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Sample records for allosteric potentiating ligand

  1. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    PubMed

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

  2. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  3. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  4. Anisotropic energy flow and allosteric ligand binding in albumin

    NASA Astrophysics Data System (ADS)

    Li, Guifeng; Magana, Donny; Dyer, R. Brian

    2014-01-01

    Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

  5. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  6. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

    PubMed

    Li, Jingjing; Gierach, Izabela; Gillies, Alison R; Warden, Charles D; Wood, David W

    2011-11-15

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  7. Allosteric Ligand Binding and Anisotropic Energy Flow in Albumin

    NASA Astrophysics Data System (ADS)

    Dyer, Brian

    2014-03-01

    Protein allostery usually involves propagation of local structural changes through the protein to a remote site. Coupling of structural changes at remote sites is thought to occur through anisotropic energy transport, but the nature of this process is poorly understood. We have studied the relationship between allosteric interactions of remote ligand binding sites of the protein and energy flow through the structure of bovine serum albumin (BSA). We applied ultrafast infrared spectroscopy to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic flow through the protein structure following input of thermal energy into the flexible ligand binding sites. We also observe anisotropic heat flow through the structure, without local heating of the rigid helix bundles that connect these sites. We will discuss the implications of this efficient energy transport mechanism with regard to the allosteric propagation of binding energy through the connecting helix structures.

  8. Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators.

    PubMed

    Gregory, Karen J; Velagaleti, Ranganadh; Thal, David M; Brady, Ryan M; Christopoulos, Arthur; Conn, P Jeffrey; Lapinsky, David J

    2016-07-15

    G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively "tuning" GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand-GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu5 NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu5 via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu5-bound probe via tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu5 and negative cooperativity with glutamate, interacted with the "common allosteric-binding site," displayed slow binding kinetics, and could irreversibly label mGlu5 following UV exposure. We depleted the number of functional mGlu5 receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes via click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu5 allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily. PMID:27115427

  9. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    SciTech Connect

    Williams,R.; Holyoak, T.; McDonald, G.; Gui, C.; Fenton, A.

    2006-01-01

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

  10. Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes.

    PubMed Central

    Soukup, G A; DeRose, E C; Koizumi, M; Breaker, R R

    2001-01-01

    Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches. PMID:11345431

  11. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  12. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  13. Exploration of Gated Ligand Binding Recognizes an Allosteric Site for Blocking FABP4-Protein Interaction

    PubMed Central

    Li, Yan; Li, Xiang; Dong, Zigang

    2015-01-01

    Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level. PMID:26580122

  14. G Protein-Coupled Receptor Heteromerization: A Role in Allosteric Modulation of Ligand BindingS⃞

    PubMed Central

    Gomes, Ivone; IJzerman, Adriaan P.; Ye, Kai; Maillet, Emeline L.

    2011-01-01

    It is becoming increasingly recognized that G protein-coupled receptors physically interact. These interactions may provide a mechanism for allosteric modulation of receptor function. In this study, we examined this possibility by using an established model system of a receptor heteromer consisting of μ and δ opioid receptors. We examined the effect of a number of μ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled δ receptor agonist, [3H]deltorphin II. We also examined the effect of δ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled μ receptor agonist, [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin ([3H]DAMGO). We show that μ receptor ligands are capable of allosterically enhancing δ receptor radioligand binding and vice versa. Thus, there is strong positive cooperativity between the two receptor units with remarkable consequences for ligand pharmacology. We find that the data can be simulated by adapting an allosteric receptor model previously developed for small molecules, suggesting that the ligand-occupied protomers function as allosteric modulators of the partner receptor's activity. PMID:21415307

  15. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

    PubMed

    Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K; Zhu, Hu; Chen, Xin; Moy, Sheryl S; Saddoris, Kara A; Nikolova, Viktoriya D; Farrell, Martilias S; Wang, Sheng; Mangano, Thomas J; Deshpande, Deepak A; Jiang, Alice; Penn, Raymond B; Jin, Jian; Koller, Beverly H; Kenakin, Terry; Shoichet, Brian K; Roth, Bryan L

    2015-11-26

    At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

  16. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel

    PubMed Central

    Joseph, Thomas T.

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  17. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel.

    PubMed

    Joseph, Thomas T; Mincer, Joshua S

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  18. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  19. Engineering an allosteric transcription factor to respond to new ligands.

    PubMed

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-02-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  20. Engineering an allosteric transcription factor to respond to new ligands

    PubMed Central

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-01-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  1. Dissecting allosteric effects of activator-coactivator complexes using a covalent small molecule ligand.

    PubMed

    Wang, Ningkun; Lodge, Jean M; Fierke, Carol A; Mapp, Anna K

    2014-08-19

    Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in koff. Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both kon and koff, suggesting stabilization of the binary complex.

  2. Evolution of oligomeric state through allosteric pathways that mimic ligand binding

    PubMed Central

    Perica, Tina; Kondo, Yasushi; Tiwari, Sandhya P.; McLaughlin, Stephen H.; Kemplen, Katherine R.; Zhang, Xiuwei; Steward, Annette; Reuter, Nathalie; Clarke, Jane; Teichmann, Sarah A.

    2015-01-01

    Evolution and design of protein complexes is almost always viewed through the lens of amino acid mutations at protein interfaces. We showed previously that residues not involved in the physical interaction between proteins make important contributions to oligomerisation by acting indirectly or allosterically. Here, we sought to investigate the mechanism by which allosteric mutations act using the example of the PyrR family of pyrimidine operon attenuators. In this family, a perfectly sequence-conserved helix that forms a tetrameric interface is exposed as solvent-accessible surface in dimeric orthologues. This means that mutations must be acting from a distance to destabilize the interface. We identified eleven key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. Finally, we show that the key mutations introduce conformational changes equivalent to the conformational shift between the free versus the nucleotide-bound conformations of the proteins. PMID:25525255

  3. Discovery of Potential Orthosteric and Allosteric Antagonists of P2Y1R from Chinese Herbs by Molecular Simulation Methods

