591

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the beta(2)-adrenoceptor.

PubMed

Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W

2010-06-01

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')-, (R,S')-, and (S,R')-fenoterol, to the beta(2)-adrenergic receptor (beta(2)-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human beta(2)-AR. Competitive displacement studies using [(3)H]CGP-12177 as the marker ligand were conducted at 4, 15, 25, 30 and 37 degrees C, the binding affinities calculated and the standard enthalpic (DeltaH degrees ) and standard entropic (DeltaS degrees ) contribution to the standard free energy change (DeltaG degrees ) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the beta(2)-AR, and, therefore, the results of this study are inconsistent with the previously described "thermodynamic agonist-antagonist discrimination", in which the binding of an agonist to the beta-AR is entropy-driven and the binding of an antagonist is enthalpy-driven. In addition, the data demonstrate that the chirality of the carbon atom containing the beta-hydroxyl group of the fenoterol molecule (the beta-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the beta-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process. Published by Elsevier Inc.

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

PubMed

Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

2017-12-01

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Mesd Is a Universal Inhibitor of Wnt Co-receptor LRP5/6 and Blocks Wnt/β-catenin Signaling in Cancer Cells†

PubMed Central

Lu, Wenyan; Liu, Chia-Chen; Thottassery, Jaideep V.; Bu, Guojun; Li, Yonghe

2010-01-01

Mesd is a specialized chaperone for the low-density lipoprotein receptor-related protein-5 (LRP5) and LRP6. In our previous studies, we found that Mesd binds to mature LRP6 on the cell surface and blocks the binding of Wnt antagonist Dickkopf-1(Dkk1) to LRP6. Herein, we demonstrated that Mesd also binds to LRP5 with a high affinity, and is a universal inhibitor of LRP5/6 ligands. Mesd not only blocks Wnt antagonists Dkk1 and Sclerostin binding to LRP5/6, but also inhibits Wnt3A and Rspondin1-induced Wnt/β-catenin signaling in LRP5/6 expressing cells. We also found that Mesd, Dkk1 and Sclerostin compete with one another for binding to LRP5 and LRP6 at the cell surface. More importantly, we demonstrated that Mesd is able to suppress LRP6 phosphorylation and Wnt/β-catenin signaling in prostate cancer PC-3 cells, and inhibits PC-3 cell proliferation. Our results indicate that recombinant Mesd protein is a useful tool for studying Wnt/β-catenin signaling on the cell surface, and has a potential therapeutic role in Wnt-dependent cancers. PMID:20446724

Homology modeling, binding site identification and docking study of human angiotensin II type I (Ang II-AT1) receptor.

PubMed

Vyas, Vivek K; Ghate, Manjunath; Patel, Kinjal; Qureshi, Gulamnizami; Shah, Surmil

2015-08-01

Ang II-AT1 receptors play an important role in mediating virtually all of the physiological actions of Ang II. Several drugs (SARTANs) are available, which can block the AT1 receptor effectively and lower the blood pressure in the patients with hypertension. Currently, there is no experimental Ang II-AT1 structure available; therefore, in this study we modeled Ang II-AT1 receptor structure using homology modeling followed by identification and characterization of binding sites and thereby assessing druggability of the receptor. Homology models were constructed using MODELLER and I-TASSER server, refined and validated using PROCHECK in which 96.9% of 318 residues were present in the favoured regions of the Ramachandran plots. Various Ang II-AT1 receptor antagonist drugs are available in the market as antihypertensive drug, so we have performed docking study with the binding site prediction algorithms to predict different binding pockets on the modeled proteins. The identification of 3D structures and binding sites for various known drugs will guide us for the structure-based drug design of novel compounds as Ang II-AT1 receptor antagonists for the treatment of hypertension. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

From receptor binding kinetics to signal transduction; a missing link in predicting in vivo drug-action.

PubMed

Nederpelt, Indira; Kuzikov, Maria; de Witte, Wilbert E A; Schnider, Patrick; Tuijt, Bruno; Gul, Sheraz; IJzerman, Adriaan P; de Lange, Elizabeth C M; Heitman, Laura H

2017-10-26

An important question in drug discovery is how to overcome the significant challenge of high drug attrition rates due to lack of efficacy and safety. A missing link in the understanding of determinants for drug efficacy is the relation between drug-target binding kinetics and signal transduction, particularly in the physiological context of (multiple) endogenous ligands. We hypothesized that the kinetic binding parameters of both drug and endogenous ligand play a crucial role in determining cellular responses, using the NK1 receptor as a model system. We demonstrated that the binding kinetics of both antagonists (DFA and aprepitant) and endogenous agonists (NKA and SP) have significantly different effects on signal transduction profiles, i.e. potency values, in vitro efficacy values and onset rate of signal transduction. The antagonistic effects were most efficacious with slowly dissociating aprepitant and slowly associating NKA while the combination of rapidly dissociating DFA and rapidly associating SP had less significant effects on the signal transduction profiles. These results were consistent throughout different kinetic assays and cellular backgrounds. We conclude that knowledge of the relationship between in vitro drug-target binding kinetics and cellular responses is important to ultimately improve the understanding of drug efficacy in vivo.

The HSP90 binding mode of a radicicol-like E-oxime from docking, binding free energy estimations, and NMR 15N chemical shifts

PubMed Central

Spichty, Martin; Taly, Antoine; Hagn, Franz; Kessler, Horst; Barluenga, Sofia; Winssinger, Nicolas; Karplus, Martin

2009-01-01

We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of back-bone 15N provides an additional evaluation criteria. As a last test we check the binding modes against available structure-activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex. PMID:19482409

Pharmacological characterization of the cysteinyl-leukotriene antagonists CGP 45715A (iralukast) and CGP 57698 in human airways in vitro

PubMed Central

Capra, Valérie; Bolla, Manlio; Angelo Belloni, Pier; Mezzetti, Maurizio; Carlo Folco, G; Nicosia, Simonetta; Enrico Rovati, G

1998-01-01

Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. We investigated the pharmacological profile of the cysteinyl-LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. Competition curves vs [3H]-LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]-LTD4; (b) as in all the G-protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2=16.6 nM±36% CV and Ki1= Ki2=5.7 nM±19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. In functional studies: (a) iralukast and CGP 57698 antagonized LTD4-induced contraction of human bronchi, with pA2 values of 7.77±4.3% CV and 8.51±1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. Both antagonists significantly inhibited antigen-induced contraction of human isolated bronchial strips in a concentration-dependent manner, lowering the upper plateau of the anti-IgE curves. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases. PMID:9504401

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

PubMed

Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

2011-01-01

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor. © 2011 Bentham Science Publishers

Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands.

PubMed

Bender, Aaron M; Clark, Mary J; Agius, Michael P; Traynor, John R; Mosberg, Henry I

2014-01-15

In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers.

PubMed

Gong, Ping; Hong, Huixiao; Perkins, Edward J

2015-01-01

Antagonism of ionotropic GABA receptors (iGABARs) can occur at three distinct types of receptor binding sites causing chemically induced epileptic seizures. Here we review three adverse outcome pathways, each characterized by a specific molecular initiating event where an antagonist competitively binds to active sites, negatively modulates allosteric sites or noncompetitively blocks ion channel on the iGABAR. This leads to decreased chloride conductance, followed by depolarization of affected neurons, epilepsy-related death and ultimately decreased population. Supporting evidence for causal linkages from the molecular to population levels is presented and differential sensitivity to iGABAR antagonists in different GABA receptors and organisms discussed. Adverse outcome pathways are poised to become important tools for linking mechanism-based biomarkers to regulated outcomes in next-generation risk assessment.

Boronic acid-containing CXCR1/2 antagonists: optimization of metabolic stability, in vivo evaluation, and a proposed receptor binding model

PubMed Central

Maeda, Dean Y.; Peck, Angela M.; Schuler, Aaron D.; Quinn, Mark T.; Kirpotina, Liliya N.; Wicomb, Winston N.; Auten, Richard L.; Gundla, Rambabu; Zebala, John A.

2015-01-01

Blockade of undesired neutrophil migration to sites of inflammation remains an area of substantial pharmaceutical interest. To effect this blockade, a validated therapeutic target is antagonism of the chemokine receptor CXCR2. Herein we report the discovery of 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide 6, an antagonist with activity at both CXCR1 and CXCR2 receptors (IC50 values 31 and 21 nM, respectively). Compound 6 exhibited potent inhibition of neutrophil influx in a rat model of pulmonary inflammation, and is hypothesized to interact with a unique intracellular binding site on CXCR2. Compound 6 (SX-576) is undergoing further investigation as a potential therapy for pulmonary inflammation. PMID:25933594

Pharmacological and molecular characterization of muscarinic receptor subtypes in human esophageal smooth muscle.

PubMed

Preiksaitis, H G; Krysiak, P S; Chrones, T; Rajgopal, V; Laurier, L G

2000-12-01

Esophageal peristalsis is dependent on activation of muscarinic receptors, but little is known about the roles of specific receptor subtypes in the human esophagus. We examined muscarinic receptor expression and function in human esophageal smooth muscle obtained from patients undergoing resection for cancer. [(3)H]Quinuclidinyl benzylate (QNB)-specific binding was similar in longitudinal muscle (B(max) = 106 +/- 22 fmol/mg of protein, K(d) = 68 +/- 9 pM) and circular muscle (B(max) = 81 +/- 16 fmol/mg of protein, K(d) = 79 +/- 15 pM). Subtype-selective antagonists inhibited [(3)H]QNB similarly in muscle from both layers. Further analysis of antagonist inhibition of [(3)H]QNB binding showed a major site (60-70%) with antagonist affinity profile consistent with the M2 subtype and a second site that could not be classified. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated the presence of all five known muscarinic receptor subtypes, and immunocytochemistry on acutely isolated smooth muscle cells confirmed the expression of each subtype on the muscle cells. Subtype-selective antagonists had similar inhibitory effects on carbachol-evoked contractions in longitudinal muscle and circular muscle strips with pA(2) values of 9.5 +/- 0.1 and 9.6 +/- 0.2 for 4-diphenylacetoxy-N-methylpiperidine methiodide, 7.1 +/- 0.1 and 7.0 +/- 0.2 for pirenzepine, and 6.2 +/- 0.2 and 6.4 +/- 0.2 for methoctramine, respectively. We conclude that human esophageal smooth muscle expresses muscarinic receptor subtypes M1 through M5. The antagonist sensitivity profile for muscle contraction is consistent with activation of the M3 subtype.

Exploring details about structure requirements based on novel CGRP receptor antagonists urethanamide, aspartate, succinate and pyridine derivatives by in silico methods

NASA Astrophysics Data System (ADS)

Li, Yan; He, Haoran; Wang, Jinghui; Han, Chunxiao; Feng, Jiaqi; Zhang, Shuwei; Yang, Ling

2014-09-01

The migraine never fails to afflict individuals in the world that knows no lack of such cases. CGRP (calcitonin gene-related peptide) is found closely related to migraine and olcegepant (BIBN4096) is effective in alleviating the pain. In our work, the combination of ligand- and receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) studies along with molecular docking was applied to provide us insights about how urethanamide, pyridine and aspartate and succinate derivatives (novel CGRP receptor antagonists) play a part in inhibiting the activity of CGRP receptor. The optimal CoMSIA model shows the Q2 of 0.505, R2ncv of 0.992 and its accurate predictive ability was confirmed by checking out an independent test set which gave R2pred value of 0.885. Besides, the 3D contour maps help us identify how different groups affect the antagonist activity while connecting to some key positions. In addition, the docking analysis shows the binding site emerging as the distorted “V” shape and including two binding pockets: one of them is hydrophobic, fixing the structural part 3 of compound 80, the other anchors the part 1 of compound 80. The docking analysis also shows the interaction mechanism between compound 80 and CGRP receptor, similar to the interaction between olcegepant and CGRP receptor. The findings derived from this work reveal the mechanism of related antagonists and facilitate the future rational design of novel antagonists with higher potency.

Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

PubMed

Shlamkovich, Tomer; Aharon, Lidan; Barton, William A; Papo, Niv

2017-05-16

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

PubMed

Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

2004-06-01

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

The structure and binding mode of citrate in the stabilization of gold nanoparticles

NASA Astrophysics Data System (ADS)

Al-Johani, Hind; Abou-Hamad, Edy; Jedidi, Abdesslem; Widdifield, Cory M.; Viger-Gravel, Jasmine; Sangaru, Shiv Shankar; Gajan, David; Anjum, Dalaver H.; Ould-Chikh, Samy; Hedhili, Mohamed Nejib; Gurinov, Andrei; Kelly, Michael J.; El Eter, Mohamad; Cavallo, Luigi; Emsley, Lyndon; Basset, Jean-Marie

2017-09-01

Elucidating the binding mode of carboxylate-containing ligands to gold nanoparticles (AuNPs) is crucial to understand their stabilizing role. A detailed picture of the three-dimensional structure and coordination modes of citrate, acetate, succinate and glutarate to AuNPs is obtained by 13C and 23Na solid-state NMR in combination with computational modelling and electron microscopy. The binding between the carboxylates and the AuNP surface is found to occur in three different modes. These three modes are simultaneously present at low citrate to gold ratios, while a monocarboxylate monodentate (1κO1) mode is favoured at high citrate:gold ratios. The surface AuNP atoms are found to be predominantly in the zero oxidation state after citrate coordination, although trace amounts of Auδ+ are observed. 23Na NMR experiments show that Na+ ions are present near the gold surface, indicating that carboxylate binding occurs as a 2e- L-type interaction for each oxygen atom involved. This approach has broad potential to probe the binding of a variety of ligands to metal nanoparticles.

LIV-1 and Zn Transporters: Establishing a Link Between Hyperprolactinemia and Breast Cancer

DTIC Science & Technology

2008-10-14

depression (e.g. dopamine antagonists) secondarily increase prolactin levels, potentially increasing breast cancer risk in an extraordinary number of...this relationship lies in the fact that drugs which induce hyperprolactinemia secondarily to the principal mode of action (e.g. dopamine antagonists...developmental disorders, mood disorders and autism . In many cases, these drugs are prescribed for patients as young as 3 years of age and are chronically

How to deal with multiple binding poses in alchemical relative protein-ligand binding free energy calculations.

PubMed

Kaus, Joseph W; Harder, Edward; Lin, Teng; Abel, Robert; McCammon, J Andrew; Wang, Lingle

2015-06-09

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the ligands. This improved the root-mean-square error (RMSE) for the predicted binding free energy from 1.9 kcal/mol with the original partial charges to 1.3 kcal/mol with the corrected partial charges.

How To Deal with Multiple Binding Poses in Alchemical Relative Protein–Ligand Binding Free Energy Calculations

PubMed Central

2016-01-01

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the ligands. This improved the root-mean-square error (RMSE) for the predicted binding free energy from 1.9 kcal/mol with the original partial charges to 1.3 kcal/mol with the corrected partial charges. PMID:26085821

Effects of an orally active vasopressin V1 receptor antagonist.

PubMed

Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I

1993-05-01

1. This paper reports on the in vitro and in vivo characteristics of a non-peptide vasopressin V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2( 1H)- quinolinone (OPC-21268). 2. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, [125I]-[d(CH2)5, sarcosine7]AVP from vasopressin V1 receptors in rat liver and kidney membranes, inhibitory concentration of 50% (IC50) 4 x 10(-8), 0.3 mol/L liver and 1.5 x 10(-8), 0.2 mol/L kidney. OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)-d(CH2)5[D-Ileu2, Ileu4]AVP binding to V2 receptors in renal membranes (IC50 > 10(-4) mol/L). 3. After oral administration to rats, OPC-21268 was an effective V1 antagonist to both liver and kidney V1 receptors, in a dose-dependent manner. 4. These studies confirm that OPC-21268 is a potent non-peptide, orally effective V1 vasopressin receptor antagonist.

Functional genomic approaches for understanding the mode of action of Bacillus sp biocontrol strains

USDA-ARS?s Scientific Manuscript database

Complete genome sequencing of several Bacillus sp. strains has shed new light on the mode of action of these antagonists of plant pathogens. The use of genomic data mining tools provided the ability to quickly determine the potential of these strains to produce bioactive secondary metabolites. Our B...

The EED protein–protein interaction inhibitor A-395 inactivates the PRC2 complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Yupeng; Selvaraju, Sujatha; Curtin, Michael L.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed inmore » vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein–protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.« less

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Multi-functional norrin is a ligand for the LGR4 receptor

PubMed Central

Deng, Cheng; Reddy, Pradeep; Cheng, Yuan; Luo, Ching-Wei; Hsiao, Chih-Lun; Hsueh, Aaron J. W.

2013-01-01

Summary Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways. PMID:23444378

The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

PubMed

He, Yupeng; Selvaraju, Sujatha; Curtin, Michael L; Jakob, Clarissa G; Zhu, Haizhong; Comess, Kenneth M; Shaw, Bailin; The, Juliana; Lima-Fernandes, Evelyne; Szewczyk, Magdalena M; Cheng, Dong; Klinge, Kelly L; Li, Huan-Qiu; Pliushchev, Marina; Algire, Mikkel A; Maag, David; Guo, Jun; Dietrich, Justin; Panchal, Sanjay C; Petros, Andrew M; Sweis, Ramzi F; Torrent, Maricel; Bigelow, Lance J; Senisterra, Guillermo; Li, Fengling; Kennedy, Steven; Wu, Qin; Osterling, Donald J; Lindley, David J; Gao, Wenqing; Galasinski, Scott; Barsyte-Lovejoy, Dalia; Vedadi, Masoud; Buchanan, Fritz G; Arrowsmith, Cheryl H; Chiang, Gary G; Sun, Chaohong; Pappano, William N

2017-04-01

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

Multi-functional norrin is a ligand for the LGR4 receptor.