    PubMed Central

    Lu, Fang; Jiang, Lu-di; Qiao, Lian-sheng; Xiang, Yu-hong

    2016-01-01

    P2Y1 receptor (P2Y1R), which belongs to G protein-coupled receptors (GPCRs), is an important target in ADP-induced platelet aggregation. The crystal structure of P2Y1R has been solved recently, which revealed orthosteric and allosteric ligand-binding sites with the details of ligand-protein binding modes. And it suggests that P2Y1R antagonists, which recognize two distinct sites, could potentially provide an efficacious and safe antithrombotic profile. In present paper, 2D similarity search, pharmacophore based screening, and molecular docking were used to explore the potential natural P2Y1R antagonists. 2D similarity search was used to classify orthosteric and allosteric antagonists of P2Y1R. Based on the result, pharmacophore models were constructed and validated by the test set. Optimal models were selected to discover potential P2Y1R antagonists of orthosteric and allosteric sites from Traditional Chinese Medicine (TCM). And the hits were filtered by Lipinski's rule. Then molecular docking was used to refine the results of pharmacophore based screening and analyze the binding mode of the hits and P2Y1R. Finally, two orthosteric and one allosteric potential compounds were obtained, which might be used in future P2Y1R antagonists design. This work provides a reliable guide for discovering natural P2Y1R antagonists acting on two distinct sites from TCM.

  4. Discovery of Potential Orthosteric and Allosteric Antagonists of P2Y1R from Chinese Herbs by Molecular Simulation Methods

    PubMed Central

    Lu, Fang; Jiang, Lu-di; Qiao, Lian-sheng; Xiang, Yu-hong

    2016-01-01

    P2Y1 receptor (P2Y1R), which belongs to G protein-coupled receptors (GPCRs), is an important target in ADP-induced platelet aggregation. The crystal structure of P2Y1R has been solved recently, which revealed orthosteric and allosteric ligand-binding sites with the details of ligand-protein binding modes. And it suggests that P2Y1R antagonists, which recognize two distinct sites, could potentially provide an efficacious and safe antithrombotic profile. In present paper, 2D similarity search, pharmacophore based screening, and molecular docking were used to explore the potential natural P2Y1R antagonists. 2D similarity search was used to classify orthosteric and allosteric antagonists of P2Y1R. Based on the result, pharmacophore models were constructed and validated by the test set. Optimal models were selected to discover potential P2Y1R antagonists of orthosteric and allosteric sites from Traditional Chinese Medicine (TCM). And the hits were filtered by Lipinski's rule. Then molecular docking was used to refine the results of pharmacophore based screening and analyze the binding mode of the hits and P2Y1R. Finally, two orthosteric and one allosteric potential compounds were obtained, which might be used in future P2Y1R antagonists design. This work provides a reliable guide for discovering natural P2Y1R antagonists acting on two distinct sites from TCM. PMID:27635149

  5. Discovery of Potential Orthosteric and Allosteric Antagonists of P2Y1R from Chinese Herbs by Molecular Simulation Methods.

    PubMed

    Zhang, Xu; Lu, Fang; Chen, Yan-Kun; Luo, Gang-Gang; Jiang, Lu-di; Qiao, Lian-Sheng; Zhang, Yan-Ling; Xiang, Yu-Hong

    2016-01-01

    P2Y1 receptor (P2Y1R), which belongs to G protein-coupled receptors (GPCRs), is an important target in ADP-induced platelet aggregation. The crystal structure of P2Y1R has been solved recently, which revealed orthosteric and allosteric ligand-binding sites with the details of ligand-protein binding modes. And it suggests that P2Y1R antagonists, which recognize two distinct sites, could potentially provide an efficacious and safe antithrombotic profile. In present paper, 2D similarity search, pharmacophore based screening, and molecular docking were used to explore the potential natural P2Y1R antagonists. 2D similarity search was used to classify orthosteric and allosteric antagonists of P2Y1R. Based on the result, pharmacophore models were constructed and validated by the test set. Optimal models were selected to discover potential P2Y1R antagonists of orthosteric and allosteric sites from Traditional Chinese Medicine (TCM). And the hits were filtered by Lipinski's rule. Then molecular docking was used to refine the results of pharmacophore based screening and analyze the binding mode of the hits and P2Y1R. Finally, two orthosteric and one allosteric potential compounds were obtained, which might be used in future P2Y1R antagonists design. This work provides a reliable guide for discovering natural P2Y1R antagonists acting on two distinct sites from TCM. PMID:27635149

  6. Allosteric modulation of ligand binding to [3H](+)pentazocine-defined sigma recognition sites by phenytoin.

    PubMed

    DeHaven-Hudkins, D L; Ford-Rice, F Y; Allen, J T; Hudkins, R L

    1993-01-01

    The allosteric modulation of sigma recognition sites by phenytoin (diphenylhydantoin) has been demonstrated by the ability of phenytoin to stimulate binding of various [3H] sigma ligands, as well as to slow dissociation from sigma sites and to shift sigma sites from a low- to a high-affinity state. Phenytoin stimulated the binding of the sigma 1- selective ligand [3H](+)pentazocine in a dose-dependent manner. Stimulation of binding at a final concentration of 250 microM phenytoin was associated with a decrease in the KD. The affinities of the sigma reference compounds caramiphen, dextromethorphan, dextrophan, (+)3-PPP and (+)SKF-10,047 were three- to eight-fold higher, while the affinities of benzetimide, BMY-14802, carbetapentane, DTG and haloperidol were unchanged in the presence of 250 microM phenytoin. The relative sensitivity of sigma compounds to allosteric modulation by phenytoin is not a property of all sigma ligands, and may provide an in vitro basis for distinguishing actions of sigma compounds and predicting sigma effects in vivo. PMID:8515681

  7. Identification and characterization of mGlu3 ligands using a high throughput FLIPR assay for detection of agonists, antagonists, and allosteric modulators.

    PubMed

    Pratt, Steven D; Mezler, Mario; Geneste, Hervé; Bakker, Margot H M; Hajduk, Philip J; Gopalakrishnan, Sujatha M

    2011-08-01

    When targeting G-protein coupled receptors (GPCRs) in early stage drug discovery, or for novel targets, the type of ligand most likely to produce the desired therapeutic effect may be unknown. Therefore, it can be desirable to identify potential lead compounds from multiple categories: agonists, antagonists, and allosteric modulators. In this study, we developed a triple addition calcium flux assay using FLIPR Tetra to identify multiple ligand classes for the metabotropic glutamate receptor 3 (mGlu3), using a cell line stably co-expressing the human G-protein-coupled mGlu3 receptor, a promiscuous G-protein (G(α16)), and rat Glast, a glutamate transporter. Compounds were added to the cells followed by stimulation with EC(10) and then EC(80) concentration of glutamate, the physiological agonist for mGlu receptors. This format produced a robust assay, facilitating the identification of agonists, positive allosteric modulators and antagonists/negative allosteric modulators. Follow up experiments were conducted to exclude false positives. Using this approach, we screened a library of approximately 800,000 compounds using FLIPR Tetra and identified viable leads for all three ligand classes. Further characterization revealed the selectivity of individual ligands. PMID:21534916

  8. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    PubMed

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

  9. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    PubMed

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins. PMID:27526031

  10. Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer.

    PubMed

    Steffen, Jamin D; Tholey, Renee M; Langelier, Marie-France; Planck, Jamie L; Schiewer, Matthew J; Lal, Shruti; Bildzukewicz, Nikolai A; Yeo, Charles J; Knudsen, Karen E; Brody, Jonathan R; Pascal, John M

    2014-01-01

    PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

  11. Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

    PubMed Central

    Krieger, James; Bahar, Ivet; Greger, Ingo H.

    2015-01-01

    Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

  12. Are AMPA receptor positive allosteric modulators potential pharmacotherapeutics for addiction?

    PubMed

    Watterson, Lucas R; Olive, M Foster

    2013-01-01

    Positive allosteric modulators (PAMs) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF) in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications. PMID:24380895

  13. Are AMPA Receptor Positive Allosteric Modulators Potential Pharmacotherapeutics for Addiction?

    PubMed Central

    Watterson, Lucas R.; Olive, M. Foster

    2013-01-01

    Positive allosteric modulators (PAMs) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF) in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications. PMID:24380895

  14. Toward understanding the molecular basis for chemical allosteric modulator design.

    PubMed

    Wang, Qi; Zheng, Mingyue; Huang, Zhimin; Liu, Xinyi; Zhou, Huchen; Chen, Yingyi; Shi, Ting; Zhang, Jian

    2012-09-01

    Among the regulation mechanisms of cellular function, allosteric regulation is the most direct, rapid and efficient. Due to the wider receptor selectivity and lower target-based toxicity, compared with orthosteric ligands, allosteric modulators are expected to play a larger role in pharmaceutical research and development. However, current difficulties, such as a low affinity and unknown structural features of potential allosteric small-molecules, usually obstruct the discovery of allosteric modulators. In this study, we compared known allosteric modulators with various compounds from different databases to unveil the structural and qualitative characteristics of allosteric modulators. The results show that allosteric modulators generally contain more hydrophobic scaffolds and have a higher structural rigidity, i.e., less rotatable bonds and more rings. Based on this analysis, an empirical rule was defined to determine the structural requirements for an allosteric modulator. It was found that a large proportion of allosteric modulators (80%) can be successfully retrieved by this "allosteric-like" filter, which shows good discriminatory power in identifying allosteric modulators. Therefore, the study provides deeper insight into the chemical properties of allosteric modulators and has a good potential for the design or optimization of allosteric compounds. PMID:23085171

  15. Orthosteric and Allosteric Ligands of Nicotinic Acetylcholine Receptors for Smoking Cessation

    PubMed Central

    Mohamed, Tasnim S.; Jayakar, Selwyn S.; Hamouda, Ayman K.

    2015-01-01

    Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide per year, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR) in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity. PMID:26635524

  16. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors – A Structural Perspective of Ligands and Mutants

    PubMed Central

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G.; Weiss, Dahlia; Arsova, Angela; Marshall, Fiona H.; Bräuner-Osborne, Hans; Gloriam, David E.

    2015-01-01

    The metabotropic glutamate receptors have a wide range of modulatory functions in the central nervous system. They are among the most highly pursued drug targets, with relevance for several neurological diseases, and a number of allosteric modulators have entered clinical trials. However, so far this has not led to a marketed drug, largely because of the difficulties in achieving subtype-selective compounds with desired properties. Very recently the first crystal structures were published for the transmembrane domain of two metabotropic glutamate receptors in complex with negative allosteric modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different mGlu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs. PMID:26359761

  17. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    PubMed Central

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  18. Discovery and Characterization of Novel Allosteric Potentiators of M1 Muscarinic Receptors Reveals Multiple Modes of Activity

    PubMed Central

    Marlo, Joy E.; Niswender, Colleen M.; Days, Emily L.; Bridges, Thomas M.; Xiang, Yun; Rodriguez, Alice L.; Shirey, Jana K.; Brady, Ashley E.; Nalywajko, Tasha; Luo, Qingwei; Austin, Cheryl A.; Williams, Michael Baxter; Kim, Kwangho; Williams, Richard; Orton, Darren; Brown, H. Alex; Lindsley, Craig W.; Weaver, C. David; Conn, P. Jeffrey

    2009-01-01

    Activators of M1 muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M1-active compounds has resulted in nonselective activation of the highly related M2 to M5 mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M1 with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N′-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M1 that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M1 activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M1 receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M1 agonist, 1-[1′-(2-tolyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M1-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M1 is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M1 potentiators may act by distinct mechanisms and differentially regulate receptor

  19. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

    PubMed Central

    Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

    2015-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

  20. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  1. Metabotropic glutamate receptor ligands as potential therapeutics for addiction

    PubMed Central

    Olive, M. F.