PubMed

Deng, Cheng; Reddy, Pradeep; Cheng, Yuan; Luo, Ching-Wei; Hsiao, Chih-Lun; Hsueh, Aaron J W

2013-05-01

Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

PubMed Central

Kuznetsova, Irina M.; Sulatskaya, Anna I.; Uversky, Vladimir N.; Turoverov, Konstantin K.

2012-01-01

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates. PMID:22383971

Analyzing thioflavin T binding to amyloid fibrils by an equilibrium microdialysis-based technique.

PubMed

Kuznetsova, Irina M; Sulatskaya, Anna I; Uversky, Vladimir N; Turoverov, Konstantin K

2012-01-01

A new approach for the determination of the amyloid fibril - thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates.

Human La binds mRNAs through contacts to the poly(A) tail.

PubMed

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-05-04

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.

Structural Diversity of Ligand-Binding Androgen Receptors Revealed by Microsecond Long Molecular Dynamics Simulations and Enhanced Sampling.

PubMed

Duan, Mojie; Liu, Na; Zhou, Wenfang; Li, Dan; Yang, Minghui; Hou, Tingjun

2016-09-13

Androgen receptor (AR) plays important roles in the development of prostate cancer (PCa). The antagonistic drugs, which suppress the activity of AR, are widely used in the treatment of PCa. However, the molecular mechanism of antagonism about how ligands affect the structures of AR remains elusive. To better understand the conformational variability of ARs bound with agonists or antagonists, we performed long time unbiased molecular dynamics (MD) simulations and enhanced sampling simulations for the ligand binding domain of AR (AR-LBD) in complex with various ligands. Based on the simulation results, we proposed an allosteric pathway linking ligands and helix 12 (H12) of AR-LBD, which involves the interactions among the ligands and the residues W741, H874, and I899. The interaction pathway provides an atomistic explanation of how ligands affect the structure of AR-LBD. A repositioning of H12 was observed, but it is facilitated by the C-terminal of H12, instead of by the loop between helix 11 (H11) and H12. The bias-exchange metadynamics simulations further demonstrated the above observations. More importantly, the free energy profiles constructed by the enhanced sampling simulations revealed the transition process between the antagonistic form and agonistic form of AR-LBD. Our results would be helpful for the design of more efficient antagonists of AR to combat PCa.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds

PubMed Central

Ballet, Steven; Marczak, Ewa D.; Feytens, Debby; Salvadori, Severo; Sasaki, Yusuke; Abell, Andrew D.; Lazarus, Lawrence H.; Balboni, Gianfranco; Tourwé, Dirk

2010-01-01

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced δ/μ antagonists, as determined by the functional [S35]GTPγS binding assay, the dimerization of potent Aia-based ‘parent’ ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds. PMID:20137938

Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature.

PubMed

Reilly, Regina M; McDonald, Heath A; Puttfarcken, Pamela S; Joshi, Shailen K; Lewis, LaGeisha; Pai, Madhavi; Franklin, Pamela H; Segreti, Jason A; Neelands, Torben R; Han, Ping; Chen, Jun; Mantyh, Patrick W; Ghilardi, Joseph R; Turner, Teresa M; Voight, Eric A; Daanen, Jerome F; Schmidt, Robert G; Gomtsyan, Arthur; Kort, Michael E; Faltynek, Connie R; Kym, Philip R

2012-08-01

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca(2+) flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Agonist and antagonist modulation of [35S]-GTPγS binding in transfected CHO cells expressing the neurotensin receptor

PubMed Central

Hermans, Emmanuel; Geurts, Muriel; Maloteaux, Jean-Marie

1997-01-01

The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. The agonist-induced binding of [35S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3±0.9 nM). The stimulation of [35S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC50 value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21±6 nM). The NT-induced [35S]-GTPγS binding was competitively inhibited by SR48692 (pA2 value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]-GTPγS. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor. PMID:9283723

Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

PubMed Central

Tognolini, Massimiliano; Incerti, Matteo; Mohamed, Iftiin Hassan; Giorgio, Carmine; Russo, Simonetta; Bruni, Renato; Lelli, Barbara; Bracci, Luisa; Noberini, Roberta; Pasquale, Elena B.; Barocelli, Elisabetta; Vicini, Paola; Mor, Marco

2012-01-01

The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions. PMID:22529030

Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension.

PubMed

Li, Wencheng; Sullivan, Michelle N; Zhang, Sheng; Worker, Caleb J; Xiong, Zhenggang; Speth, Robert C; Feng, Yumei

2015-02-01

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension. © 2014 American Heart Association, Inc.

Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

PubMed

Song, J; Doucette, C; Hanniford, D; Hunady, K; Wang, N; Sherf, B; Harrington, J J; Brunden, K R; Stricker-Krongrad, A

2005-06-01

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

Preserved pharmacological activity of hepatocytes-treated extracts of valerian and St. John's wort.

PubMed

Simmen, Urs; Saladin, Caroline; Kaufmann, Priska; Poddar, Manisha; Wallimann, Christine; Schaffner, Willi

2005-07-01

The two herbal extracts valerian (Valeriana officinalis L.) and St. John's wort (Hypericum perforatum L.) were studied for their metabolic changes upon incubation with freshly prepared rat hepatocytes and subsequently analysed phytochemically as well as pharmacologically in vitro. Quantitative HPLC analysis of valerian extracts revealed considerable metabolic activities with regard to sesquiterpenes and iridoids. The amount of acetoxyvalerenic acid decreased 9-fold, while that of hydroxyvalerenic acid correspondingly increased 9-fold due to O-deacetylation. The valepotriates didrovaltrate, isovaltrate and valtrate decreased 2-, 18- and 16-fold, respectively. However, the binding affinities of the incubated extracts to the benzodiazepine and picrotoxin binding site of the GABA (A) receptor were quite similar to those of the non-incubated extracts. Neither valerenic acids nor valepotriates exhibited any significant effect on the two binding sites when tested as single compounds. Therefore, either other constituents represent the active ones or multiple compounds are necessary for the observed inhibitory and allosteric effects at the GABA (A) receptor. Extracts of St. John's wort were less potently metabolised than valerian. The amount of pseudohypericin and the main flavonoids (hyperoside, rutin, isoquercitrin, quercitrin, quercetin and I3,II8-biapigenin) slightly decreased during the 4-h incubation period. Both the antagonist effect at the corticotropin-releasing factor (CRF) type 1 receptor and the binding inhibition at the 5-HT transporter were attenuated during the metabolic treatment. The reduced antagonist effect correlates with the decreasing amount of pseudohypericin known to be a CRF (1) receptor antagonist. In conclusion, the incubation of plant extracts with freshly prepared rat hepatocytes represents a useful approach to study the pharmacological action of metabolised plant extracts. The consistent pharmacological activity of both valerian and St. John's wort is concordant with the known clinical efficacy of pharmacological activities.

Intracerebroventricular Infusion of the (Pro)renin Receptor Antagonist PRO20 Attenuates Deoxycorticosterone Acetate-Salt–Induced Hypertension

PubMed Central

Li, Wencheng; Sullivan, Michelle N.; Zhang, Sheng; Worker, Caleb J.; Xiong, Zhenggang; Speth, Robert C.; Feng, Yumei

2016-01-01

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT1 receptor–dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension. PMID:25421983

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Lack of activity of 15-epi-lipoxin A₄ on FPR2/ALX and CysLT1 receptors in interleukin-8-driven human neutrophil function.

PubMed

Planagumà, A; Domenech, T; Jover, I; Ramos, I; Sentellas, S; Malhotra, R; Miralpeix, M

2013-08-01

Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15-epi-lipoxin (LX)A interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti-inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15-epi-LXA₄ analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK-571 binds to FPR2/ALX, so cross-reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15-epi-LXA4 are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15-epi-LXA₄-mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX-mediated signalling, enhancing guanosine triphosphate-gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15-epi-LXA₄ was inactive. Furthermore, 15-epi-LXA4 showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15-epi-LXA₄ showed a moderate reduction of interleukin (IL)-8-mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15-epi-LXA₄ is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL-8-induced neutrophil survival and produces only moderate inhibition in IL-8-mediated neutrophil migration. Our data do not support an anti-inflammatory role of 15-epi-LXA₄- FPR2/ALX interaction in IL-8-induced neutrophil inflammation. © 2013 British Society for Immunology.

CD and NMR conformational studies of a peptide encompassing the Mid Loop interface of Ship2-Sam.

PubMed

Mercurio, Flavia A; Scognamiglio, Pasqualina L; Di Natale, Concetta; Marasco, Daniela; Pellecchia, Maurizio; Leone, Marilisa

2014-11-01

The lipid phosphatase Ship2 is a protein that intervenes in several diseases such as diabetes, cancer, neurodegeneration, and atherosclerosis. It is made up of a catalytic domain and several protein docking modules such as a C-terminal Sam (Sterile alpha motif) domain. The Sam domain of Ship2 (Ship2-Sam) binds to the Sam domains of the EphA2 receptor (EphA2-Sam) and the PI3K effector protein Arap3 (Arap3-Sam). These heterotypic Sam-Sam interactions occur through formation of dimers presenting the canonical "Mid Loop/End Helix" binding mode. The central region of Ship2-Sam, spanning the C-terminal end of α2, the α3 and α4 helices together with the α2α3 and α3α4 interhelical loops, forms the Mid Loop surface that is needed to bind partners Sam domains. A peptide encompassing most of the Ship2-Sam Mid Loop interface (Shiptide) capable of binding to both EphA2-Sam and Arap3-Sam, was previously identified. Here we investigated the conformational features of this peptide, through solution CD and NMR studies in different conditions. These studies reveal that the peptide is highly flexible in aqueous buffer, while it adopts a helical conformation in presence of 2,2,2-trifluoroethanol. The discovered structural insights and in particular the identification of a helical motif, may lead to the design of more constrained and possibly cell permeable Shiptide analogs that could work as efficient antagonists of Ship2-Sam heterotypic interactions and embrace therapeutic applications. © 2014 Wiley Periodicals, Inc.

Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

PubMed

Byrne, C; Miclet, E; Broutin, I; Gallo, D; Pelekanou, V; Kampa, M; Castanas, E; Leclercq, G; Jacquot, Y

2013-10-01

Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation. © 2013 Wiley Periodicals, Inc.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms

PubMed Central

Lee, Sang-Min; Hay, Debbie L.; Pioszak, Augen A.

2016-01-01

Receptor activity-modifying proteins (RAMP1–3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8–37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. PMID:26895962

Identification of both NK1 and NK2 receptors in guinea-pig airways.

PubMed Central

McKee, K. T.; Millar, L.; Rodger, I. W.; Metters, K. M.

1993-01-01

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7694756

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh

2013-10-01

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα bindingmore » affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function. • The results have potential applications to green chemistry. • Models are publicly available for virtual screening via a web portal.« less

3D-QSAR comparative molecular field analysis on opioid receptor antagonists: pooling data from different studies.

PubMed

Peng, Youyi; Keenan, Susan M; Zhang, Qiang; Kholodovych, Vladyslav; Welsh, William J

2005-03-10

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) on a series of opioid receptor antagonists. To obtain statistically significant and robust CoMFA models, a sizable data set of naltrindole and naltrexone analogues was assembled by pooling biological and structural data from independent studies. A process of "leave one data set out", similar to the traditional "leave one out" cross-validation procedure employed in partial least squares (PLS) analysis, was utilized to study the feasibility of pooling data in the present case. These studies indicate that our approach yields statistically significant and highly predictive CoMFA models from the pooled data set of delta, mu, and kappa opioid receptor antagonists. All models showed excellent internal predictability and self-consistency: q(2) = 0.69/r(2) = 0.91 (delta), q(2) = 0.67/r(2) = 0.92 (mu), and q(2) = 0.60/r(2) = 0.96 (kappa). The CoMFA models were further validated using two separate test sets: one test set was selected randomly from the pooled data set, while the other test set was retrieved from other published sources. The overall excellent agreement between CoMFA-predicted and experimental binding affinities for a structurally diverse array of ligands across all three opioid receptor subtypes gives testimony to the superb predictive power of these models. CoMFA field analysis demonstrated that the variations in binding affinity of opioid antagonists are dominated by steric rather than electrostatic interactions with the three opioid receptor binding sites. The CoMFA steric-electrostatic contour maps corresponding to the delta, mu, and kappa opioid receptor subtypes reflected the characteristic similarities and differences in the familiar "message-address" concept of opioid receptor ligands. Structural modifications to increase selectivity for the delta over mu and kappa opioid receptors have been predicted on the basis of the CoMFA contour maps. The structure-activity relationships (SARs) together with the CoMFA models should find utility for the rational design of subtype-selective opioid receptor antagonists.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.

PubMed

Jeeva, Subbiah; Pador, Sean; Voss, Brittany; Ganaie, Safder Saieed; Mir, Mohammad Ayoub

2017-01-01

Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.

Binding Mode Analyses and Pharmacophore Model Development for Stilbene Derivatives as a Novel and Competitive Class of α-Glucosidase Inhibitors

PubMed Central

Kim, Jun Young; Arooj, Mahreen; Kim, Siu; Hwang, Swan; Kim, Byeong-Woo; Park, Ki Hun; Lee, Keun Woo

2014-01-01

Stilbene urea derivatives as a novel and competitive class of non-glycosidic α-glucosidase inhibitors are effective for the treatment of type II diabetes and obesity. The main purposes of our molecular modeling study are to explore the most suitable binding poses of stilbene derivatives with analyzing the binding affinity differences and finally to develop a pharmacophore model which would represents critical features responsible for α-glucosidase inhibitory activity. Three-dimensional structure of S. cerevisiae α-glucosidase was built by homology modeling method and the structure was used for the molecular docking study to find out the initial binding mode of compound 12, which is the most highly active one. The initial structure was subjected to molecular dynamics (MD) simulations for protein structure adjustment at compound 12-bound state. Based on the adjusted conformation, the more reasonable binding modes of the stilbene urea derivatives were obtained from molecular docking and MD simulations. The binding mode of the derivatives was validated by correlation analysis between experimental Ki value and interaction energy. Our results revealed that the binding modes of the potent inhibitors were engaged with important hydrogen bond, hydrophobic, and π-interactions. With the validated compound 12-bound structure obtained from combining approach of docking and MD simulation, a proper four featured pharmacophore model was generated. It was also validated by comparison of fit values with the Ki values. Thus, these results will be helpful for understanding the relationship between binding mode and bioactivity and for designing better inhibitors from stilbene derivatives. PMID:24465730

The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition.

PubMed

Tamayo, Joel V; Teramoto, Takamasa; Chatterjee, Seema; Hall, Traci M Tanaka; Gavis, Elizabeth R

2017-04-04

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo's RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Drosophila hnRNP F/H homolog glorund uses two distinct RNA-binding modes to diversify target recognition

DOE PAGES

Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema; ...

2017-04-04

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subsetmore » of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Lastly, our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.« less

The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subsetmore » of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.« less

The Drosophila hnRNP F/H homolog glorund uses two distinct RNA-binding modes to diversify target recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subsetmore » of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Lastly, our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.« less

Dopamine receptor antagonists as new mode-of-action insecticide leads for control of Aedes and Culex mosquito vectors.

PubMed

Nuss, Andrew B; Ejendal, Karin F K; Doyle, Trevor B; Meyer, Jason M; Lang, Emma G; Watts, Val J; Hill, Catherine A

2015-03-01

New mode-of-action insecticides are sought to provide continued control of pesticide resistant arthropod vectors of neglected tropical diseases (NTDs). We previously identified antagonists of the AaDOP2 D1-like dopamine receptor (DAR) from the yellow fever mosquito, Aedes aegypti, with toxicity to Ae. aegypti larvae as leads for novel insecticides. To extend DAR-based insecticide discovery, we evaluated the molecular and pharmacological characteristics of an orthologous DAR target, CqDOP2, from Culex quinquefasciatus, the vector of lymphatic filariasis and West Nile virus. CqDOP2 has 94.7% amino acid identity to AaDOP2 and 28.3% identity to the human D1-like DAR, hD1. CqDOP2 and AaDOP2 exhibited similar pharmacological responses to biogenic amines and DAR antagonists in cell-based assays. The antagonists amitriptyline, amperozide, asenapine, chlorpromazine and doxepin were between 35 to 227-fold more selective at inhibiting the response of CqDOP2 and AaDOP2 in comparison to hD1. Antagonists were toxic to both C. quinquefasciatus and Ae. aegypti larvae, with LC50 values ranging from 41 to 208 μM 72 h post-exposure. Orthologous DOP2 receptors identified from the African malaria mosquito, Anopheles gambiae, the sand fly, Phlebotomus papatasi and the tsetse fly, Glossina morsitans, had high sequence similarity to CqDOP2 and AaDOP2. DAR antagonists represent a putative new insecticide class with activity against C. quinquefasciatus and Ae. aegypti, the two most important mosquito vectors of NTDs. There has been limited change in the sequence and pharmacological properties of the DOP2 DARs of these species since divergence of the tribes Culicini and Aedini. We identified antagonists selective for mosquito versus human DARs and observed a correlation between DAR pharmacology and the in vivo larval toxicity of antagonists. These data demonstrate that sequence similarity can be predictive of target potential. On this basis, we propose expanded insecticide discovery around orthologous DOP2 targets from additional dipteran vectors.

Interaction of thioflavin T with amyloid fibrils: stoichiometry and affinity of dye binding, absorption spectra of bound dye.