    2009-01-01

    There is now compelling evidence that the excitatory amino acid neurotransmitter glutamate plays a pivotal role in drug addiction and alcoholism. As a result, there has been increasing interest in developing glutamate-based therapies for the treatment of addictive disorders. Receptors for glutamate are primarily divided into two classes: ionotropic glutamate receptors (iGluRs) that mediate fast excitatory glutamate transmission, and metabotropic glutamate receptors (mGluRs), which are G-protein coupled receptors that mediate slower, modulatory glutamate transmission. Most iGluR antagonists, while showing some efficacy in animal models of addiction, exhibit serious side effects when tested in humans. mGluR ligands, on the other hand, which have been advanced to testing in clinical trials for various medical conditions, have demonstrated the ability to reduce drug reward, reinforcement, and relapse-like behaviors in animal studies. mGluR ligands that have been shown to be primarily effective are Group I (mGluR1 and mGluR5) negative allosteric modulators and Group II (mGluR2 and mGluR3) orthosteric presynaptic autoreceptor agonists. In this review, we will summarize findings from animal studies suggesting that these mGluR ligands may be of potential benefit in reducing on-going drug self-administration and may aid in the prevention of relapse. The neuroanatomical distribution of mGluR1, mGluR2/3, and mGluR5 receptors and the pharmacological properties of Group I negative allosteric modulators and Group II agonists will also be overviewed. Finally, we will discuss the current status of mGluR ligands in human clinical trials. PMID:19630739

  2. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    PubMed Central

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, Thanasis; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45′ (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  3. Chemical, Target, and Bioactive Properties of Allosteric Modulation

    PubMed Central

    van Westen, Gerard J. P.; Gaulton, Anna; Overington, John P.

    2014-01-01

    Allosteric modulators are ligands for proteins that exert their effects via a different binding site than the natural (orthosteric) ligand site and hence form a conceptually distinct class of ligands for a target of interest. Here, the physicochemical and structural features of a large set of allosteric and non-allosteric ligands from the ChEMBL database of bioactive molecules are analyzed. In general allosteric modulators are relatively smaller, more lipophilic and more rigid compounds, though large differences exist between different targets and target classes. Furthermore, there are differences in the distribution of targets that bind these allosteric modulators. Allosteric modulators are over-represented in membrane receptors, ligand-gated ion channels and nuclear receptor targets, but are underrepresented in enzymes (primarily proteases and kinases). Moreover, allosteric modulators tend to bind to their targets with a slightly lower potency (5.96 log units versus 6.66 log units, p<0.01). However, this lower absolute affinity is compensated by their lower molecular weight and more lipophilic nature, leading to similar binding efficiency and surface efficiency indices. Subsequently a series of classifier models are trained, initially target class independent models followed by finer-grained target (architecture/functional class) based models using the target hierarchy of the ChEMBL database. Applications of these insights include the selection of likely allosteric modulators from existing compound collections, the design of novel chemical libraries biased towards allosteric regulators and the selection of targets potentially likely to yield allosteric modulators on screening. All data sets used in the paper are available for download. PMID:24699297

  4. Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.

    PubMed

    Mony, Laetitia; Kew, James N C; Gunthorpe, Martin J; Paoletti, Pierre

    2009-08-01

    N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.

  5. Mutations in the GABAA receptor that mimic the allosteric ligand, etomidate

    PubMed Central

    Forman, Stuart A.; Stewart, Deirdre

    2012-01-01

    Etomidate is a hydrophobic molecule, a potent general anesthetic, and the best understood drug in this group. Etomidate’s target molecules are GABAA receptors, its site of action has been identified with photolabeling, and a quantitative allosteric co-agonist model has emerged for etomidate effects on GABAA receptors. We have shown that when methionine residues that are thought to be adjacent to the etomidate site are mutated to tryptophan, that the bulky hydrophobic side-chains alter mutant GABAA receptor function in ways that mimic the effects of etomidate binding to wild-type receptors. Furthermore, these mutations reduce receptor modulation by etomidate. Both of these observations support the hypothesis that these methionine residues form part of the etomidate binding pocket. PMID:22052498

  6. A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase.

    PubMed

    Velyvis, Algirdas; Yang, Ying R; Schachman, Howard K; Kay, Lewis E

    2007-05-22

    The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, (1)H,(13)C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L') between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is "invisible" in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R-T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R-T equilibrium, consistent with the Monod-Wyman-Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons.

  7. Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

    PubMed Central

    Nys, Mieke; Wijckmans, Eveline; Farinha, Ana; Yoluk, Özge; Andersson, Magnus; Brams, Marijke; Spurny, Radovan; Peigneur, Steve; Tytgat, Jan; Lindahl, Erik; Ulens, Chris

    2016-01-01

    Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8–β9 loop in the extracellular ligand-binding domain. The β8–β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8–β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors. PMID:27791038

  8. Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor

    PubMed Central

    Jiang, Jianxiong; Ganesh, Thota; Du, Yuhong; Thepchatri, Pahk; Rojas, Asheebo; Lewis, Iestyn; Kurtkaya, Serdar; Li, Lian; Qui, Min; Serrano, Geidy; Shaw, Renee; Sun, Aiming; Dingledine, Ray

    2010-01-01

    Activation of the Gαs-coupled EP2 receptor for prostaglandin E2 (PGE2) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 μM (i) potentiated the cAMP response to a low concentration of PGE2 by > 50%; (ii) had no effect on EP4 or β2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE2 on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE2 on EP2 receptors 4- to 5-fold at 10 to 20 μM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE2 receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE2 released in a cell-injury setting is neuroprotective. PMID:20080612

  9. Recent Progress on the Identification of Metabotropic Glutamate 4 Receptor Ligands and Their Potential Utility as CNS Therapeutics

    PubMed Central

    2011-01-01

    This Review describes recent activity in the advancement of ligands for the metabotropic glutamate 4 receptor subtype and their potential utility as central nervous system (CNS) therapeutics. Until recently, there was a paucity of compounds with suitable selectivity and druglike properties to elucidate the value of this target. The search for selective entities has led several groups to the investigation of allosteric modulators as a path to optimization of potential ligands. Recent efforts, discussed here, have afforded a variety of derivatives with improvements in potency, solubility, and pharmacokinetic properties that garner support for continued investigation and optimization. PMID:22860170

  10. Allosteric Modulation of Metabotropic Glutamate Receptors: Structural Insights and Therapeutic Potential

    PubMed Central

    Gregory, Karen J.; Dong, Elizabeth N.; Meiler, Jens; Conn, P. Jeffrey

    2010-01-01

    Allosteric modulation of G protein-coupled receptors (GPCRs) represents a novel approach to the development of probes and therapeutics that is expected to enable subtype-specific regulation of central nervous system target receptors. The metabotropic glutamate receptors (mGlus) are class C GPCRs that play important neuromodulatory roles throughout the brain, as such they are attractive targets for therapeutic intervention for a number of psychiatric and neurological disorders including anxiety, depression, Fragile X Syndrome, Parkinson’s disease and schizophrenia. Over the last fifteen years, selective allosteric modulators have been identified for many members of the mGlu family. The vast majority of these allosteric modulators are thought to bind within the transmembrane-spanning domains of the receptors to enhance or inhibit functional responses. A combination of mutagenesis-based studies and pharmacological approaches are beginning to provide a better understanding of mGlu allosteric sites. Collectively, when mapped onto a homology model of the different mGlu subtypes based on the β2-adrenergic receptor, the previous mutagenesis studies suggest commonalities in the location of allosteric sites across different members of the mGlu family. In addition, there is evidence for multiple allosteric binding pockets within the transmembrane region that can interact to modulate one another. In the absence of a class C GPCR crystal structure, this approach has shown promise with respect to the interpretation of mutagenesis data and understanding structure-activity relationships of allosteric modulator pharmacophores. PMID:20637216

  11. ASBench: benchmarking sets for allosteric discovery.

    PubMed

    Huang, Wenkang; Wang, Guanqiao; Shen, Qiancheng; Liu, Xinyi; Lu, Shaoyong; Geng, Lv; Huang, Zhimin; Zhang, Jian

    2015-08-01

    Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a 'Core set' with 235 unique allosteric sites and a 'Core-Diversity set' with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand-protein interactions to guide allosteric drug design.

  12. Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor.

    PubMed

    Burford, Neil T; Clark, Mary J; Wehrman, Tom S; Gerritz, Samuel W; Banks, Martyn; O'Connell, Jonathan; Traynor, John R; Alt, Andrew

    2013-06-25

    μ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the μ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a β-arrestin-recruitment assay. PMID:23754417

  13. Cholinergic modulation by opioid receptor ligands: potential application to Alzheimer's disease.

    PubMed

    Motel, William C; Coop, Andrew; Cunningham, Christopher W

    2013-03-01

    Morphinans have a storied history in medicinal chemistry as pain management drugs but have received attention as modulators of cholinergic signaling for the treatment of Alzheimer's Disease (AD). Galantamine is a reversible, competitive acetylcholinesterase (AChE) inhibitor and allosteric potentiating ligand of nicotinic acetylcholine receptors (nAChR-APL) that shares many common structural elements with morphinan-based opioids. The structurally diverse opioids codeine and eseroline, like galantamine, are also nAChR-APL that have greatly diminished affinity for AChE, representing potential lead compounds for selective nAChR-APL development. In accordance with the emerging repurposing trend of evaluating known compounds for novel pharmacological activity, ongoing research on augmentation of cholinergic signaling that has been aided by the use of opioids will be reviewed. PMID:22931533

  14. Allosteric regulation of the discriminative responsiveness of retinoic acid receptor to natural and synthetic ligands by retinoid X receptor and DNA.

    PubMed

    Mouchon, A; Delmotte, M H; Formstecher, P; Lefebvre, P

    1999-04-01

    Transcriptional activation by retinoids is mediated through two families of nuclear receptors, all-trans-retinoic acid (RARs) and 9-cis retinoic acid receptors (RXRs). Conformationally restricted retinoids are used to achieve selective activation of RAR isotype alpha, beta or gamma, which reduces side effects in therapeutical applications. Synthetic retinoids mimic some of all-trans retinoic acid biological effects in vivo but interact differently with the ligand binding domain of RARalpha and induce distinct structural transitions of the receptor. In this report, we demonstrate that RAR-selective ligands have distinct quantitative activation properties which are reflected by their abilities to promote interaction of DNA-bound human RXRalpha (hRXRalpha)-hRARalpha heterodimers with the nuclear receptor coactivator (NCoA) SRC-1 in vitro. The hormone response element core motifs spacing defined the relative affinity of liganded heterodimers for two NCoAs, SRC-1 and RIP140. hRXRalpha activating function 2 was critical to confer hRARalpha full responsiveness but not differential sensitivity of hRARalpha to natural or synthetic retinoids. We also provide evidence showing that lysines located in helices 3 and 4, which define part of hRARalpha NCoA binding surface, contribute differently to (i) the transcriptional activity and (ii) the interaction of RXR-RAR heterodimers with SRC-1, when challenged by either natural or RAR-selective retinoids. Thus, ligand structure, DNA, and RXR exert allosteric regulations on hRARalpha conformation organized as a DNA-bound heterodimer. We suggest that the use of physically distinct NCoA binding interfaces may be important in controlling specific genes by conformationally restricted ligands.

  15. Structure-Based Statistical Mechanical Model Accounts for the Causality and Energetics of Allosteric Communication.

    PubMed

    Guarnera, Enrico; Berezovsky, Igor N

    2016-03-01

    Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design. PMID:26939022

  16. Structure-Based Statistical Mechanical Model Accounts for the Causality and Energetics of Allosteric Communication.

    PubMed

    Guarnera, Enrico; Berezovsky, Igor N

    2016-03-01

    Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design.

  17. Structure-Based Statistical Mechanical Model Accounts for the Causality and Energetics of Allosteric Communication

    PubMed Central

    Guarnera, Enrico; Berezovsky, Igor N.