PubMed

Sulatskaya, Anna I; Kuznetsova, Irina M; Turoverov, Konstantin K

2011-10-06

The fluorescence of the benzothiazole dye thioflavin T (ThT) is a well-known test for amyloid fibril formation. It has now become evident that ThT can also be used for structural investigations of amyloid fibrils and even for the treatment of amyloid diseases. In this case, one of the most urgent problems is an accurate determination of ThT-amyloid fibril binding parameters: the number of binding modes, stoichiometry, and binding constant for each mode. To obtain information concerning the ThT-amyloid fibril binding parameters, we propose to use absorption spectrophotometry of solutions prepared by equilibrium microdialysis. This approach is inherently designed for the determination of dye-receptor binding parameters. However, it has been very rarely used in the study of dye-protein interactions and has never been used to study the binding parameters of ThT or its analogues to amyloid fibrils. We showed that, when done in corpore, this approach enables the determination of not only binding parameters but also the absorption spectrum and molar extinction coefficient of ThT bound to sites of different binding modes. The proposed approach was used for the examination of lysozyme amyloid fibrils. Two binding modes were found for the ThT-lysozyme amyloid fibril interaction. These binding modes have significantly different binding constants (K(b1) = 7.5 × 10(6) M(-1), K(b2) = 5.6 × 10(4) M(-1)) and a different number of dye binding sites on the amyloid fibrils per protein molecule (n(1) = 0.11, n(2) = 0.24). The absorption spectra of ThT bound to sites of different modes differ from each other (ε(b1,max) = 5.1 × 10(4) M(-1) cm(-1), ε(b2,max) = 6.7 × 10(4) M(-1)cm(-1), λ(max) = 449 nm) and significantly differ from that of free ThT in aqueous solution (ε(max) = 3.2 × 10(4) M(-1)cm(-1), λ(max) = 412 nm). © 2011 American Chemical Society

Detection and characterization of nonspecific, sparsely-populated binding modes in the early stages of complexation

PubMed Central

Cardone, A.; Bornstein, A.; Pant, H. C.; Brady, M.; Sriram, R.; Hassan, S. A.

2015-01-01

A method is proposed to study protein-ligand binding in a system governed by specific and non-specific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultra-weak associations lead instead to broader distributions, a manifestation of non-specific, sparsely-populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first-encounter modes are metastable states from which stable (pre-relaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide-protein complex. The peptide adopts several low-population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated. PMID:25782918

Serotonin 2A receptor agonist binding in the human brain with [11C]Cimbi-36

PubMed Central

Ettrup, Anders; da Cunha-Bang, Sophie; McMahon, Brenda; Lehel, Szabolcs; Dyssegaard, Agnete; Skibsted, Anine W; Jørgensen, Louise M; Hansen, Martin; Baandrup, Anders O; Bache, Søren; Svarer, Claus; Kristensen, Jesper L; Gillings, Nic; Madsen, Jacob; Knudsen, Gitte M

2014-01-01

[11C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT2A) receptor agonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [11C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT2A receptors with [11C]Cimbi-36 PET. The two-tissue compartment model using arterial input measurements provided the most optimal quantification of cerebral [11C]Cimbi-36 binding. Reference tissue modeling was feasible as it induced a negative but predictable bias in [11C]Cimbi-36 PET outcome measures. In five subjects, pretreatment with the 5-HT2A receptor antagonist ketanserin before a second PET scan significantly decreased [11C]Cimbi-36 binding in all cortical regions with no effects in cerebellum. These results confirm that [11C]Cimbi-36 binding is selective for 5-HT2A receptors in the cerebral cortex and that cerebellum is an appropriate reference tissue for quantification of 5-HT2A receptors in the human brain. Thus, we here describe [11C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT2A receptors in the human brain. PMID:24780897

Efficacy and putative mode of action of native and commercial antagonistic yeasts against postharvest pathogens of pear.

PubMed

Lutz, M Cecilia; Lopes, Christian A; Rodriguez, M Eugenia; Sosa, M Cristina; Sangorrín, Marcela P

2013-06-17

Putative mechanisms of action associated with the biocontrol capacity of four yeast strains (Cryptoccocus albidus NPCC 1248, Pichia membranifaciens NPCC 1250, Cryptoccocus victoriae NPCC 1263 and NPCC 1259) against Penicillium expansum and Botrytis cinerea were studied by means of in vitro and in situ assays. C. albidus(YP), a commercial yeast was also evaluated for comparative purposes. The yeast strains exhibited a variety of different mechanisms including: wound colonization, germination inhibition, biofilm formation, secretion of killer toxins, competition for nutrient and secretion of hydrolytic enzymes (protease, chitinase and glucanase). The relationship between strains (and their associated antagonist mechanisms) and in situ antagonist activity was also evaluated. Results indicate that mechanisms such as production of hydrolytic enzymes, the ability for colonization of wounds, production of killer toxin and inhibition of germination are the most important for biocontrol activity. Our study indicate that multiple modes of action may explain why P. membranifaciens NPCC 1250 and C. victoriae NPCC 1263 provided excellent control of postharvest pears disease. Copyright © 2013 Elsevier B.V. All rights reserved.

Understanding the molecular basis of agonist/antagonist mechanism of GPER1/GPR30 through structural and energetic analyses.

PubMed

Méndez-Luna, David; Bello, Martiniano; Correa-Basurto, José

2016-04-01

The G-protein coupled receptors (GPCRs) represent the largest superfamily of membrane proteins in charge to pass the cell signaling after binding with their cognate ligands to the cell interior. In breast cancer, a GPCR named GPER1 plays a key role in the process of growth and the proliferation of cancer cells. In a previous study, theoretical methods were applied to construct a model of GPER1, which later was submitted to molecular dynamics (MD) simulations to perform a docking calculation. Based on this preceding work, it is known that GPER1 is sensitive to structural differences in its binding site. However, due to the nature of that past study, conformational changes linked to the ligand binding were not observed. Therefore, in this study, in order to explore the conformational changes coupled to the agonist/antagonist binding, MD simulations of about 0.25μs were performed for the free and bound states, summarizing 0.75μs of MD simulation in total. For the bound states, one agonist (G-1) and antagonist (G-15) were chosen since is widely known that these two molecules cause an impact on GPER1 mobility. Based on the conformational ensemble generated through MD simulations, we found that despite G-1 and G-15 being stabilized by similar map of residues, the structural differences between both ligands impact the hydrogen bond pattern not only at the GPER1 binding site but also along the seven-helix bundle, causing significant differences in the conformational mobility along the extracellular and cytoplasmic domain, and to a lesser degree in the curvatures of helix 2, helix 3 and helix 7 between the free and bound states, which is in agreement with reported literature, and might be linked to microscopic characteristics of the activated-inactivated transition. Furthermore, binding free energy calculations using the MM/GBSA method for the bound states, followed by an alanine scanning analysis allowed us to identify some important residues for the complex stabilization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tracing binding modes in hit-to-lead optimization: chameleon-like poses of aspartic protease inhibitors.

PubMed

Kuhnert, Maren; Köster, Helene; Bartholomäus, Ruben; Park, Ah Young; Shahim, Amir; Heine, Andreas; Steuber, Holger; Klebe, Gerhard; Diederich, Wibke E

2015-02-23

Successful lead optimization in structure-based drug discovery depends on the correct deduction and interpretation of the underlying structure-activity relationships (SAR) to facilitate efficient decision-making on the next candidates to be synthesized. Consequently, the question arises, how frequently a binding mode (re)-validation is required, to ensure not to be misled by invalid assumptions on the binding geometry. We present an example in which minor chemical modifications within one inhibitor series lead to surprisingly different binding modes. X-ray structure determination of eight inhibitors derived from one core scaffold resulted in four different binding modes in the aspartic protease endothiapepsin, a well-established surrogate for e.g. renin and β-secretase. In addition, we suggest an empirical metrics that might serve as an indicator during lead optimization to qualify compounds as candidates for structural revalidation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

PubMed Central

West, Graham M.; Willard, Francis S.; Sloop, Kyle W.; Showalter, Aaron D.; Pascal, Bruce D.; Griffin, Patrick R.

2014-01-01

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands. PMID:25180755

Gold-Catalyzed Cycloisomerization and Diels-Alder Reaction of 1,4,9-Dienyne Esters to 3 a,6-Methanoisoindole Esters with Pro-Inflammatory Cytokine Antagonist Activity.

PubMed

Susanti, Dewi; Liu, Li-Juan; Rao, Weidong; Lin, Sheng; Ma, Dik-Lung; Leung, Chung-Hang; Chan, Philip Wai Hong

2015-06-15

A synthetic method to prepare 3a,6-methanoisoindole esters efficiently by gold(I)-catalyzed tandem 1,2-acyloxy migration/Nazarov cyclization followed by Diels-Alder reaction of 1,4,9-dienyne esters is described. We also report the ability of one example to inhibit binding of tumor necrosis factor-α (TNF-α) to the tumor necrosis factor receptor 1 (TNFR1) site and TNF-α-induced nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation in cell at a half-maximal inhibitory concentration (IC50 ) value of 6.6 μM. Along with this is a study showing the isoindolyl derivative to exhibit low toxicity toward human hepatocellular liver carcinoma (HepG2) cells and its possible mode of activity based on molecular modeling analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Boronic acid-containing CXCR1/2 antagonists: Optimization of metabolic stability, in vivo evaluation, and a proposed receptor binding model.

PubMed

Maeda, Dean Y; Peck, Angela M; Schuler, Aaron D; Quinn, Mark T; Kirpotina, Liliya N; Wicomb, Winston N; Auten, Richard L; Gundla, Rambabu; Zebala, John A

2015-06-01

Blockade of undesired neutrophil migration to sites of inflammation remains an area of substantial pharmaceutical interest. To effect this blockade, a validated therapeutic target is antagonism of the chemokine receptor CXCR2. Herein we report the discovery of 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide 6, an antagonist with activity at both CXCR1 and CXCR2 receptors (IC50 values 31 and 21 nM, respectively). Compound 6 exhibited potent inhibition of neutrophil influx in a rat model of pulmonary inflammation, and is hypothesized to interact with a unique intracellular binding site on CXCR2. Compound 6 (SX-576) is undergoing further investigation as a potential therapy for pulmonary inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

The clinical development of p53-reactivating drugs in sarcomas - charting future therapeutic approaches and understanding the clinical molecular toxicology of Nutlins.

PubMed

Biswas, Swethajit; Killick, Emma; Jochemsen, Aart G; Lunec, John

2014-05-01

The majority of human sarcomas, particularly soft tissue sarcomas, are relatively resistant to traditional cytotoxic therapies. The proof-of-concept study by Ray-Coquard et al., using the Nutlin human double minute (HDM)2-binding antagonist RG7112, has recently opened a new chapter in the molecular targeting of human sarcomas. In this review, the authors discuss the challenges and prospective remedies for minimizing the significant haematological toxicities of the cis-imidazole Nutlin HDM2-binding antagonists. Furthermore, they also chart the future direction of the development of p53-reactivating (p53-RA) drugs in 12q13-15 amplicon sarcomas and as potential chemopreventative therapies against sarcomagenesis in germ line mutated TP53 carriers. Drawing lessons from the therapeutic use of Imatinib in gastrointestinal tumours, the authors predict the potential pitfalls, which may lie in ahead for the future clinical development of p53-RA agents, as well as discussing potential non-invasive methods to identify the development of drug resistance. Medicinal chemistry strategies, based on structure-based drug design, are required to re-engineer cis-imidazoline Nutlin HDM2-binding antagonists into less haematologically toxic drugs. In silico modelling is also required to predict toxicities of other p53-RA drugs at a much earlier stage in drug development. Whether p53-RA drugs will be therapeutically effective as a monotherapy remains to be determined.

Human La binds mRNAs through contacts to the poly(A) tail

PubMed Central

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-01-01

Abstract In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3’OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3’OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail. PMID:29447394

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Identification of spinal 5-HT sub 3 receptors and their role in the modulation of nociceptive responses in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glaum, S.R.

1988-01-01

The project consisted of two related studies: (1) the characterization of serotonin binding sites in crude and purified synaptic membranes prepared from the rat spinal cord, and (2) the association of serotonin binding sites with functional 5-HT receptor responses in the modulation of nociceptive information at the level of the spinal cord. The first series of experiments involved the preparation of membranes from the dorsal and ventral halves of the rat spinal cord and the demonstration of specific ({sup 3}H)serotonin binding to these membranes. High affinity binding sites which conformed to the 5-HT{sub 3} subtype were identified in dorsal, butmore » not ventral spinal cord synaptic membranes. These experiments also confirmed the presence of high affinity ({sup 3}H)5-HT binding sites in dorsal spinal cord synaptic membranes of the 5-HT{sub 1} subtype. The second group of studies demonstrated the ability of selective 5-HT{sub 3} antagonists to inhibit the antinociceptive response to intrathecally administered 5-HT, as measured by a change in tail flick and hot plate latencies. Intrathecal pretreatment with the selective 5-HT{sub 3} antagonists ICS 205-930 or MDL 72222 abolished the antinociceptive effects of 5-HT. Furthermore, the selective 5-HT{sub 3} agonist 2-methyl-5-HT mimicked the antinociceptive effects of 5-HT.« less

Role of adipocyte lipid-binding protein (ALBP) and acyl-coA binding protein (ACBP) in PPAR-mediated transactivation.

PubMed

Helledie, Torben; Jørgensen, Claus; Antonius, Marianne; Krogsdam, Ann M; Kratchmarova, Irina; Kristiansen, Karsten; Mandrup, Susanne

2002-10-01

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84.

PubMed

Mahmud, Zobaer Al; Jenkins, Laura; Ulven, Trond; Labéguère, Frédéric; Gosmini, Romain; De Vos, Steve; Hudson, Brian D; Tikhonova, Irina G; Milligan, Graeme

2017-12-20

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine 172 , located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [ 3 H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine 172 . These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

Studies of New Fused Benzazepine as Selective Dopamine D3 Receptor Antagonists Using 3D-QSAR, Molecular Docking and Molecular Dynamics

PubMed Central

Liu, Jing; Li, Yan; Zhang, Shuwei; Xiao, Zhengtao; Ai, Chunzhi

2011-01-01

In recent years, great interest has been paid to the development of compounds with high selectivity for central dopamine (DA) D3 receptors, an interesting therapeutic target in the treatment of different neurological disorders. In the present work, based on a dataset of 110 collected benzazepine (BAZ) DA D3 antagonists with diverse kinds of structures, a variety of in silico modeling approaches, including comparative molecular field analysis (CoMFA), comparative similarity indices analysis (CoMSIA), homology modeling, molecular docking and molecular dynamics (MD) were carried out to reveal the requisite 3D structural features for activity. Our results show that both the receptor-based (Q2 = 0.603, R2ncv = 0.829, R2pre = 0.690, SEE = 0.316, SEP = 0.406) and ligand-based 3D-QSAR models (Q2 = 0.506, R2ncv =0.838, R2pre = 0.794, SEE = 0.316, SEP = 0.296) are reliable with proper predictive capacity. In addition, a combined analysis between the CoMFA, CoMSIA contour maps and MD results with a homology DA receptor model shows that: (1) ring-A, position-2 and R3 substituent in ring-D are crucial in the design of antagonists with higher activity; (2) more bulky R1 substituents (at position-2 of ring-A) of antagonists may well fit in the binding pocket; (3) hydrophobicity represented by MlogP is important for building satisfactory QSAR models; (4) key amino acids of the binding pocket are CYS101, ILE105, LEU106, VAL151, PHE175, PHE184, PRO254 and ALA251. To our best knowledge, this work is the first report on 3D-QSAR modeling of the new fused BAZs as DA D3 antagonists. These results might provide information for a better understanding of the mechanism of antagonism and thus be helpful in designing new potent DA D3 antagonists. PMID:21541053

Studies of new fused benzazepine as selective dopamine D3 receptor antagonists using 3D-QSAR, molecular docking and molecular dynamics.

PubMed

Liu, Jing; Li, Yan; Zhang, Shuwei; Xiao, Zhengtao; Ai, Chunzhi

2011-02-18

In recent years, great interest has been paid to the development of compounds with high selectivity for central dopamine (DA) D3 receptors, an interesting therapeutic target in the treatment of different neurological disorders. In the present work, based on a dataset of 110 collected benzazepine (BAZ) DA D3 antagonists with diverse kinds of structures, a variety of in silico modeling approaches, including comparative molecular field analysis (CoMFA), comparative similarity indices analysis (CoMSIA), homology modeling, molecular docking and molecular dynamics (MD) were carried out to reveal the requisite 3D structural features for activity. Our results show that both the receptor-based (Q(2) = 0.603, R(2) (ncv) = 0.829, R(2) (pre) = 0.690, SEE = 0.316, SEP = 0.406) and ligand-based 3D-QSAR models (Q(2) = 0.506, R(2) (ncv) =0.838, R(2) (pre) = 0.794, SEE = 0.316, SEP = 0.296) are reliable with proper predictive capacity. In addition, a combined analysis between the CoMFA, CoMSIA contour maps and MD results with a homology DA receptor model shows that: (1) ring-A, position-2 and R(3) substituent in ring-D are crucial in the design of antagonists with higher activity; (2) more bulky R(1) substituents (at position-2 of ring-A) of antagonists may well fit in the binding pocket; (3) hydrophobicity represented by MlogP is important for building satisfactory QSAR models; (4) key amino acids of the binding pocket are CYS101, ILE105, LEU106, VAL151, PHE175, PHE184, PRO254 and ALA251. To our best knowledge, this work is the first report on 3D-QSAR modeling of the new fused BAZs as DA D3 antagonists. These results might provide information for a better understanding of the mechanism of antagonism and thus be helpful in designing new potent DA D3 antagonists.

Profile of netupitant/palonosetron (NEPA) fixed dose combination and its potential in the treatment of chemotherapy-induced nausea and vomiting (CINV)

PubMed Central

Navari, Rudolph M

2015-01-01

Chemotherapy-induced nausea and vomiting (CINV) is associated with a significant deterioration in quality of life. The emetogenicity of the chemotherapeutic agents, repeated chemotherapy cycles, and patient risk factors significantly influence CINV. The use of a combination of a 5-hydroxytryptamine-3 (5-HT3) receptor antagonists, dexamethasone, and a neurokinin-1 (NK-1) receptor antagonist has significantly improved the control of acute and delayed emesis in single-day chemotherapy. Palonosetron, a second generation 5-HT3 receptor antagonist with a different half-life, different binding capacity, and a different mechanism of action than the first generation 5-HT3 receptor antagonists, appears to be the most effective agent in its class. Netupitant, is a new NK-1 receptor antagonist with a high binding affinity, a long half-life of 90 hours, is metabolized by CYP3A4, and is an inhibitor of CYP3A4. NEPA is an oral fixed-dose combination of netupitant and palonosetron which has recently been employed in Phase II and Phase III clinical trials for the prevention of CINV in patients receiving moderately and highly emetogenic chemotherapy (MEC and HEC). The clinical trials demonstrated that NEPA (300 mg of netupitant plus 0.50 mg of palonosetron) significantly improved the prevention of CINV compared to the use of palonosetron alone in patients receiving either HEC or MEC. The clinical efficacy was maintained over multiple cycles of chemotherapy. NEPA (Akynzeo®) has recently been approved by the Food and Drug Administration (FDA) to treat nausea and vomiting in patients undergoing cancer chemotherapy. PMID:25552904

Importance of D1 and D2 receptor stimulation for the induction and expression of cocaine-induced behavioral sensitization in preweanling rats

PubMed Central

McDougall, Sanders A.; Rudberg, Krista N.; Veliz, Ana; Dhargalkar, Janhavi M.; Garcia, Aleesha S.; Romero, Loveth C.; Gonzalez, Ashley E.; Mohd-Yusof, Alena; Crawford, Cynthia A.

2017-01-01

The behavioral manifestations of psychostimulant-induced sensitization vary markedly between young and adult rats, suggesting that the neural mechanisms mediating this phenomenon differ across ontogeny. In this project we examined the importance of D1 and D2 receptors for the induction and expression of cocaine-induced behavioral sensitization during the preweanling period. In the behavioral experiments, rats were injected with reversible D1 and/or D2 antagonists (SCH23390 and/or raclopride) or an irreversible receptor antagonist (EEDQ) either before cocaine administration on the pretreatment day (induction) or before cocaine challenge on the test day (expression). In the EEDQ experiments, receptor specificity was assessed by using selective dopamine antagonists to protect D1 and/or D2 receptors from inactivation. Receptor binding assays showed that EEDQ caused substantial reductions in dorsal striatal D1 and D2 binding sites, while SCH23390 and raclopride fully protected D1 and D2 receptors from EEDQ-induced alkylation. Behavioral results showed that neither D1 nor D2 receptor stimulation was necessary for the induction of cocaine sensitization in preweanling rats. EEDQ disrupted the sensitization process, suggesting that another receptor type sensitive to EEDQ alkylation was necessary for the induction process. Expression of the sensitized response was prevented by an acute injection of a D1 receptor antagonist. The pattern of DA antagonist-induced effects described for preweanling rats is, with few exceptions, similar to what is observed when the same drugs are administered to adult rats. Thus, it appears that maturational changes in D1 and D2 receptor systems are not responsible for ontogenetic differences in the behavioral manifestation of cocaine sensitization. PMID:28284952

Importance of D1 and D2 receptor stimulation for the induction and expression of cocaine-induced behavioral sensitization in preweanling rats.

PubMed

McDougall, Sanders A; Rudberg, Krista N; Veliz, Ana; Dhargalkar, Janhavi M; Garcia, Aleesha S; Romero, Loveth C; Gonzalez, Ashley E; Mohd-Yusof, Alena; Crawford, Cynthia A

2017-05-30

The behavioral manifestations of psychostimulant-induced sensitization vary markedly between young and adult rats, suggesting that the neural mechanisms mediating this phenomenon differ across ontogeny. In this project we examined the importance of D1 and D2 receptors for the induction and expression of cocaine-induced behavioral sensitization during the preweanling period. In the behavioral experiments, rats were injected with reversible D1 and/or D2 antagonists (SCH23390 and/or raclopride) or an irreversible receptor antagonist (EEDQ) either before cocaine administration on the pretreatment day (induction) or before cocaine challenge on the test day (expression). In the EEDQ experiments, receptor specificity was assessed by using selective dopamine antagonists to protect D1 and/or D2 receptors from inactivation. Receptor binding assays showed that EEDQ caused substantial reductions in dorsal striatal D1 and D2 binding sites, while SCH23390 and raclopride fully protected D1 and D2 receptors from EEDQ-induced alkylation. Behavioral results showed that neither D1 nor D2 receptor stimulation was necessary for the induction of cocaine sensitization in preweanling rats. EEDQ disrupted the sensitization process, suggesting that another receptor type sensitive to EEDQ alkylation was necessary for the induction process. Expression of the sensitized response was prevented by an acute injection of a D1 receptor antagonist. The pattern of DA antagonist-induced effects described for preweanling rats is, with few exceptions, similar to what is observed when the same drugs are administered to adult rats. Thus, it appears that maturational changes in D1 and D2 receptor systems are not responsible for ontogenetic differences in the behavioral manifestation of cocaine sensitization. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and Validation of a Computational Model for Androgen Receptor Activity

PubMed Central

2016-01-01

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can more rapidly and inexpensively identify potential androgen-active chemicals. We integrated 11 HTS ToxCast/Tox21 in vitro assays into a computational network model to distinguish true AR pathway activity from technology-specific assay interference. The in vitro HTS assays probed perturbations of the AR pathway at multiple points (receptor binding, coregulator recruitment, gene transcription, and protein production) and multiple cell types. Confirmatory in vitro antagonist assay data and cytotoxicity information were used as additional flags for potential nonspecific activity. Validating such alternative testing strategies requires high-quality reference data. We compiled 158 putative androgen-active and -inactive chemicals from a combination of international test method validation efforts and semiautomated systematic literature reviews. Detailed in vitro assay information and results were compiled into a single database using a standardized ontology. Reference chemical concentrations that activated or inhibited AR pathway activity were identified to establish a range of potencies with reproducible reference chemical results. Comparison with existing Tier 1 AR binding data from the U.S. EPA Endocrine Disruptor Screening Program revealed that the model identified binders at relevant test concentrations (<100 μM) and was more sensitive to antagonist activity. The AR pathway model based on the ToxCast/Tox21 assays had balanced accuracies of 95.2% for agonist (n = 29) and 97.5% for antagonist (n = 28) reference chemicals. Out of 1855 chemicals screened in the AR pathway model, 220 chemicals demonstrated AR agonist or antagonist activity and an additional 174 chemicals were predicted to have potential weak AR pathway activity. PMID:27933809

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luthin, G.R.; Wolfe, B.B.

Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum bindingmore » values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.« less

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

Predicting bioactive conformations and binding modes of macrocycles

NASA Astrophysics Data System (ADS)

Anighoro, Andrew; de la Vega de León, Antonio; Bajorath, Jürgen

2016-10-01

Macrocyclic compounds experience increasing interest in drug discovery. It is often thought that these large and chemically complex molecules provide promising candidates to address difficult targets and interfere with protein-protein interactions. From a computational viewpoint, these molecules are difficult to treat. For example, flexible docking of macrocyclic compounds is hindered by the limited ability of current docking approaches to optimize conformations of extended ring systems for pose prediction. Herein, we report predictions of bioactive conformations of macrocycles using conformational search and binding modes using docking. Conformational ensembles generated using specialized search technique of about 70 % of the tested macrocycles contained accurate bioactive conformations. However, these conformations were difficult to identify on the basis of conformational energies. Moreover, docking calculations with limited ligand flexibility starting from individual low energy conformations rarely yielded highly accurate binding modes. In about 40 % of the test cases, binding modes were approximated with reasonable accuracy. However, when conformational ensembles were subjected to rigid body docking, an increase in meaningful binding mode predictions to more than 50 % of the test cases was observed. Electrostatic effects did not contribute to these predictions in a positive or negative manner. Rather, achieving shape complementarity at macrocycle-target interfaces was a decisive factor. In summary, a combined computational protocol using pre-computed conformational ensembles of macrocycles as a starting point for docking shows promise in modeling binding modes of macrocyclic compounds.

A calixpyrrole derivative acts as an antagonist to GPER, a G-protein coupled receptor: mechanisms and models

PubMed Central

Lappano, Rosamaria; Rosano, Camillo; Pisano, Assunta; Santolla, Maria Francesca; De Francesco, Ernestina Marianna; De Marco, Paola; Dolce, Vincenza; Ponassi, Marco; Felli, Lamberto; Cafeo, Grazia; Kohnke, Franz Heinrich; Abonante, Sergio; Maggiolini, Marcello

2015-01-01

ABSTRACT Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERβ. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells. PMID:26183213

An Overview of the Mechanism of Action of the Monoclonal Antibody Vedolizumab.

PubMed

Wyant, Tim; Fedyk, Eric; Abhyankar, Brihad

2016-12-01

Vedolizumab is a novel therapeutic monoclonal antibody recently approved for the treatment of moderately to severely active ulcerative colitis and Crohn's disease in adults who have failed at least one conventional therapy. An integrin antagonist, vedolizumab binds to the α 4 β 7 integrin which is expressed specifically by a subset of gastrointestinal-homing T lymphocytes. The binding of α 4 β 7 integrin to mucosal addressin cell adhesion molecule-1 expressed on the surface of mucosal endothelial cells is a crucial component of the gut-selective homing mechanism for lymphocytes.In contrast, other monoclonal antibodies approved for the treatment of inflammatory bowel diseases, such as tumour necrosis factor α antagonists and the integrin antagonist natalizumab, act systemically or on multiple targets to reduce inflammation.The unique gut selectivity of vedolizumab may contribute to the favourable benefit-risk profile observed in vedolizumab clinical trials. In this review, we summarise data from the preclinical development of vedolizumab and describe the current understanding of the mechanism of action as it relates to other biological therapies for inflammatory bowel disease. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast.

PubMed

Sridharan, Rajashri; Connelly, Sara M; Naider, Fred; Dumont, Mark E

2016-11-11

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Function-oriented development of CXCR4 antagonists as selective human immunodeficiency virus (HIV)-1 entry inhibitors.

PubMed

Wu, Chien-Huang; Wang, Chuan-Jen; Chang, Chun-Ping; Cheng, Yung-Chi; Song, Jen-Shin; Jan, Jiing-Jyh; Chou, Ming-Chen; Ke, Yi-Yu; Ma, Jing; Wong, Ying-Chieh; Hsieh, Tsung-Chih; Tien, Yun-Chen; Gullen, Elizabeth A; Lo, Chen-Fu; Cheng, Chia-Yi; Liu, Yu-Wei; Sadani, Amit A; Tsai, Chia-Hua; Hsieh, Hsin-Pang; Tsou, Lun K; Shia, Kak-Shan

2015-02-12

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy.

PubMed

Bot, Ilze; Ortiz Zacarías, Natalia V; de Witte, Wilhelmus E A; de Vries, Henk; van Santbrink, Peter J; van der Velden, Daniël; Kröner, Mara J; van der Berg, Dirk-Jan; Stamos, Dean; de Lange, Elizabeth C M; Kuiper, Johan; IJzerman, Adriaan P; Heitman, Laura H

2017-03-03

CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were performed to determine affinity and binding kinetics of 15a on murine CCR2. To assess the in vivo efficacy, western-type diet fed apoE -/- mice were treated daily with 15a or vehicle as control. Treatment with 15a reduced the amount of circulating CCR2 + monocytes and the size of the atherosclerotic plaques in both the carotid artery and the aortic root. We then showed that the long pharmacokinetic half-life of 15a combined with the high drug concentrations ensured prolonged CCR2 occupancy. These data render 15a a promising compound for drug development and confirms high receptor occupancy as a key parameter when targeting chemokine receptors.

Effects of Static and Dynamic Stretching on the Isokinetic Peak Torques and Electromyographic Activities of the Antagonist Muscles

PubMed Central

Serefoglu, Abdullah; Sekir, Ufuk; Gür, Hakan; Akova, Bedrettin

2017-01-01

The aim of this study was to investigate if static and dynamic stretching exercises of the knee muscles (quadriceps and hamstring muscles) have any effects on concentric and eccentric isokinetic peak torques and electromyographic amplitudes (EMG) of the antagonist muscles. Twenty healthy male athletes (age between 18-30 years) voluntarily participated in this study. All of the subjects visited the laboratory to complete the following intervention in a randomized order on 5 separate days; (a) non-stretching (control), (b) static stretching of the quadriceps muscles, (c) static stretching of the hamstring muscles, (d) dynamic stretching of the quadriceps muscles, and (e) dynamic stretching of the hamstring muscles. Static stretching exercises either for the quadriceps or the hamstring muscles were carried out at the standing and sitting positions. Subjects performed four successive repetitions of each stretching exercises for 30 seconds in both stretching positions. Similar to static stretching exercises two different stretching modes were designed for dynamic stretching exercises. Concentric and eccentric isokinetic peak torque for the non-stretched antagonist quadriceps or hamstring muscles at angular velocities of 60°/sec and 240°/sec and their concurrent electromyographic (EMG) activities were measured before and immediately after the intervention. Isokinetic peak torques of the non-stretched agonist hamstring and quadriceps muscles did not represent any significant (p > 0.05) differences following static and dynamic stretching of the antagonist quadriceps and hamstring muscles, respectively. Similarly, the EMG activities of the agonist muscles exhibited no significant alterations (p > 0.05) following both stretching exercises of the antagonist muscles. According to the results of the present study it is possible to state that antagonist stretching exercises either in the static or dynamic modes do not affect the isokinetic peak torques and the EMG activities of the non-stretched agonist quadriceps or hamstring muscles. Key points The effects of dynamic stretching of the antagonist muscles on strength performance are unknown. We showed that both static and dynamic stretching of the antagonist muscle does not influence strength and EMG activities in the agonist muscles. Further research should focus on the effects of antagonist stretching using other techniques like PNF or ballistic stretching and/or different volumes of stretching. PMID:28344445

Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

PubMed

Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

2015-11-01

In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cash, Jennifer N.; Rejon, Carlis A.; McPherron, Alexandra C.

2009-09-29

Myostatin is a member of the transforming growth factor-{beta} (TGF-{beta}) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-{beta} class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuousmore » electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.« less

Nucleic acids encoding human trithorax protein

DOEpatents

Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

2001-01-01

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Differential modulation of FXR activity by chlorophacinone and ivermectin analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, Chia-Wen

Chemicals that alter normal function of farnesoid X receptor (FXR) have been shown to affect the homeostasis of bile acids, glucose, and lipids. Several structural classes of environmental chemicals and drugs that modulated FXR transactivation were previously identified by quantitative high-throughput screening (qHTS) of the Tox21 10 K chemical collection. In the present study, we validated the FXR antagonist activity of selected structural classes, including avermectin anthelmintics, dihydropyridine calcium channel blockers, 1,3-indandione rodenticides, and pyrethroid pesticides, using in vitro assay and quantitative structural-activity relationship (QSAR) analysis approaches. (Z)-Guggulsterone, chlorophacinone, ivermectin, and their analogs were profiled for their ability to altermore » CDCA-mediated FXR binding using a panel of 154 coregulator motifs and to induce or inhibit transactivation and coactivator recruitment activities of constitutive androstane receptor (CAR), liver X receptor alpha (LXRα), or pregnane X receptor (PXR). Our results showed that chlorophacinone and ivermectin had distinct modes of action (MOA) in modulating FXR-coregulator interactions and compound selectivity against the four aforementioned functionally-relevant nuclear receptors. These findings collectively provide mechanistic insights regarding compound activities against FXR and possible explanations for in vivo toxicological observations of chlorophacinone, ivermectin, and their analogs. - Highlights: • A subset of Tox21 chemicals was investigated for FXR antagonism. • In vitro and computational approaches were used to evaluate FXR antagonists. • Chlorophacinone and ivermectin had distinct patterns in modulating FXR activity.« less

Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

PubMed

Istyastono, Enade P; Nijmeijer, Saskia; Lim, Herman D; van de Stolpe, Andrea; Roumen, Luc; Kooistra, Albert J; Vischer, Henry F; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

2011-12-08

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

Coarse-Grained MD Simulations and Protein-Protein Interactions: The Cohesin-Dockerin System.

PubMed

Hall, Benjamin A; Sansom, Mark S P

2009-09-08

Coarse-grained molecular dynamics (CG-MD) may be applied as part of a multiscale modeling approach to protein-protein interactions. The cohesin-dockerin interaction provides a valuable test system for evaluation of the use of CG-MD, as structural (X-ray) data indicate a dual binding mode for the cohesin-dockerin pair. CG-MD simulations (of 5 μs duration) of the association of cohesin and dockerin identify two distinct binding modes, which resemble those observed in X-ray structures. For each binding mode, ca. 80% of interfacial residues are predicted correctly. Furthermore, each of the binding modes identified by CG-MD is conformationally stable when converted to an atomistic model and used as the basis of a conventional atomistic MD simulation of duration 20 ns.

Confirmation of high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays.