    2016-01-01

    Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design. PMID:26939022

  18. Preclinical evaluation of the antipsychotic potential of the mGlu2-positive allosteric modulator JNJ-40411813

    PubMed Central

    Lavreysen, Hilde; Langlois, Xavier; Donck, Luc Ver; Nuñez, José María Cid; Pype, Stefan; Lütjens, Robert; Megens, Anton

    2015-01-01

    JNJ-40411813/ADX71149 (1-butyl-3-chloro-4-(4-phenylpiperidin-1-yl) pyridin-2(1H)-one) is a positive allosteric modulator (PAM) of the mGlu2 receptor, which also displays 5-Hydroxytryptamine (5HT2A) antagonism after administration in rodents due to a rodent-specific metabolite. JNJ-40411813 was compared with the orthosteric mGlu2/3 agonist LY404039 (4-amino-2-thiabicyclo [3.1.0] hexane-4,6-dicarboxylic acid 2,2-dioxide), the selective mGlu2 PAM JNJ-42153605 (3-(cyclopropylmethyl)-7-(4-phenylpiperidin-1-yl)-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine) and the 5HT2A antagonist ritanserin in rodent models for antipsychotic activity and potential side effects, attempting to differentiate between the various compounds and mechanisms of action. In mice, JNJ-40411813, JNJ-42153605, and LY404039 inhibited spontaneous locomotion and phencyclidine- and scopolamine-induced but not d-amphetamine-induced hyperlocomotion; the 5HT2A antagonist ritanserin inhibited only spontaneous locomotion and phencyclidine-induced hyperlocomotion. As measured by 2-deoxyglucose uptake, all compounds reversed memantine-induced brain activation in mice. The two mGlu2 PAMs and LY404039, but not ritanserin, inhibited conditioned avoidance behavior in rats. Like ritanserin, the mGlu2 ligands antagonized 2,5-dimethoxy-4-methylamphetamine-induced head twitches in rats. LY404039 but not the mGlu2 PAMs impaired rotarod performance in rats and increased the acoustic startle response in mice. Our results show that although 5HT2A antagonism has effect in some models, mGlu2 receptor activation is sufficient for activity in several animal models of antipsychotic activity. The mGlu2 PAMs mimicked the in vivo pharmacodynamic effects observed with LY404039 except for effects on the rotarod and acoustic startle, suggesting that they produce a primary activity profile similar to that of the mGlu2/3 receptor agonist while they can be differentiated based on their secondary activity profile. The results are

  19. Discovery and SAR of a novel series of metabotropic glutamate receptor 5 positive allosteric modulators with high ligand efficiency

    PubMed Central

    Turlington, Mark; Noetzel, Meredith J.; Bridges, Thomas M.; Vinson, Paige N.; Steckler, Thomas; Lavreysen, Hilde; Mackie, Claire; Bartolomé-Nebreda, José M.; Conde-Ceide, Susana; Tong, Han Min; Macdonald, Gregor J.; Daniels, J. Scott; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    We report the optimization of a series of novel metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from a 5,6-bicyclic class of dihydropyrazolo[1,5-a]pyridin-4(5H)-ones containing a phenoxymethyl linker. Studies focused on a survey of nonamide containing hydrogen bond accepting (HBA) pharmacophore replacements. A highly potent and selective PAM, 2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyridin-4(5H)-one 11, VU0462054), bearing a simple ketone moiety, was identified (LE = 0.52, LELP = 3.2). In addition, hydroxyl, difluoro, ether, and amino variations were examined. Despite promising lead properties and exploration of alternative core heterocycles, linkers, and ketone replacements, oxidative metabolism and in vivo clearance remained problematic for the series. PMID:24961642

  20. Using NMR to Develop New Allosteric and Allo-Network Drugs.

    PubMed

    Smith, Robert E; Tran, Kevin; Richards, Kristy M; Luo, Rensheng

    2015-01-01

    NMR is becoming an important tool for developing new allosteric and allo-network drugs that bind to allosteric sites on enzymes, partially inhibiting them and causing fewer side effects than drugs already developed that target active sites. This is based on systems thinking, in which active enzymes and other proteins are known to be flexible and interact with each other. In other words, proteins can exist in an ensemble of different conformations whose populations are tunable. NMR is being used to find the pathways through which the effects of binding of an allosteric ligand propagate. There are NMR screening assays for studying ligand binding. This includes determining the changes in the spin lattice relaxation due to changes in the mobility of atoms involved in the binding, measuring magnetization transfer from the protein to the ligand by saturation difference transfer NMR (STD-NMR) and the transfer of bulk magnetization to the ligand by water-Ligand Observed via Gradient Spectroscopy, or waterLOGSY. The chemical shifts of (1)H and (15)N of some of the atoms in amino acids change when an allosteric ligand binds to a protein. So, (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra can be used to identify key amino acids and ligand binding sites. The NMR chemical shifts of amino acids affected by ligand binding form a network that can be characterized. Allosteric networks can be identified by chemical shift covariance analysis (CHESCA). This approach has been used recently to study the binding of new molecular entities (NMEs) to potentially therapeutic drug targets. PMID:26577663

  1. Temperature-induced inversion of allosteric phenomena.

    PubMed

    Braxton, B L; Tlapak-Simmons, V L; Reinhart, G D

    1994-01-01

    Two instances, involving the enzymes carbamoyl-phosphate synthetase from Escherichia coli and phosphofructokinase from Bacillus stearothermophilus, respectively, are described in which increasing temperature alone causes the actions of an allosteric ligand to change from inhibition to activation. In neither case are these effects due to a change in the activation energy of the enzyme catalyzed reaction induced by the allosteric ligand. Rather, they are due to temperature-dependent changes in the extent to which the binding of allosteric ligand modifies the affinity of the enzyme for substrate. The data can be readily explained by an analysis of the apparent delta H and delta S components of the coupling free energy, which quantitatively describe the actions of allosteric ligands that act in this manner. These observations underscore the shortcomings of expecting to explain the actions of an allosteric ligand solely by the structural perturbations that accompany the binding of an allosteric ligand such as those often revealed by x-ray crystallography. PMID:8276837

  2. Activation mechanism of AMPA receptors illuminated by complexes with cone snail toxin, allosteric potentiator and orthosteric agonists

    PubMed Central

    Chen, Lei; Dürr, Katharina L.; Gouaux, Eric

    2014-01-01

    AMPA-sensitive glutamate receptors are crucial to the structural and dynamical properties of the brain, to the development and function of the central nervous system and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. Here we determine multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator and orthosteric agonists at 3.8 – 4.1 Å resolution. We show how the toxin acts like a ‘straight jacket’ on the ligand-binding domain (LBD) “gating ring”, restraining the domains via both intra and interdimer cross links such that agonist-induced closure of the LBD “clamshells” is transduced into an iris-like expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in ‘pulling’ forces on the M3 helices that, in turn, are coupled to ion channel gating. PMID:25103405