PubMed

Mahapatra, Debabrata; Franzosa, Jill A; Roell, Kyle; Kuenemann, Melaine Agnes; Houck, Keith A; Reif, David M; Fourches, Denis; Kullman, Seth W

2018-06-11

High throughput screening (HTS) programs have demonstrated that the Vitamin D receptor (VDR) is activated and/or antagonized by a wide range of structurally diverse chemicals. In this study, we examined the Tox21 qHTS data set generated against VDR for reproducibility and concordance and elucidated functional insights into VDR-xenobiotic interactions. Twenty-one potential VDR agonists and 19 VDR antagonists were identified from a subset of >400 compounds with putative VDR activity and examined for VDR functionality utilizing select orthogonal assays. Transient transactivation assay (TT) using a human VDR plasmid and Cyp24 luciferase reporter construct revealed 20/21 active VDR agonists and 18/19 active VDR antagonists. Mammalian-2-hybrid assay (M2H) was then used to evaluate VDR interactions with co-activators and co-regulators. With the exception of a select few compounds, VDR agonists exhibited significant recruitment of co-regulators and co-activators whereas antagonists exhibited considerable attenuation of recruitment by VDR. A unique set of compounds exhibiting synergistic activity in antagonist mode and no activity in agonist mode was identified. Cheminformatics modeling of VDR-ligand interactions were conducted and revealed selective ligand VDR interaction. Overall, data emphasizes the molecular complexity of ligand-mediated interactions with VDR and suggest that VDR transactivation may be a target site of action for diverse xenobiotics.

Comparison of cannabinoid binding sites in guinea-pig forebrain and small intestine

PubMed Central

Ross, Ruth A; Brockie, Heather C; Fernando, Susanthi R; Saha, Bijali; Razdan, Raj K; Pertwee, Roger G

1998-01-01

We have investigated the nature of cannabinoid receptors in guinea-pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1-selective antagonist SR141716A, the CB2-selective antagonist SR144528, the novel cannabinoid receptor ligand, 6′-azidohex-2′-yne-Δ8-tetrahydrocannabinol (O-1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212-2, which shows marginal CB2 selectivity.[3H]-CP55940 (1 nM) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus-longitudinal muscle of guinea-pig small intestine (65.2%).Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg−1) than in intestinal membranes (2092 fmol mg−1). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nM respectively). Nor did the Ki values for its displacement by CP55940, WIN55212-2, O-1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nM respectively).The Bmax values of [3H]-CP55940 and [3H]-SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg−1) but were both greater than the Bmax of [3H]-WIN55212-2 (2032 fmol mg−1).O-1184 (10 or 100 nM) produced parallel dextral shifts in the log concentration-response curves of WIN55212-2 and CP55940 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation, its KD values being 0.20 nM (against WIN55212-2) and 0.89 nM (against CP55940).We conclude that cannabinoid binding sites in guinea-pig small intestine closely resemble CB1 binding sites of guinea-pig brain and that O-1184 behaves as a cannabinoid receptor antagonist in the guinea-pig myenteric plexus-longitudinal muscle preparation. PMID:9863666

Effect of beta-antagonists on isoprenaline-induced secretion of fluid, amylase and protein by the parotid gland of the red kangaroo, Macropus rufus.

PubMed

Beal, A M

2000-02-01

Selective and non-selective beta-adrenoceptor antagonists were used to block the increases in fluid, protein and amylase secretion caused by sympathomimetic stimulation of the parotid gland of red kangaroos during intracarotid infusion of isoprenaline. ICI118551 at antagonist/agonist ratios up to 300:1 caused increasing but incomplete blockade of fluid secretion, and protein/amylase release. Atenolol at antagonist/agonist ratios up to 300:1 was only marginally more potent than ICI118551 at blocking the fluid, protein and amylase responses. Propranolol at antagonist/agonist ratios of 30:1 was as effective at blocking fluid and protein secretion as the highest ratios of either atenolol or ICI118551. Simultaneous administration of atenolol (30:1) with ICI118551 (30:1) was not as potent as propranolol (30:1). Thus, the beta-adrenoceptor/s in the acini of the kangaroo parotid gland appear to have antagonist-binding affinities atypical of those found for eutherian tissues. The data are consistent with the gland possessing either a single anomalous beta-adrenoceptor or functional beta(2)-receptors in addition to the beta(1)-receptors which are characteristic of eutherian salivary glands.

Structure of the BTB Domain of Keap1 and Its Interaction with the Triterpenoid Antagonist CDDO

PubMed Central

Cleasby, Anne; Yon, Jeff; Day, Philip J.; Richardson, Caroline; Tickle, Ian J.; Williams, Pamela A.; Callahan, James F.; Carr, Robin; Concha, Nestor; Kerns, Jeffrey K.; Qi, Hongwei; Sweitzer, Thomas; Ward, Paris; Davies, Thomas G.

2014-01-01

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface. PMID:24896564

Antagonistic Actions of HLH/bHLH Proteins Are Involved in Grain Length and Weight in Rice

PubMed Central

Heang, Dany; Sassa, Hidenori

2012-01-01

Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1. PMID:22363621

AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barrett, R.J.; Appell, K.C.; Kilpatrick, B.F.

1991-01-01

In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) (3H)ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADPmore » (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) (3H)nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.« less

Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide.

PubMed Central

Okada, Miki; Takezawa, Daisuke; Tachibanaki, Shuji; Kawamura, Satoru; Tokumitsu, Hiroshi; Kobayashi, Ryoji

2003-01-01

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK ( Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 microM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 microM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K(ATP) channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists. PMID:12844348

CEP-26401 (irdabisant), a potent and selective histamine H₃ receptor antagonist/inverse agonist with cognition-enhancing and wake-promoting activities.

PubMed

Raddatz, Rita; Hudkins, Robert L; Mathiasen, Joanne R; Gruner, John A; Flood, Dorothy G; Aimone, Lisa D; Le, Siyuan; Schaffhauser, Hervé; Duzic, Emir; Gasior, Maciej; Bozyczko-Coyne, Donna; Marino, Michael J; Ator, Mark A; Bacon, Edward R; Mallamo, John P; Williams, Michael

2012-01-01

CEP-26401 [irdabisant; 6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one HCl] is a novel, potent histamine H₃ receptor (H₃R) antagonist/inverse agonist with drug-like properties. High affinity of CEP-26401 for H₃R was demonstrated in radioligand binding displacement assays in rat brain membranes (K(i) = 2.7 ± 0.3 nM) and recombinant rat and human H₃R-expressing systems (K(i) = 7.2 ± 0.4 and 2.0 ± 1.0 nM, respectively). CEP-26401 displayed potent antagonist and inverse agonist activities in [³⁵S]guanosine 5'-O-(γ-thio)triphosphate binding assays. After oral dosing of CEP-26401, occupancy of H₃R was estimated by the inhibition of ex vivo binding in rat cortical slices (OCC₅₀ = 0.1 ± 0.003 mg/kg), and antagonism of the H₃R agonist R-α-methylhistamine- induced drinking response in the rat dipsogenia model was demonstrated in a similar dose range (ED₅₀ = 0.06 mg/kg). CEP-26401 improved performance in the rat social recognition model of short-term memory at doses of 0.01 to 0.1 mg/kg p.o. and was wake-promoting at 3 to 30 mg/kg p.o. In DBA/2NCrl mice, CEP-26401 at 10 and 30 mg/kg i.p. increased prepulse inhibition (PPI), whereas the antipsychotic risperidone was effective at 0.3 and 1 mg/kg i.p. Coadministration of CEP-26401 and risperidone at subefficacious doses (3 and 0.1 mg/kg i.p., respectively) increased PPI. These results demonstrate potent behavioral effects of CEP-26401 in rodent models and suggest that this novel H₃R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. CEP-26401 may also have therapeutic utility in treating schizophrenia or as adjunctive therapy to approved antipsychotics.

Epigenetic Programming of Breast Cancer and Nutrition Prevention

DTIC Science & Technology

2011-05-01

is to test the role of xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin -like and...tumor promoter 2,3,7,8 tetrachlorobenzo-p- dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XRE...phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonizes at physiologically relevant doses (1  mol /L) the TCDD-induced

FMRFamide: low affinity inhibition of opioid binding to rabbit brain membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, X.Z.; Raffa, R.B.

1986-03-05

FMRFamide (Phe-Met-Arg-Phe-NH/sub 2/) was first isolated from the ganglia of molluscs by Price and Greenberg in 1977. The peptide was subsequently shown to have diverse actions on various types of molluscan and mammalian tissues. The presence of immunoreactive FMRFamide-like material (irFMRF) in multiple areas of rat brain, spinal cord, and gastrointestinal tract suggests that irFMRF may have a physiological role in mammals. Tang, Yang and Costa recently demonstrated that FMRFamide attenuates morphine antinociception in rats and postulated, based on this and several other lines of evidence, that irFMRF might be an endogenous opioid antagonist. In the present study, they testedmore » the ability of FMRFamide to inhibit the binding of opioid receptor ligands to rabbit membrane preparations. FMRFamide inhibited the specific binding of both /sup 3/(H)-dihydromorphine and /sup 3/(H)-ethylketocyclazocine (IC/sub 50/ = 14 ..mu..M and 320 ..mu..M, respectively) in a dose-related manner, suggesting that FMRFamide may affect binding to at least two types of opioid receptors (mu and kappa). These data are consistent with the concept that irFMRF might act as an endogenous opioid antagonist. However, the low affinity of FMRFamide leaves open the possibility of another mechanism of opioid antagonism, such as neuromodulation.« less

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

Druggability of methyl-lysine binding sites

NASA Astrophysics Data System (ADS)

Santiago, C.; Nguyen, K.; Schapira, M.

2011-12-01

Structural modules that specifically recognize—or read—methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions.

Depolarizing Effects of Daikenchuto on Interstitial Cells of Cajal from Mouse Small Intestine

PubMed Central

Kim, Hyungwoo; Kim, Hyun Jung; Yang, Dongki; Jung, Myeong Ho; Kim, Byung Joo

2017-01-01

Background: Daikenchuto (DKT; TJ-100, TU-100), a traditional herbal medicineis used in modern medicine to treat gastrointestinal (GI) functional disorders. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the GI tract and play important roles in the regulation of GI motility. Objective: The objective of this study was to investigate the effects of DKT on the pacemaker potentials (PPs) of cultured ICCs from murine small intestine. Materials and Methods: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record ICC PPs (current clamp mode). All experiments were performed at 30-32°C. Results: In current-clamp modeDKT depolarized and concentration-dependently decreased the amplitudes of PPs. Y25130 (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT4 receptor antagonist) did. Methoctramine (a muscarinic M2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-diphenylacetoxy-N-methylpiperidine methiodide (a muscarinic M3 receptor antagonist) facilitated blockade of DKT-induced PP depolarization. Pretreatment with an external Ca2+-free solution or thapsigargin abolished PPsand under these conditions, DKT did not induce PP depolarization. Furthermore Ginseng radix and Zingiberis rhizomes depolarized PPs, whereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Conclusion: These results suggest that DKT depolarizes ICC PPs in an internal or external Ca2+-dependent manner by stimulating 5-HT4 and M3 receptors. Furthermore, the authors suspect that the component in DKT largely responsible for depolarization is probably also a component of Ginseng radix and Zingiberis rhizomes. SUMMARY Daikenchuto (DKT) depolarized and concentration-dependently decreased the amplitudes of pacemaker potentials (PPs)Y25130 (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT4 receptor antagonist) didMethoctramine (a muscarinic M2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-DAMP (a muscarinic M3 receptor antagonist) facilitated blockade of DKT-induced PP depolarizationGinseng radix and Zingiberis rhizomes depolarized PPswhereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Abbreviation used: DKT: Daikenchuto, GI: Gastrointestinal, ICCs: Interstitial cells of Cajal, PPs: Pacemaker Potentials. PMID:28216898

Depolarizing Effects of Daikenchuto on Interstitial Cells of Cajal from Mouse Small Intestine.

PubMed

Kim, Hyungwoo; Kim, Hyun Jung; Yang, Dongki; Jung, Myeong Ho; Kim, Byung Joo

2017-01-01

Daikenchuto (DKT; TJ-100, TU-100), a traditional herbal medicineis used in modern medicine to treat gastrointestinal (GI) functional disorders. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the GI tract and play important roles in the regulation of GI motility. The objective of this study was to investigate the effects of DKT on the pacemaker potentials (PPs) of cultured ICCs from murine small intestine. Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record ICC PPs (current clamp mode). All experiments were performed at 30-32°C. In current-clamp modeDKT depolarized and concentration-dependently decreased the amplitudes of PPs. Y25130 (a 5-HT 3 receptor antagonist) or SB269970 (a 5-HT 7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT 4 receptor antagonist) did. Methoctramine (a muscarinic M 2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-diphenylacetoxy-N-methylpiperidine methiodide (a muscarinic M 3 receptor antagonist) facilitated blockade of DKT-induced PP depolarization. Pretreatment with an external Ca 2+ -free solution or thapsigargin abolished PPsand under these conditions, DKT did not induce PP depolarization. Furthermore Ginseng radix and Zingiberis rhizomes depolarized PPs, whereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. These results suggest that DKT depolarizes ICC PPs in an internal or external Ca 2+ -dependent manner by stimulating 5-HT 4 and M 3 receptors. Furthermore, the authors suspect that the component in DKT largely responsible for depolarization is probably also a component of Ginseng radix and Zingiberis rhizomes. Daikenchuto (DKT) depolarized and concentration-dependently decreased the amplitudes of pacemaker potentials (PPs)Y25130 (a 5-HT 3 receptor antagonist) or SB269970 (a 5-HT 7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT 4 receptor antagonist) didMethoctramine (a muscarinic M 2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-DAMP (a muscarinic M 3 receptor antagonist) facilitated blockade of DKT-induced PP depolarizationGinseng radix and Zingiberis rhizomes depolarized PPswhereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Abbreviation used: DKT: Daikenchuto, GI: Gastrointestinal, ICCs: Interstitial cells of Cajal, PPs: Pacemaker Potentials.

Positron emission tomographic evaluation of the putative dopamine-D3 receptor ligand, [11C]RGH-1756 in the monkey brain.

PubMed

Sóvágó, Judit; Farde, Lars; Halldin, Christer; Langer, Oliver; Laszlovszky, István; Kiss, Béla; Gulyás, Balázs

2004-10-01

The dopamine-D3 receptor is of special interest due to its postulated role in the pathophysiology and treatment of schizophrenia and Parkinson's Disease. Increasing evidences support the assumption that the D3 receptors are occupied to a high degree by dopamine at physiological conditions. Research on the functional role of the D3 receptors in brain has however been hampered by the lack of D3 selective ligands. In the present Positron Emission Tomography (PET) study the binding of the novel, putative dopamine-D3 receptor ligand, [11C]RGH-1756 was characterized in the cynomolgus monkey brain. [11C]RGH-1756 was rather homogenously distributed in brain and the regional binding potential (BP) values ranged between 0.17 and 0.48. Pretreatment with unlabelled RGH-1756 decreased radioligand binding to the level of the cerebellum in most brain areas. The regional BP values were lower after intravenous injection of a higher mass of RGH-1756, indicating saturable binding of [11C]RGH-1756. The D2/D3 antagonist raclopride partly inhibited the binding of [11C]RGH-1756 in several brain areas, including the striatum, mesencephalon and neocortex, whereas the 5HT(1A) antagonist WAY-100635 had no evident effect on [11C]RGH-1756 binding. Despite the promising binding characteristics of RGH-1756 in vitro the present PET-study indicates that [11C]RGH-1756 provides a low signal for specific binding to the D3 receptor in vivo. One explanation is that the favorable binding characteristics of RGH-1756 in vitro are not manifested in vivo. Alternatively, the results may support the hypothesis that the dopamine-D3 receptors are indeed occupied to a high extent by dopamine in vivo and thus not available for radioligand binding.

Interaction of a radiolabeled agonist with cardiac muscarinic cholinergic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harden, T.K.; Meeker, R.B.; Martin, M.W.

The interaction of a radiolabeled muscarinic cholinergic receptor agonist, (methyl-/sup 3/H)oxotremorine acetate ((/sup 3/H)OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of (/sup 3/H)OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of (/sup 3/H)OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist (/sup 3/H)quinuclidinyl benzilate.more » Both muscarinic receptor agonists and antagonists inhibited the binding of (/sup 3/H)OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of (/sup 3/H)OXO. This effect was on the maximum binding (Bmax) of (/sup 3/H)OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The (/sup 3/H)OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with (/sup 3/H)OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, (/sup 3/H)OXO-labeled complex resulted in dissociation of (/sup 3/H)OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited (/sup 3/H)OXO binding by 85% with no change in the Kd of (/sup 3/H)OXO, and with no effect on (/sup 3/H)quinuclidinyl benzilate binding.« less

Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses

PubMed Central

Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D

1999-01-01

The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents. PMID:10204994

How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

NASA Astrophysics Data System (ADS)

Dudev, Todor; Grauffel, Cédric; Lim, Carmay

2017-02-01

Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

Synthesis and characterization of potent and selective mu-opioid receptor antagonists, [Dmt(1), D-2-Nal(4)]endomorphin-1 (Antanal-1) and [Dmt(1), D-2-Nal(4)]endomorphin-2 (Antanal-2).

PubMed

Fichna, Jakub; do-Rego, Jean-Claude; Chung, Nga N; Lemieux, Carole; Schiller, Peter W; Poels, Jeroen; Broeck, Jozef Vanden; Costentin, Jean; Janecka, Anna

2007-02-08

To synthesize potent antagonists of the mu-opioid receptor, we prepared a series of endomorphin-1 and endomorphin-2 analogues with 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal) in position 4. Some of these analogues displayed weak antagonist properties. We tried to strengthen these properties by introducing the structurally modified tyrosine residue 2,6-dimethyltyrosine (Dmt) in place of Tyr1. Among the synthesized compounds, [Dmt1, d-2-Nal4]endomorphin-1, designated antanal-1, and [Dmt1, d-2-Nal4]endomorphin-2, designated antanal-2, turned out to be highly potent and selective mu-opioid receptor antagonists, as judged on the basis of two functional assays, the receptor binding assay and the hot plate test of analgesia. Interestingly, another analogue of this series, [Dmt1, d-1-Nal4]endomorphin-1, turned out to be a moderately potent mixed mu-agonist/delta-antagonist.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovtun, Oleg; Ross, Emily J.; Tomlinson, Ian D.

Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). Our anticipation is that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

In Vitro Binding of [³H]PSB-0413 to P2Y₁₂ Receptors.

PubMed

Dupuis, Arnaud; Heim, Véronique; Ohlmann, Philippe; Gachet, Christian

2015-12-08

The P2Y₁₂/ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y₁₂ receptor defect and for evaluating P2Y₁₂ receptor agonists and antagonists. In the absence of suitable anti-P2Y₁₂ antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assessing P2Y₁₂ receptors with [³H]PSB-0413, a selective antagonist for this site. The saturation and competition assays described herein make possible the determination of P2Y₁₂ receptor density on cells, as well as the potencies and affinities of test agents at this site. Copyright © 2015 John Wiley & Sons, Inc.

A radiogallium-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging.

PubMed

Sano, Kohei; Masuda, Ryo; Hisada, Hayato; Oishi, Shinya; Shimokawa, Kenta; Ono, Masahiro; Fujii, Nobutaka; Saji, Hideo; Mukai, Takahiro

2014-03-01

We have developed a novel radiogallium (Ga)-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging. A CXCR4 imaging probe with two CXCR4 antagonists (Ac-TZ14011) on Ga-DOTA core, Ga-DOTA-TZ2, was synthesized, and the affinity and binding to CXCR4 was evaluated in CXCR4 expressing cells in vitro. The affinity of Ga-DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga-DOTA-TZ1. (67)Ga-DOTA-TZ2 showed the significantly higher accumulation in CXCR4-expressing tumor cells compared with (67)Ga-DOTA-TZ1, suggesting the bivalent effect enhances its binding to CXCR4. The incorporation of two CXCR4 antagonists to Ga-DOTA could be effective in detecting CXCR4-expressing tumors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Guanidine-acylguanidine bioisosteric approach in the design of radioligands: synthesis of a tritium-labeled N(G)-propionylargininamide ([3H]-UR-MK114) as a highly potent and selective neuropeptide Y Y1 receptor antagonist.

PubMed

Keller, Max; Pop, Nathalie; Hutzler, Christoph; Beck-Sickinger, Annette G; Bernhardt, Günther; Buschauer, Armin

2008-12-25

Synthesis and characterization of (R)-N(alpha)-(2,2-diphenylacetyl)-N-(4-hydroxybenzyl)-N(omega)-([2,3-(3)H]-propanoyl)argininamide ([(3)H]-UR-MK114), an easily accessible tritium-labeled NPY Y(1) receptor (Y(1)R) antagonist (K(B): 0.8 nM, calcium assay, HEL cells) derived from the (R)-argininamide BIBP 3226, is reported. The radioligand binds with high affinity (K(D), saturation: 1.2 nM, kinetic experiments: 1.1 nM, SK-N-MC cells) and selectivity for Y(1)R over Y(2), Y(4), and Y(5) receptors. The title compound is a useful pharmacological tool for the determination of Y(1)R ligand affinities, quantification of Y(1)R binding sites, and autoradiography.

Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.

1991-03-11

The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{supmore » 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.« less

Novel 18F-Labeled κ-Opioid Receptor Antagonist as PET Radiotracer: Synthesis and In Vivo Evaluation of 18F-LY2459989 in Nonhuman Primates.

PubMed

Li, Songye; Cai, Zhengxin; Zheng, Ming-Qiang; Holden, Daniel; Naganawa, Mika; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Kapinos, Michael; Lara-Jaime, Teresa; Navarro, Antonio; Huang, Yiyun

2018-01-01

The κ-opioid receptor (KOR) has been implicated in depression, addictions, and other central nervous system disorders and, thus, is an important target for drug development. We previously developed several 11 C-labeled PET radiotracers for KOR imaging in humans. Here we report the synthesis and evaluation of 18 F-LY2459989 as the first 18 F-labeled KOR antagonist radiotracer in nonhuman primates and its comparison with 11 C-LY2459989. Methods: The novel radioligand 18 F-LY2459989 was synthesized by 18 F displacement of a nitro group or an iodonium ylide. PET scans in rhesus monkeys were obtained on a small-animal scanner to assess the pharmacokinetic and in vivo binding properties of the ligand. Metabolite-corrected arterial activity curves were measured and used as input functions in the analysis of brain time-activity curves and the calculation of binding parameters. Results: With the iodonium ylide precursor, 18 F-LY2459989 was prepared at high radiochemical yield (36% ± 7% [mean ± SD]), radiochemical purity (>99%), and mean molar activity (1,175 GBq/μmol; n = 6). In monkeys, 18 F-LY2459989 was metabolized at a moderate rate, with a parent fraction of approximately 35% at 30 min after injection. Fast and reversible kinetics were observed, with a regional peak uptake time of less than 20 min. Pretreatment with the selective KOR antagonist LY2456302 (0.1 mg/kg) decreased the activity level in regions with high levels of binding to that in the cerebellum, thus demonstrating the binding specificity and selectivity of 18 F-LY2459989 in vivo. Regional time-activity curves were well fitted by the multilinear analysis 1 kinetic model to derive reliable estimates of regional distribution volumes. With the cerebellum as the reference region, regional binding potentials were calculated and ranked as follows: cingulate cortex > insula > caudate/putamen > frontal cortex > temporal cortex > thalamus, consistent with the reported KOR distribution in the monkey brain. Conclusion: The evaluation of 18 F-LY2459989 in nonhuman primates demonstrated many attractive imaging properties: fast tissue kinetics, specific and selective binding to the KOR, and high specific binding signals. A side-by-side comparison of 18 F-LY2459989 and 11 C-LY2459989 indicated similar kinetic and binding profiles for the 2 radiotracers. Taken together, the results indicated that 18 F-LY2459989 appears to be an excellent PET radiotracer for the imaging and quantification of the KOR in vivo. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

PubMed Central

Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

2012-01-01

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism

PubMed Central

Hughes, Travis S.; Chalmers, Michael J.; Novick, Scott; Kuruvilla, Dana S.; Chang, Mi Ra; Kamenecka, Theodore M.; Rance, Mark; Johnson, Bruce A.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2011-01-01

SUMMARY Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution NMR studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators. PMID:22244763

Effects of Static and Dynamic Stretching on the Isokinetic Peak Torques and Electromyographic Activities of the Antagonist Muscles.

PubMed

Serefoglu, Abdullah; Sekir, Ufuk; Gür, Hakan; Akova, Bedrettin

2017-03-01

The aim of this study was to investigate if static and dynamic stretching exercises of the knee muscles (quadriceps and hamstring muscles) have any effects on concentric and eccentric isokinetic peak torques and electromyographic amplitudes (EMG) of the antagonist muscles. Twenty healthy male athletes (age between 18-30 years) voluntarily participated in this study. All of the subjects visited the laboratory to complete the following intervention in a randomized order on 5 separate days; (a) non-stretching (control), (b) static stretching of the quadriceps muscles, (c) static stretching of the hamstring muscles, (d) dynamic stretching of the quadriceps muscles, and (e) dynamic stretching of the hamstring muscles. Static stretching exercises either for the quadriceps or the hamstring muscles were carried out at the standing and sitting positions. Subjects performed four successive repetitions of each stretching exercises for 30 seconds in both stretching positions. Similar to static stretching exercises two different stretching modes were designed for dynamic stretching exercises. Concentric and eccentric isokinetic peak torque for the non-stretched antagonist quadriceps or hamstring muscles at angular velocities of 60°/sec and 240°/sec and their concurrent electromyographic (EMG) activities were measured before and immediately after the intervention. Isokinetic peak torques of the non-stretched agonist hamstring and quadriceps muscles did not represent any significant (p > 0.05) differences following static and dynamic stretching of the antagonist quadriceps and hamstring muscles, respectively. Similarly, the EMG activities of the agonist muscles exhibited no significant alterations (p > 0.05) following both stretching exercises of the antagonist muscles. According to the results of the present study it is possible to state that antagonist stretching exercises either in the static or dynamic modes do not affect the isokinetic peak torques and the EMG activities of the non-stretched agonist quadriceps or hamstring muscles.

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12.

PubMed

Smith, Lorna J; Gunsteren, Wilfred F Van; Allison, Jane R

2013-01-01

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein-ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein-ligand atom-atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force-field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen-bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein-ligand interactions in complexes where NMR data provide insufficient restraints. Copyright © 2012 The Protein Society.

A selective estrogen receptor modulator for the treatment of hot flushes.

PubMed

Wallace, Owen B; Lauwers, Kenneth S; Dodge, Jeffrey A; May, Scott A; Calvin, Joel R; Hinklin, Ronald; Bryant, Henry U; Shetler, Pamela K; Adrian, Mary D; Geiser, Andrew G; Sato, Masahiko; Burris, Thomas P

2006-02-09

A selective estrogen receptor modulator (SERM) for the potential treatment of hot flushes is described. (R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol, LSN2120310, potently binds ERalpha and ERbeta and is an antagonist in MCF-7 breast adenocarcinoma and Ishikawa uterine cancer cell lines. The compound is a potent estrogen antagonist in the rat uterus. In ovariectomized rats, the compound lowers cholesterol, maintains bone mineral density, and is efficacious in a morphine dependent rat model of hot flush efficacy.

(−) Arctigenin and (+) Pinoresinol Are Antagonists of the Human Thyroid Hormone Receptor β

PubMed Central

2015-01-01

Lignans are important biologically active dietary polyphenolic compounds. Consumption of foods that are rich in lignans is associated with positive health effects. Using modeling tools to probe the ligand-binding pockets of molecular receptors, we found that lignans have high docking affinity for the human thyroid hormone receptor β. Follow-up experimental results show that lignans (−) arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor β. The modeled complexes show key plausible interactions between the two ligands and important amino acid residues of the receptor. PMID:25383984

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens.

PubMed

Cordero-Bueso, Gustavo; Mangieri, Nicola; Maghradze, David; Foschino, Roberto; Valdetara, Federica; Cantoral, Jesús M; Vigentini, Ileana

2017-01-01

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius , and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species ( Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae , and Pichia kluyveri ) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action.

Wild Grape-Associated Yeasts as Promising Biocontrol Agents against Vitis vinifera Fungal Pathogens

PubMed Central

Cordero-Bueso, Gustavo; Mangieri, Nicola; Maghradze, David; Foschino, Roberto; Valdetara, Federica; Cantoral, Jesús M.; Vigentini, Ileana

2017-01-01

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action. PMID:29163377

An alternate binding site for PPARγ ligands

PubMed Central

Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2014-01-01

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

A Unique Human Chorionic Gonadotropin Antagonist Suppresses Ovarian Hyperstimulation Syndrome in Rats

PubMed Central

Vardhana, Pratibhasri A.; Julius, Martin A.; Pollak, Susan V.; Lustbader, Evan G.; Trousdale, Rhonda K.; Lustbader, Joyce W.

2009-01-01

Ovarian hyperstimulation syndrome (OHSS) is a complication of in vitro fertilization associated with physiological changes after hCG administration to induce final oocyte maturation. It presents as widespread increases in vascular permeability and, in rare cases, results in cycle cancellation, multi-organ dysfunction, and pregnancy termination. These physiological changes are due primarily to activation of the vascular endothelial growth factor (VEGF) system in response to exogenous human chorionic gonadotropin (hCG). An hCG antagonist (hCG-Ant) could attenuate these effects by competitively binding to the LH/CG receptor, thereby blocking LH activity in vivo. We expressed a form of hCG that lacks three of its four N-linked glycosylation sites and tested its efficacy as an antagonist. The hCG-Ant binds the LH receptor with an affinity similar to native hCG and inhibits cAMP response in vitro. In a rat model for ovarian stimulation, hCG-Ant dramatically reduces ovulation and steroid hormone production. In a well-established rat OHSS model, vascular permeability and vascular endothelial growth factor (VEGF) expression are dramatically reduced after hCG-Ant treatment. Finally, hCG-Ant does not appear to alter blastocyst development when given after hCG in mice. These studies demonstrate that removing specific glycosylation sites on native hCG can produce an hCG-Ant that is capable of binding without activating the LH receptor and blocking the actions of hCG. Thus hCG-Ant will be investigated as a potential therapy for OHSS. PMID:19443574

In vitro pharmacological characterization of CJ-042794, a novel, potent, and selective prostaglandin EP(4) receptor antagonist.

PubMed

Murase, Akio; Taniguchi, Yasuhito; Tonai-Kachi, Hiroko; Nakao, Kazunari; Takada, Junji

2008-01-16

Activation of the prostaglandin E(2) (PGE(2)) EP(4) receptor, a G-protein-coupled receptor (GPCR), results in increases in intracellular cyclic AMP (cAMP) levels via stimulation of adenylate cyclase. Here we describe the in vitro pharmacological characterization of a novel EP(4) receptor antagonist, CJ-042794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl}carbonyl)amino]ethyl}benzoic acid). CJ-042794 inhibited [(3)H]-PGE(2) binding to the human EP(4) receptor with a mean pK(i) of 8.5, a binding affinity that was at least 200-fold more selective for the human EP(4) receptor than other human EP receptor subtypes (EP(1), EP(2), and EP(3)). CJ-042794 did not exhibit any remarkable binding to 65 additional proteins, including GPCRs, enzymes, and ion channels, suggesting that CJ-042794 is highly selective for the EP(4) receptor. CJ-042794 competitively inhibited PGE(2)-evoked elevations of intracellular cAMP levels in HEK293 cells overexpressing human EP(4) receptor with a mean pA(2) value of 8.6. PGE(2) inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha) in human whole blood (HWB); CJ-042794 reversed the inhibitory effects of PGE(2) on LPS-induced TNFalpha production in a concentration-dependent manner. These results suggest that CJ-042794, a novel, potent, and selective EP(4) receptor antagonist, has excellent pharmacological properties that make it a useful tool for exploring the physiological role of EP(4) receptors.

Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer

PubMed Central

Singhal, Hari; Greene, Marianne E.; Tarulli, Gerard; Zarnke, Allison L.; Bourgo, Ryan J.; Laine, Muriel; Chang, Ya-Fang; Ma, Shihong; Dembo, Anna G.; Raj, Ganesh V.; Hickey, Theresa E.; Tilley, Wayne D.; Greene, Geoffrey L.

2016-01-01

The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER+ (estrogen receptor–positive)/PR+ human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak phenotypic agonist of estrogen action. However, in the presence of both hormones, progestin behaves as a phenotypic estrogen antagonist. PR remodels nucleosomes to noncompetitively redirect ER genomic binding to distal enhancers enriched for BRCA1 binding motifs and sites that link PR and ER/PR complexes. When both hormones are present, progestin modulates estrogen action, such that responsive transcriptomes, cellular processes, and ER/PR recruitment to genomic sites correlate with those observed with PR alone, but not ER alone. Despite this overall correlation, the transcriptome patterns modulated by dual treatment are sufficiently different from individual treatments, such that antagonism of oncogenic processes is both predicted and observed. Combination therapies using the selective PR modulator/antagonist (SPRM) CDB4124 in combination with tamoxifen elicited 70% cytotoxic tumor regression of T47D tumor xenografts, whereas individual therapies inhibited tumor growth without net regression. Our findings demonstrate that PR redirects ER chromatin binding to antagonize estrogen signaling and that SPRMs can potentiate responses to antiestrogens, suggesting that cotargeting of ER and PR in ER+/PR+ breast cancers should be explored. PMID:27386569

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

PubMed

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

PubMed

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Understanding bimolecular machines: Theoretical and experimental approaches

NASA Astrophysics Data System (ADS)

Goler, Adam Scott

This dissertation concerns the study of two classes of molecular machines from a physical perspective: enzymes and membrane proteins. Though the functions of these classes of proteins are different, they each represent important test-beds from which new understanding can be developed by the application of different techniques. HIV1 Reverse Transcriptase is an enzyme that performs multiple functions, including reverse transcription of RNA into an RNA/DNA duplex, RNA degradation by the RNaseH domain, and synthesis of dsDNA. These functions allow for the incorporation of the retroviral genes into the host genome. Its catalytic cycle requires repeated large-scale conformational changes fundamental to its mechanism. Motivated by experimental work, these motions were studied theoretically by the application of normal mode analysis. It was observed that the lowest order modes correlate with largest amplitude (low-frequency) motion, which are most likely to be catalytically relevant. Comparisons between normal modes obtained via an elastic network model to those calculated from the essential dynamics of a series of all-atom molecular dynamics simulations show the self-consistency between these calculations. That similar conformational motions are seen between independent theoretical methods reinforces the importance of large-scale subdomain motion for the biochemical action of DNA polymerases in general. Moreover, it was observed that the major subunits of HIV1 Reverse Transcriptase interact quasi-harmonically. The 5HT3A Serotonin receptor and P2X1 receptor, by contrast, are trans-membrane proteins that function as ligand gated ion channels. Such proteins feature a central pore, which allows for the transit of ions necessary for cellular function across a membrane. The pore is opened by the ligation of binding sites on the extracellular portion of different protein subunits. In an attempt to resolve the individual subunits of these membrane proteins beyond the diffraction limit, a super-localization microscope capable of reconstructing super-resolution images was constructed. This novel setup allows for the study of discrete state kinetic mechanisms with spatial resolution good enough to distinguish individual binding sites of these membrane proteins. Further use of this technique may allow for the study of allostery and subunit specific stoichiometry in the presence of agonist or antagonist ligands relevant to pharmacology.