  3. An allosteric photoredox catalyst inspired by photosynthetic machinery.

    PubMed

    Lifschitz, Alejo M; Young, Ryan M; Mendez-Arroyo, Jose; Stern, Charlotte L; McGuirk, C Michael; Wasielewski, Michael R; Mirkin, Chad A

    2015-03-30

    Biological photosynthetic machinery allosterically regulate light harvesting via conformational and electronic changes at the antenna protein complexes as a response to specific chemical inputs. Fundamental limitations in current approaches to regulating inorganic light-harvesting mimics prevent their use in catalysis. Here we show that a light-harvesting antenna/reaction centre mimic can be regulated by utilizing a coordination framework incorporating antenna hemilabile ligands and assembled via a high-yielding, modular approach. As in nature, allosteric regulation is afforded by coupling the conformational changes to the disruptions in the electrochemical landscape of the framework upon recognition of specific coordinating analytes. The hemilabile ligands enable switching using remarkably mild and redox-inactive inputs, allowing one to regulate the photoredox catalytic activity of the photosynthetic mimic reversibly and in situ. Thus, we demonstrate that bioinspired regulatory mechanisms can be applied to inorganic light-harvesting arrays displaying switchable catalytic properties and with potential uses in solar energy conversion and photonic devices.

  4. Molecular Recognition of the Catalytic Zinc(II) Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies

    PubMed Central

    Fischer, Thomas; Riedl, Rainer

    2016-01-01

    Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91). PMID:26938528

  5. Potentiating mGluR5 function with a positive allosteric modulator enhances adaptive learning.

    PubMed

    Xu, Jian; Zhu, Yongling; Kraniotis, Stephen; He, Qionger; Marshall, John J; Nomura, Toshihiro; Stauffer, Shaun R; Lindsley, Craig W; Conn, P Jeffrey; Contractor, Anis

    2013-08-01

    Metabotropic glutamate receptor 5 (mGluR5) plays important roles in modulating neural activity and plasticity and has been associated with several neuropathological disorders. Previous work has shown that genetic ablation or pharmacological inhibition of mGluR5 disrupts fear extinction and spatial reversal learning, suggesting that mGluR5 signaling is required for different forms of adaptive learning. Here, we tested whether ADX47273, a selective positive allosteric modulator (PAM) of mGluR5, can enhance adaptive learning in mice. We found that systemic administration of the ADX47273 enhanced reversal learning in the Morris Water Maze, an adaptive task. In addition, we found that ADX47273 had no effect on single-session and multi-session extinction, but administration of ADX47273 after a single retrieval trial enhanced subsequent fear extinction learning. Together these results demonstrate a role for mGluR5 signaling in adaptive learning, and suggest that mGluR5 PAMs represent a viable strategy for treatment of maladaptive learning and for improving behavioral flexibility.

  6. Potentiating mGluR5 function with a positive allosteric modulator enhances adaptive learning

    PubMed Central

    Xu, Jian; Zhu, Yongling; Kraniotis, Stephen; He, Qionger; Marshall, John J.; Nomura, Toshihiro; Stauffer, Shaun R.; Lindsley, Craig W.; Conn, P. Jeffrey; Contractor, Anis

    2013-01-01

    Metabotropic glutamate receptor 5 (mGluR5) plays important roles in modulating neural activity and plasticity and has been associated with several neuropathological disorders. Previous work has shown that genetic ablation or pharmacological inhibition of mGluR5 disrupts fear extinction and spatial reversal learning, suggesting that mGluR5 signaling is required for different forms of adaptive learning. Here, we tested whether ADX47273, a selective positive allosteric modulator (PAM) of mGluR5, can enhance adaptive learning in mice. We found that systemic administration of the ADX47273 enhanced reversal learning in the Morris Water Maze, an adaptive task. In addition, we found that ADX47273 had no effect on single-session and multi-session extinction, but administration of ADX47273 after a single retrieval trial enhanced subsequent fear extinction learning. Together these results demonstrate a role for mGluR5 signaling in adaptive learning, and suggest that mGluR5 PAMs represent a viable strategy for treatment of maladaptive learning and for improving behavioral flexibility. PMID:23869026

  7. Allosteric modulation of caspases.

    PubMed

    Häcker, Hans-Georg; Sisay, Mihiret Tekeste; Gütschow, Michael

    2011-11-01

    Caspases are proteolytic enzymes mainly involved in the induction and execution phases of apoptosis. This type of programmed cell death is an essential regulatory process required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis is attributed a key role in many human diseases, including neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. Allosteric modulation of the function of a protein occurs when the regulatory trigger, such as the binding of a small effector or inhibitor molecule, takes place some distance from the protein's active site. In recent years, several caspases have been identified that possess allosteric sites and binding of small molecule to these sites resulted in the modulation of enzyme activities. Regulation of caspase activity by small molecule allosteric modulators is believed to be of great therapeutic importance. In this review we give brief highlights on recent developments in identifying and characterizing natural and synthetic allosteric inhibitors as well as activators of caspases and discuss their potential in drug discovery and protein engineering. PMID:21807025

  8. Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

    PubMed Central

    2015-01-01

    Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs. PMID:26529344

  9. Allosteric Modulation of Purine and Pyrimidine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Göblyös, Anikó; IJzerman, Adriaan P.

    2011-01-01

    Among the purine and pyrimidine receptors, the discovery of small molecular allosteric modulators has been most highly advanced for the A1 and A3 ARs. These AR modulators have allosteric effects that are structurally separated from the orthosteric effects in SAR studies. The benzoylthiophene derivatives tend to act as allosteric agonists, as well as selective positive allosteric modulators (PAMs) of the A1 AR. A 2-amino-3-aroylthiophene derivative T-62 has been under development as a PAM of the A1 AR for the treatment of chronic pain. Several structurally distinct classes of allosteric modulators of the human A3 AR have been reported: 3-(2-pyridinyl)isoquinolines, 2,4-disubstituted quinolines, 1H-imidazo-[4,5-c]quinolin-4-amines, endocannabinoid 2-arachidonylglycerol and the food dye Brilliant Black BN. Site-directed mutagenesis of A1 and A3 ARs has identified residues associated with the allosteric effect, distinct from those that affect orthosteric binding. A few small molecular allosteric modulators have been reported for several of the P2X ligand-gated ion channels and the G protein-coupled P2Y receptor nucleotides. Metal ion modulation of the P2X receptors has been extensively explored. The allosteric approach to modulation of purine and pyrimidine receptors looks promising for development of drugs that are event-specific and site-specific in action. PMID:21586360