Structural insight into the antagonistic action of diarylheptanoid on human estrogen receptor alpha.

PubMed

Geetha Rani, Yuvaraj; Lakshmi, Baddireddi Subhadra

2018-03-30

Estrogen receptor α (ER α) is an important therapeutic target in the regulation of ligand dependent signaling in breast cancer. The current study investigates the anti-estrogenic potential of the Diarylheptanoid, 5-hydroxy-7-(4-hydroxy-3 methoxyphenyl)-1-phenyl-3-heptanone (DAH) in silico. Rigid Docking analysis of DAH at the ligand binding domain (LBD) of ER α showed hydrogen bond interactions with Arg394 and Glu353 at the active site, similar to the positive controls 4-Hydroxy Tamoxifen (4-OHT) and Fulvestrant (FUL). The protein and the protein-DAH complexes were further analyzed using molecular dynamics simulations for a time scale of 50 ns using GROMACS. Root mean square fluctuation (RMSF) analysis showed large fluctuations at the N-terminal region of Helices (H) 3, 9 and at the C-terminal region of H11, which could be involved in the antagonistic conformational change. Interestingly, H12 appeared to move away from the ligand binding pocket and occupy the co-activator binding groove at the LBD of ER α. Secondary structure analysis of the protein upon binding of DAH and CUR showed structural change from α-helix to Turn conformation at H4. We hypothesize that this structural change at H4, similar to the positive control, could hinder the activity of AF-2 by blocking the binding of co-activator. These conformational changes in ER α indicate an anti-estrogenic and therapeutic potential of the DAH.

Blonanserin extensively occupies rat dopamine D3 receptors at antipsychotic dose range.

PubMed

Baba, Satoko; Enomoto, Takeshi; Horisawa, Tomoko; Hashimoto, Takashi; Ono, Michiko

2015-03-01

Antagonism of the dopamine D3 receptor has been hypothesized to be beneficial for schizophrenia cognitive deficits, negative symptoms and extrapyramidal symptoms. However, recent animal and human studies have shown that most antipsychotics do not occupy D3 receptors in vivo, despite their considerable binding affinity for this receptor in vitro. In the present study, we investigated the D3 receptor binding of blonanserin, a dopamine D2/D3 and serotonin 5-HT2A receptors antagonist, in vitro and in vivo. Blonanserin showed the most potent binding affinity for human D3 receptors among the tested atypical antipsychotics (risperidone, olanzapine and aripiprazole). Our GTPγS-binding assay demonstrated that blonanserin acts as a potent full antagonist for human D3 receptors. All test-drugs exhibited antipsychotic-like efficacy in methamphetamine-induced hyperactivity in rats. Treatment with blonanserin at its effective dose blocked the binding of [(3)H]-(+)-PHNO, a D2/D3 receptor radiotracer, both in the D2 receptor-rich region (striatum) and the D3 receptor-rich region (cerebellum lobes 9 and 10). On the other hand, the occupancies of other test-drugs for D3 receptors were relatively low. In conclusion, we have shown that blonanserin, but not other tested antipsychotics, extensively occupies D3 receptors in vivo in rats. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

IL-27 Regulates IL-18 binding protein in skin resident cells.

PubMed

Wittmann, Miriam; Doble, Rosella; Bachmann, Malte; Pfeilschifter, Josef; Werfel, Thomas; Mühl, Heiko

2012-01-01

IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48-72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts.

Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors.

PubMed

Molgó, Jordi; Marchot, Pascale; Aráoz, Rómulo; Benoit, Evelyne; Iorga, Bogdan I; Zakarian, Armen; Taylor, Palmer; Bourne, Yves; Servent, Denis

2017-08-01

We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

PubMed

Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

2015-10-01

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

Odorant Receptor Modulation: Ternary Paradigm for Mode of Action of Insect Repellents

DTIC Science & Technology

2012-01-01

Introduction Perception of chemicals in the environment by insects begins when compounds activate ionotropic receptors , gustatory recep- tors and odorant...Author’s personal copy Odorant receptor modulation: Ternary paradigm for mode of action of insect repellents Jonathan D. Bohbot, Joseph C. Dickens...Received in revised form 12 December 2011 Accepted 9 January 2012 Keywords: Odorant receptor Insect repellent Insurmountable antagonist Aedes aegypti

Structural basis for binding of fluorinated glucose and galactose to Trametes multicolor pyranose 2-oxidase variants with improved galactose conversion.

PubMed

Tan, Tien Chye; Spadiut, Oliver; Gandini, Rosaria; Haltrich, Dietmar; Divne, Christina

2014-01-01

Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the α-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the β-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the β-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose α-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.

Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

PubMed Central

Gandini, Rosaria; Haltrich, Dietmar; Divne, Christina

2014-01-01

Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the α-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the β-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the β-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose α-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts. PMID:24466218

In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor.

PubMed

Chinta, Gopichand; Ramya Chandar Charles, Mariasoosai; Klopčič, Ivana; Sollner Dolenc, Marija; Periyasamy, Latha; Selvaraj Coumar, Mohane

2015-07-01

Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future. Georg Thieme Verlag KG Stuttgart · New York.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

PubMed Central

Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

2003-01-01

Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539

Quantum transport in alkane molecular wires: Effects of binding modes and anchoring groups

NASA Astrophysics Data System (ADS)

Sheng, W.; Li, Z. Y.; Ning, Z. Y.; Zhang, Z. H.; Yang, Z. Q.; Guo, H.

2009-12-01

Effects of binding modes and anchoring groups on nonequilibrium electronic transport properties of alkane molecular wires are investigated from atomic first-principles based on density functional theory and nonequilibrium Green's function formalism. Four typical binding modes, top, bridge, hcp-hollow, and fcc-hollow, are considered at one of the two contacts. For wires with three different anchoring groups, dithiol, diamine, or dicarboxylic acid, the low bias conductances resulting from the four binding modes are all found to have either a high or a low value, well consistent with recent experimental observations. The trend can be rationalized by the behavior of electrode-induced gap states at small bias. When bias increases to higher values, states from the anchoring groups enter into the bias window and contribute significantly to the tunneling process so that transport properties become more complicated for the four binding modes. Other low bias behaviors including the values of the inverse length scale for tunneling characteristic, contact resistance, and the ratios of the high/low conductance values are also calculated and compared to experimental results. The conducting capabilities of the three anchoring groups are found to decrease from dithiol, diamine to dicarboxylic-acid, largely owing to a decrease in binding strength to the electrodes. Our results give a clear microscopic picture to the transport physics and provide reasonable qualitative explanations for the corresponding experimental data.

Miraxanthin-V, Liriodenin and Chitranone are Hepcidin Antagonist In silico for Iron Deficiency Anemia

NASA Astrophysics Data System (ADS)

Yotriana, S.; Suselo, YH; Muthmainah; Indarto, D.

2018-03-01

Anemia is one of the greatest nutrition problem in the world that is commonly found in children, pregnant women and reproductive women. This disorder is predominantly caused by iron deficiency. Hepcidin, a hepatic hormone, regulates iron metabolism and high serum levels of this hormone are detected in patients with iron deficiency anemia (IDA). Anticalin is a sintetic compound which is able to interacts with hepcidin leading to inhibition of ferroportin-hepcidin binding complexes but its therapeutic effects are still under investigation. Indonesia has various herbal plants which are potentially developed to treat some human diseases. Therefore, the purpose of this study was to identify phytochemicals derived from Indonesian plants that is able to inhibit hepcidin-ferroportin interaction. A bioinformatics study with molecular docking method was used in this study. Three-dimensional structures of human hepcidin and anticalin were obtained from the Protein Data Bank (ID: 1M4F and 4QAE respectively). Because their molecular size was big, each molecule was cut into 2 parts of its binding sites. All phytochemicals structures were obtained from HerbalDB and PubChem NCBI database. Truncated anticalin/phytochemicals were molecularly docked with truncated hepcidin by using AutoDock Vina 1.1.2. and their interactions were visualized using PyMol 1.3. Truncated Anticalin had -4.6 and -4.2 kcal/mol binding affinity to truncated human hepcidin. Truncated anticalin 1 was bound to Cys13, Cys14, Arg16 and Ser17 residues in truncated hepcidin 1 while truncated anticalin 2 was at Cy23 and Lys24 residues in truncated hepcidin 2. Miraxanthine-V, Liriodenin and Chitranone had lower binding affinity (-4.8±0.77, -4.7±0.33 and -5.01±0.30 kcal/mol respectively) than that of anticalin and occupied binding sites as same as anticalin did. There are three phytochemicals that potentially become hepcidin antagonists in silico. In vitro assays are required for verification of the antagonist effect of these phytochemicals on iron metabolism.

Tris-amidoximate uranyl complexes via η2 binding mode coordinated in aqueous solution shown by X-ray absorption spectroscopy and density functional theory methods.

PubMed

Zhang, Linjuan; Qie, Meiying; Su, Jing; Zhang, Shuo; Zhou, Jing; Li, Jiong; Wang, Yu; Yang, Shitong; Wang, Shuao; Li, Jingye; Wu, Guozhong; Wang, Jian Qiang

2018-03-01

The present study sheds some light on the long-standing debate concerning the coordination properties between uranyl ions and the amidoxime ligand, which is a key ingredient for achieving efficient extraction of uranium. Using X-ray absorption fine structure combined with theoretical simulation methods, the binding mode and bonding nature of a uranyl-amidoxime complex in aqueous solution were determined for the first time. The results show that in a highly concentrated amidoxime solution the preferred binding mode between UO 2 2+ and the amidoxime ligand is η 2 coordination with tris-amidoximate species. In such a uranyl-amidoximate complex with η 2 binding motif, strong covalent interaction and orbital hybridization between U 5f/6d and (N, O) 2p should be responsible for the excellent binding ability of the amidoximate ligand to uranyl. The study was performed directly in aqueous solution to avoid the possible binding mode differences caused by crystallization of a single-crystal sample. This work also is an example of the simultaneous study of local structure and electronic structure in solution systems using combined diagnostic tools.

Evaluation of [11C]metergoline as a PET radiotracer for 5HTR in nonhuman primates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hooker, J.M.; Hooker, J.M.; Kim, S.W.

2010-04-20

Metergoline, a serotonin receptor antagonist, was labeled with carbon-11 in order to evaluate its pharmacokinetics and distribution in non-human primates using positron emission tomography. [{sup 11}C]Metergoline had moderate brain uptake and exhibited heterogeneous specific binding, which was blocked by pretreatment with metergoline and altanserin throughout the cortex. Non-specific binding and insensitivity to changes in synaptic serotonin limit its potential as a PET radiotracer. However, the characterization of [{sup 11}C]metergoline pharmacokinetics and binding in the brain and peripheral organs using PET improves our understanding of metergoline drug pharmacology.

Application of oxime-diversification to optimize ligand interactions within a cryptic pocket of the polo-like kinase 1 polo-box domain.

PubMed

Zhao, Xue Zhi; Hymel, David; Burke, Terrence R

2016-10-15

By a process involving initial screening of a set of 87 aldehydes using an oxime ligation-based strategy, we were able to achieve a several-fold affinity enhancement over one of the most potent previously known polo-like kinase 1 (Plk1) polo-box domain (PBD) binding inhibitors. This improved binding may result by accessing a newly identified auxiliary region proximal to a key hydrophobic cryptic pocket on the surface of the protein. Our findings could have general applicability to the design of PBD-binding antagonists. Published by Elsevier Ltd.

Vasopressin V1 receptor in rat hippocampus is regulated by adrenocortical functions.

PubMed

Saito, R; Ishiharada, N; Ban, Y; Honda, K; Takano, Y; Kamiya, H

1994-05-16

Two subtypes of arginine vasopressin (AVP) receptors (V1 and V2) have been distinguished. In this study, we examined the characteristics of AVP binding in rat hippocampus and the effects of bilateral adrenalectomy and adrenal steroids on its [3H]AVP binding. [3H]AVP binding to rat liver and the hippocampal membranes was strongly inhibited by the V1 antagonist, OPC-21268. ADX resulted in a significant decrease in the Bmax of AVP binding in the hippocampus. Chronic treatment with aldosterone and corticosterone restored the ADX-induced reduction, but treatment with dexamethasone did not. These results suggest that the AVP V1 receptor in the hippocampus is regulated by adrenocortical neuroregulatory functions.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

PubMed

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W; Vanden Broeck, Jozef; Tourwé, Dirk

2011-04-14

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

PubMed

Krogsgaard-Larsen, Niels; Delgar, Claudia G; Koch, Karina; Brown, Patricia M G E; Møller, Charlotte; Han, Liwei; Huynh, Tri H V; Hansen, Stinne W; Nielsen, Birgitte; Bowie, Derek; Pickering, Darryl S; Kastrup, Jette Sandholm; Frydenvang, Karla; Bunch, Lennart

2017-01-12

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (K i = 4 μM). In a functional assay, 1b displayed full antagonist activity with IC 50 = 6 ± 2 μM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

A calixpyrrole derivative acts as an antagonist to GPER, a G-protein coupled receptor: mechanisms and models.

PubMed

Lappano, Rosamaria; Rosano, Camillo; Pisano, Assunta; Santolla, Maria Francesca; De Francesco, Ernestina Marianna; De Marco, Paola; Dolce, Vincenza; Ponassi, Marco; Felli, Lamberto; Cafeo, Grazia; Kohnke, Franz Heinrich; Abonante, Sergio; Maggiolini, Marcello

2015-10-01

Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERβ. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells. © 2015. Published by The Company of Biologists Ltd.

TNF binding protein of variola virus acts as a TNF antagonist at epicutaneous application.

PubMed

Gileva, Irina P; Viazovaia, Elena A; Toporkova, Ludmila B; Tsyrendorzhiev, Dondok D; Shchelkunov, Sergei N; Orlovskaya, Irina A

2015-01-01

VARV-CrmB is a TNF binding protein of variola virus. VARV-CrmB protein was previously shown to be active as a TNF-antagonist in a number of in vivo and in vitro models. Here we investigated the epicutaneous effect of recombinant VARV-CrmB protein using an experimental model of muTNFinduced migration of skin leukocytes as well as colony forming activity of bone marrow cells (BMC). Epiсutaneous applications of muTNF enhanced the number of cells migrating from skin flaps of BALB/c mice, whereas subsequent applications of VARV-CrmB protein in 30 min after muTNF, abolished that effect. Epicutaneously applied muTNF influenced the activity of committed hematopoietic progenitors causing a reduction of erythroid (BFUe+CFUe) colonies and increase of granulocyte-macrophage (CFU-GM) colonies in the colony-forming tests. VARV-CrmB, applied in combination with muTNF, demonstrated an ability to reverse this effect, namely, to increase BFUe+CFUe and reduce CFU-GM back to the control levels. Taking together, these data demonstrate the TNF-blocking properties of VARV-CrmB in vivo at epicutaneous applications. As effective TNF antagonist VARV-CrmB protein might be conceded as a beneficial candidate for future research and development of therapeutic approaches in the field of inflammatory skin diseases.

In vivo effects of a GPR30 antagonist.

PubMed

Dennis, Megan K; Burai, Ritwik; Ramesh, Chinnasamy; Petrie, Whitney K; Alcon, Sara N; Nayak, Tapan K; Bologa, Cristian G; Leitao, Andrei; Brailoiu, Eugen; Deliu, Elena; Dun, Nae J; Sklar, Larry A; Hathaway, Helen J; Arterburn, Jeffrey B; Oprea, Tudor I; Prossnitz, Eric R

2009-06-01

Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30 (also known as GPER), in addition to classical nuclear estrogen receptors (ER and ER), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized G-1 (1), a selective agonist of GPR30. To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Qun-Yi; Zhang, Meng; State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai

Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K{sub i} = 13.2 nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids formore » the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a 'competitive' GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.« less

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT3 receptors

PubMed Central

Thompson, A J; Lochner, M; Lummis, S C R

2007-01-01

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors. Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT3A receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. Key Results: 5-HT3 responses were blocked with IC 50 values of 13.4 μM, 11.8 μM and 9.36 μM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA 2 values of 4.92 (K B=12.0 μM) and 4.97 (K B=16.4 μM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [3H]granisetron with K i values of 15.0, 24.2 and 35.7 μ M. Docking of quinine into a homology model of the 5-HT3 receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT3 receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT3 receptor antagonists. PMID:17502851

Interaction mechanism exploration of R-bicalutamide/S-1 with WT/W741L AR using molecular dynamics simulations.

PubMed

Liu, Hongli; An, Xiaoli; Li, Shuyan; Wang, Yuwei; Li, Jiazhong; Liu, Huanxiang

2015-12-01

R-Bicalutamide is a first generation antiandrogen used to treat prostate cancer, which inhibits androgen action by competitively binding to the androgen receptor (AR). However, R-bicalutamide was discovered to exhibit some agonistic properties in clinical application. According to reports, the W741L AR mutation may lead to resistance towards R-bicalutamide. But the mechanism of the R-bicalutamide switch from an antagonist to an agonist due to the mutation of AR W741L is still not so clear. Another molecule, S-1, owing to a very similar structure to R-bicalutamide, is always agonistic to both the wild type and W741L AR. The main difference between these two chemicals is that S-1 has an ether linkage while R-bicalutamide has a sulfonyl group. To study the drug-resistant mechanism caused by W741L mutation and the opposite effects arising from subtle structure differences, molecular dynamics (MD) simulations and molecular mechanics generalized Born surface area (MM-GBSA) calculations were employed to explore the interaction mechanisms between R-bicalutamide/S-1 and WT/W741L AR. The calculated binding free energies are in accordance with the reported experimental values. The obtained results indicate that M895 and W741 are vital amino acids in the antagonism of R-bicalutamide. The bulkier substitution of sulfonyl and tryptophan push aside M895, together with helix 12 (H12), to expose the ligand-binding domain resulting in the antagonistic conformation of the AR. If W741 is mutated to L741, the B-ring of these two chemicals would shift toward L741. At the same time, M895 dragging helix H12, would also move closer to L741. So H12 tends to cover the AR ligand-binding domain to a certain degree, changing the androgen receptor from an antagonistic to an agonistic conformation, which may explain the agonism of R-bicalutamide to the mutant W741L AR.

Radioligand binding characterization of the bradykinin B(2) receptor in the rabbit and pig ileal smooth muscle.

PubMed

Meini, Stefania; Cucchi, Paola; Catalani, Claudio; Bellucci, Francesca; Santicioli, Paolo; Giuliani, Sandro; Maggi, Carlo Alberto

2010-06-10

Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and

Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bencherif, M.; Lukas, R.J.

1991-06-01

Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10more » microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.« less

A flexible docking scheme to explore the binding selectivity of PDZ domains.

PubMed

Gerek, Z Nevin; Ozkan, S Banu

2010-05-01

Modeling of protein binding site flexibility in molecular docking is still a challenging problem due to the large conformational space that needs sampling. Here, we propose a flexible receptor docking scheme: A dihedral restrained replica exchange molecular dynamics (REMD), where we incorporate the normal modes obtained by the Elastic Network Model (ENM) as dihedral restraints to speed up the search towards correct binding site conformations. To our knowledge, this is the first approach that uses ENM modes to bias REMD simulations towards binding induced fluctuations in docking studies. In our docking scheme, we first obtain the deformed structures of the unbound protein as initial conformations by moving along the binding fluctuation mode, and perform REMD using the ENM modes as dihedral restraints. Then, we generate an ensemble of multiple receptor conformations (MRCs) by clustering the lowest replica trajectory. Using ROSETTALIGAND, we dock ligands to the clustered conformations to predict the binding pose and affinity. We apply this method to postsynaptic density-95/Dlg/ZO-1 (PDZ) domains; whose dynamics govern their binding specificity. Our approach produces the lowest energy bound complexes with an average ligand root mean square deviation of 0.36 A. We further test our method on (i) homologs and (ii) mutant structures of PDZ where mutations alter the binding selectivity. In both cases, our approach succeeds to predict the correct pose and the affinity of binding peptides. Overall, with this approach, we generate an ensemble of MRCs that leads to predict the binding poses and specificities of a protein complex accurately.

A flexible docking scheme to explore the binding selectivity of PDZ domains

PubMed Central

Gerek, Z Nevin; Ozkan, S Banu

2010-01-01

Modeling of protein binding site flexibility in molecular docking is still a challenging problem due to the large conformational space that needs sampling. Here, we propose a flexible receptor docking scheme: A dihedral restrained replica exchange molecular dynamics (REMD), where we incorporate the normal modes obtained by the Elastic Network Model (ENM) as dihedral restraints to speed up the search towards correct binding site conformations. To our knowledge, this is the first approach that uses ENM modes to bias REMD simulations towards binding induced fluctuations in docking studies. In our docking scheme, we first obtain the deformed structures of the unbound protein as initial conformations by moving along the binding fluctuation mode, and perform REMD using the ENM modes as dihedral restraints. Then, we generate an ensemble of multiple receptor conformations (MRCs) by clustering the lowest replica trajectory. Using RosettaLigand, we dock ligands to the clustered conformations to predict the binding pose and affinity. We apply this method to postsynaptic density-95/Dlg/ZO-1 (PDZ) domains; whose dynamics govern their binding specificity. Our approach produces the lowest energy bound complexes with an average ligand root mean square deviation of 0.36 Å. We further test our method on (i) homologs and (ii) mutant structures of PDZ where mutations alter the binding selectivity. In both cases, our approach succeeds to predict the correct pose and the affinity of binding peptides. Overall, with this approach, we generate an ensemble of MRCs that leads to predict the binding poses and specificities of a protein complex accurately. PMID:20196074

Fragment-Based Optimization of Small Molecule CXCL12 Inhibitors for Antagonizing the CXCL12/CXCR4 Interaction

PubMed Central

Ziarek, Joshua J.; Liu, Yan; Smith, Emmanuel; Zhang, Guolin; Peterson, Francis C.; Chen, Jun; Yu, Yongping; Chen, Yu; Volkman, Brian F.; Li, Rongshi

2013-01-01

The chemokine CXCL12 and its G protein-coupled receptor (GPCR) CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines interact with two distinct sites to bind and activate their receptors, both the GPCRs and chemokines are potential targets for small molecule inhibition. A number of chemokines have been validated as targets for drug development, but virtually all drug discovery efforts focus on the GPCRs. However, all CXCR4 receptor antagonists with the exception of MSX-122 have failed in clinical trials due to unmanageable toxicities, emphasizing the need for alternative strategies to interfere with CXCL12/CXCR4-guided metastatic homing. Although targeting the relatively featureless surface of CXCL12 was presumed to be challenging, focusing efforts at the sulfotyrosine (sY) binding pockets proved successful for procuring initial hits. Using a hybrid structure-based in silico/NMR screening strategy, we recently identified a ligand that occludes the receptor recognition site. From this initial hit, we designed a small fragment library containing only nine tetrazole derivatives using a fragment-based and bioisostere approach to target the sY binding sites of CXCL12. Compound binding modes and affinities were studied by 2D NMR spectroscopy, X-ray crystallography, molecular docking and cell-based functional assays. Our results demonstrate that the sY binding sites are conducive to the development of high affinity inhibitors with better ligand efficiency (LE) than typical protein-protein interaction inhibitors (LE ≤ 0.24). Our novel tetrazole-based fragment 18 was identified to bind the sY21 site with a Kd of 24 μM (LE = 0.30). Optimization of 18 yielded compound 25 which specifically inhibits CXCL12-induced migration with an improvement in potency over the initial hit 9. The fragment from this library that exhibited the highest affinity and ligand efficiency (11: Kd = 13 μM, LE = 0.33) may serve as a starting point for development of inhibitors targeting the sY12 site. PMID:23368099

The CB1 Neutral Antagonist Tetrahydrocannabivarin Reduces Default Mode Network and Increases Executive Control Network Resting State Functional Connectivity in Healthy Volunteers.

PubMed

Rzepa, Ewelina; Tudge, Luke; McCabe, Ciara

2015-09-10

The cannabinoid cannabinoid type 1 (CB1) neutral antagonist tetrahydrocannabivarin (THCv) has been suggested as a possible treatment for obesity, but without the depressogenic side-effects of inverse antagonists such as Rimonabant. However, how THCv might affect the resting state functional connectivity of the human brain is as yet unknown. We examined the effects of a single 10mg oral dose of THCv and placebo in 20 healthy volunteers in a randomized, within-subject, double-blind design. Using resting state functional magnetic resonance imaging and seed-based connectivity analyses, we selected the amygdala, insula, orbitofrontal cortex, and dorsal medial prefrontal cortex (dmPFC) as regions of interest. Mood and subjective experience were also measured before and after drug administration using self-report scales. Our results revealed, as expected, no significant differences in the subjective experience with a single dose of THCv. However, we found reduced resting state functional connectivity between the amygdala seed region and the default mode network and increased resting state functional connectivity between the amygdala seed region and the dorsal anterior cingulate cortex and between the dmPFC seed region and the inferior frontal gyrus/medial frontal gyrus. We also found a positive correlation under placebo for the amygdala-precuneus connectivity with the body mass index, although this correlation was not apparent under THCv. Our findings are the first to show that treatment with the CB1 neutral antagonist THCv decreases resting state functional connectivity in the default mode network and increases connectivity in the cognitive control network and dorsal visual stream network. This effect profile suggests possible therapeutic activity of THCv for obesity, where functional connectivity has been found to be altered in these regions. © The Author 2015. Published by Oxford University Press on behalf of CINP.

The dopamine D2 receptor dimer and its interaction with homobivalent antagonists: homology modeling, docking and molecular dynamics.

PubMed

Kaczor, Agnieszka A; Jörg, Manuela; Capuano, Ben

2016-09-01

In order to apply structure-based drug design techniques to G protein-coupled receptor complexes, it is essential to model their 3D structure and to identify regions that are suitable for selective drug binding. For this purpose, we have developed and tested a multi-component protocol to model the inactive conformation of the dopamine D2 receptor dimer, suitable for interaction with homobivalent antagonists. Our approach was based on protein-protein docking, applying the Rosetta software to obtain populations of dimers as present in membranes with all the main possible interfaces. Consensus scoring based on the values and frequencies of best interfaces regarding four scoring parameters, Rosetta interface score, interface area, free energy of binding and energy of hydrogen bond interactions indicated that the best scored dimer model possesses a TM4-TM5-TM7-TM1 interface, which is in agreement with experimental data. This model was used to study interactions of the previously published dopamine D2 receptor homobivalent antagonists based on clozapine,1,4-disubstituted aromatic piperidines/piperazines and arylamidoalkyl substituted phenylpiperazine pharmacophores. It was found that the homobivalent antagonists stabilize the receptor-inactive conformation by maintaining the ionic lock interaction, and change the dimer interface by disrupting a set of hydrogen bonds and maintaining water- and ligand-mediated hydrogen bonds in the extracellular and intracellular part of the interface. Graphical Abstract Structure of the final model of the dopamine D2 receptor homodimer, indicating the distancebetween Tyr37 and Tyr 5.42 in the apo form (left) and in the complex with the ligand (right).

Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor.

PubMed

Zhang, Jianyun; Zhang, Jing; Liu, Rui; Gan, Jay; Liu, Jing; Liu, Weiping

2016-01-05

Many pesticides have been identified as endocrine-disrupting chemicals (EDCs) due to their ability to bind sex-steroid hormone receptors. However, little attention has been paid to the ability of pesticides to interfere with other steroid hormone receptors such as glucocorticoid receptor (GR) that plays a critical role in metabolic, endocrine, immune, and nervous systems. In this study, the glucocorticoidic and antiglucocorticoidic effects of 34 pesticides on human GR were investigated using luciferase reporter gene assay. Surprisingly, none of the test chemicals showed GR agonistic activity, but 12 chemicals exhibited apparent antagonistic effects. Bifenthrin, λ-cyhalothrin, cypermethrin, resmethrin, o,p'-DDT, p,p'-DDT, methoxychlor, ethiofencarb, and tolylfluanid showed remarkable GR antagonistic properties with RIC20 values lower than 10(-6) M. The disruption of glucocorticoid-responsive genes in H4IIE and J774A.1 cells was further evaluated on these 12 GR antagonists. In H4IIEcells, four organochlorine insecticides, bifenthrin, and 3-PBA decreased cortisol-induced PEPCK gene expression, while o,p'-DDT and methoxychlor inhibited cortisol-stimulated Arg and TAT gene expression. Cypermethrin and tolyfluanid attenuated cortisol-induced TAT expression. In J774A.1 cells, λ-cyhalothrin, resmethrin, 3-PBA, o,p'-DDT, p,p'-DDT, p,p'-DDE, methoxychlor- and tolylfluanid-reduced cortisol-stimulated GILZ expression. Furthermore, molecular docking simulation indicated that different interactions may stabilize the binding between molecules and GR. Our findings suggest that comprehensive screening and evaluation of GR antagonists and agonists should be considered to better understand the health and ecological risks of man-made chemicals such as pesticides.

Binding of the respiratory chain inhibitor ametoctradin to the mitochondrial bc1 complex.

PubMed

Fehr, Marcus; Wolf, Antje; Stammler, Gerd

2016-03-01

Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc1 complex of oomycetes. The bc1 complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross-resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. Cross-resistance analyses, the reduction of isolated Pythium sp. bc1 complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc1 complex similarly to stigmatellin. The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross-resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates. © 2015 Society of Chemical Industry.

INTERACTION OF ORGANOPHOSPHATE PESTICIDES AND RELATED COMPOUNDS WITH THE ANDROGEN RECEPTOR

EPA Science Inventory

Identification of several environmental chemicals capable of binding to the androgen receptor (AR) and interfering with its normal function has heightened concern for adverse effects across a broad spectrum of environmental chemicals. We previously demonstrated AR antagonist act...

Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer

PubMed Central

Kim, Ari; Yen, Paul; Mroczek, Marta; Nouri, Mannan; Lien, Scott; Axerio-Cilies, Peter; Dalal, Kush; Yau, Clement; Ghaidi, Fariba; Guo, Yubin; Yamazaki, Takeshi; Lawn, Sam; Gleave, Martin E.; Gregory-Evans, Cheryl Y.

2017-01-01

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer. PMID:28465491

Exploring new scaffolds for angiotensin II receptor antagonism.

PubMed

Kritsi, Eftichia; Matsoukas, Minos-Timotheos; Potamitis, Constantinos; Karageorgos, Vlasios; Detsi, Anastasia; Magafa, Vasilliki; Liapakis, George; Mavromoustakos, Thomas; Zoumpoulakis, Panagiotis

2016-09-15

Nowadays, AT1 receptor (AT1R) antagonists (ARBs) constitute the one of the most prevalent classes of antihypertensive drugs that modulate the renin-angiotensin system (RAS). Their main uses include also treatment of diabetic nephropathy (kidney damage due to diabetes) and congestive heart failure. Towards this direction, our study has been focused on the discovery of novel agents bearing different scaffolds which may evolve as a new class of AT1 receptor antagonists. To fulfill this aim, a combination of computational approaches and biological assays were implemented. Particularly, a pharmacophore model was established and served as a 3D search query to screen the ChEMBL15 database. The reliability and accuracy of virtual screening results were improved by using molecular docking studies. In total, 4 compounds with completely diverse chemical scaffolds from potential ARBs, were picked and tested for their binding affinity to AT1 receptor. Results revealed high nanomolar to micromolar affinity (IC50) for all the compounds. Especially, compound 4 exhibited a binding affinity of 199nM. Molecular dynamics simulations were utilized in an effort to provide a molecular basis of their binding to AT1R in accordance to their biological activities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2

PubMed Central

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim

2015-01-01

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2−/− mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2−/− mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2−/− mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2−/− mice. Our results demonstrate that Eif4ebp2−/− mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. SIGNIFICANCE STATEMENT Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E-binding protein 2, leads to ASD-like behaviors and increased excitatory synaptic activity. Here we demonstrated that autistic behavioral and electrophysiological phenotypes can be treated in adult mice with antagonists of group I metabotropic glutamate receptors (mGluRs), which have been previously used in other ASD models (i.e., fragile X syndrome). These findings support the use of group I mGluR antagonists as a potential therapy that extends to autism models involving exacerbated mRNA translation initiation. PMID:26245973

Inhibition of Group I Metabotropic Glutamate Receptors Reverses Autistic-Like Phenotypes Caused by Deficiency of the Translation Repressor eIF4E Binding Protein 2.

PubMed

Aguilar-Valles, Argel; Matta-Camacho, Edna; Khoutorsky, Arkady; Gkogkas, Christos; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

2015-08-05

Exacerbated mRNA translation during brain development has been linked to autism spectrum disorders (ASDs). Deletion of the eukaryotic initiation factor 4E (eIF4E)-binding protein 2 gene (Eif4ebp2), encoding the suppressor of mRNA translation initiation 4E-BP2, leads to an imbalance in excitatory-to-inhibitory neurotransmission and ASD-like behaviors. Inhibition of group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5 reverses the autistic phenotypes in several ASD mouse models. Importantly, these receptors control synaptic physiology via activation of mRNA translation. We investigated the potential reversal of autistic-like phenotypes in Eif4ebp2(-/-) mice by using antagonists of mGluR1 (JNJ16259685) or mGluR5 (fenobam). Augmented hippocampal mGluR-induced long-term depression (LTD; or chemically induced mGluR-LTD) in Eif4ebp2(-/-) mice was rescued by mGluR1 or mGluR5 antagonists. While rescue by mGluR5 inhibition occurs through the blockade of a protein synthesis-dependent component of LTD, normalization by mGluR1 antagonists requires the activation of protein synthesis. Synaptically induced LTD was deficient in Eif4ebp2(-/-) mice, and this deficit was not rescued by group I mGluR antagonists. Furthermore, a single dose of mGluR1 (0.3 mg/kg) or mGluR5 (3 mg/kg) antagonists in vivo reversed the deficits in social interaction and repetitive behaviors (marble burying) in Eif4ebp2(-/-) mice. Our results demonstrate that Eif4ebp2(-/-) mice serve as a relevant model to test potential therapies for ASD symptoms. In addition, we provide substantive evidence that the inhibition of mGluR1/mGluR5 is an effective treatment for physiological and behavioral alterations caused by exacerbated mRNA translation initiation. Exacerbated mRNA translation during brain development is associated with several autism spectrum disorders (ASDs). We recently demonstrated that the deletion of a negative regulator of mRNA translation initiation, the eukaryotic initiation factor 4E-binding protein 2, leads to ASD-like behaviors and increased excitatory synaptic activity. Here we demonstrated that autistic behavioral and electrophysiological phenotypes can be treated in adult mice with antagonists of group I metabotropic glutamate receptors (mGluRs), which have been previously used in other ASD models (i.e., fragile X syndrome). These findings support the use of group I mGluR antagonists as a potential therapy that extends to autism models involving exacerbated mRNA translation initiation. Copyright © 2015 the authors 0270-6474/15/3511126-08$15.00/0.

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes.

PubMed

Allen, Bruce G; Phuong, Luu Lien; Farhat, Hala; Chevalier, Dominique

2003-02-01

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.