  10. Acetylcholine Receptor: An Allosteric Protein

    NASA Astrophysics Data System (ADS)

    Changeux, Jean-Pierre; Devillers-Thiery, Anne; Chemouilli, Phillippe

    1984-09-01

    The nicotine receptor for the neurotransmitter acetylcholine is an allosteric protein composed of four different subunits assembled in a transmembrane pentamer α 2β γ δ . The protein carries two acetylcholine sites at the level of the α subunits and contains the ion channel. The complete sequence of the four subunits is known. The membrane-bound protein undergoes conformational transitions that regulate the opening of the ion channel and are affected by various categories of pharmacologically active ligands.

  11. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

    PubMed Central

    Reichau, Sebastian; Blackmore, Nicola J.; Jiao, Wanting; Parker, Emily J.

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal. PMID:27128682

  12. Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

    PubMed Central

    Gregory, Karen J.

    2015-01-01

    The metabotropic glutamate (mGlu) receptors are a group of eight family C G protein–coupled receptors that are expressed throughout the central nervous system (CNS) and periphery. Within the CNS the different subtypes are found in neurons, both pre- and/or postsynaptically, where they mediate modulatory roles and in glial cells. The mGlu receptor family provides attractive targets for numerous psychiatric and neurologic disorders, with the majority of discovery programs focused on targeting allosteric sites, with allosteric ligands now available for all mGlu receptor subtypes. However, the development of allosteric ligands remains challenging. Biased modulation, probe dependence, and molecular switches all contribute to the complex molecular pharmacology exhibited by mGlu receptor allosteric ligands. In recent years we have made significant progress in our understanding of this molecular complexity coupled with an increased understanding of the structural basis of mGlu allosteric modulation. PMID:25808929

  13. Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

    PubMed Central

    Oikonomakos, N. G.; Tsitsanou, K. E.; Zographos, S. E.; Skamnaki, V. T.; Goldmann, S.; Bischoff, H.

    1999-01-01

    The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carries the Ser14(P), changes its conformation into a distorted 3(10) helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T-state GPb and produces further conformational changes, characteristic of a T'-like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (-AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 microM) with respect to glucose 1-phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 A and 2.1 A resolution, and refined to crystallographic R-values of 0.179 (R(free) = 0.230) and 0.189 (R(free) = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N

  14. Group I and group II metabotropic glutamate receptor allosteric modulators as novel potential antipsychotics

    PubMed Central

    Walker, Adam G.; Conn, P. Jeffrey

    2014-01-01

    Recently, there has been a shift in the schizophrenia field focusing on restoring glutamate signaling. Extensive preclinical data suggests that mGlu5 PAMs could have efficacy in all three symptom domains but there is concern of potential adverse effects. New insights into mechanisms underlying this toxicity may provide a path for discovery of safe mGlu5 PAMs. Genetic mutations in mGlu1 have been described in schizophrenics creating interest in this receptor as a therapeutic target. Preclinical data demonstrated the antipsychotic potential of of mGlu2/3 agonists but clinical trials were not successful. However, studies have suggested that mGlu2 is the subtype mediating antipsychotic effects and selective mGlu2 PAMs are now in clinical development. Finally, recent genetic studies suggest mGlu3 modulators may be pro-cognitive. PMID:25462291

  15. Group I and group II metabotropic glutamate receptor allosteric modulators as novel potential antipsychotics.

    PubMed

    Walker, Adam G; Conn, P Jeffrey

    2015-02-01

    Recently, there has been a shift in the schizophrenia field focusing on restoring glutamate signaling. Extensive preclinical data suggests that mGlu5 PAMs could have efficacy in all three symptom domains but there is concern of potential adverse effects. New insights into mechanisms underlying this toxicity may provide a path for discovery of safe mGlu5 PAMs. Genetic mutations in mGlu1 have been described in schizophrenics creating interest in this receptor as a therapeutic target. Preclinical data demonstrated the antipsychotic potential of mGlu2/3 agonists but clinical trials were not successful. However, studies have suggested that mGlu2 is the subtype mediating antipsychotic effects and selective mGlu2 PAMs are now in clinical development. Finally, recent genetic studies suggest mGlu3 modulators may be pro-cognitive. PMID:25462291

  16. Allosteric modulators of the extracellular calcium receptor.

    PubMed

    Nemeth, E F

    2013-01-01

    The extracellular calcium receptor (CaR) is a Family C G protein-coupled receptor that controls systemic Ca2+ homeostasis, largely by regulating the secretion of parathyroid hormone (PTH). Ligands that activate the CaR have been termed calcimimetics and are classified as either Type I (agonists) or Type II (allosteric activators) and effectively inhibit the secretion of PTH. CaR antagonists have been termed calcilytics and all act allosterically to stimulate secretion of PTH. The calcimimetic cinacalcet has been approved for treating parathyroid cancer and secondary hyperparathyroidism in patients on renal replacement therapy. Cinacalcet was the first allosteric modulator of a G proteincoupled receptor to achieve regulatory approval. This review will focus on the technologies used to discover and develop allosterically acting calcimimetics and calcilytics as novel therapies for bone and mineral-related disorders. PMID:24050279

  17. Seven transmembrane receptors as nature's prototype allosteric protein: de-emphasizing the geography of binding.

    PubMed

    Kenakin, Terry P

    2008-09-01

    The article in this issue by Redka et al. (p. 834) illustrates some interesting interactions between classified orthosteric (bind to the same recognition site as endogenous agonist) and allosteric (bind to a different site) ligands. Of particular interest are the methods used to deal with an obfuscating factor in these kinds of studies, namely the propensity of seven transmembrane receptors to form dimers and thus demonstrate allosteric effects through binding at the orthosteric site. The judicious use of kinetics to detect and quantify allosteric action also is demonstrated. The various unique properties of allosteric modulators are discussed in the context of the increasing prevalence of allosteric ligands as investigational drugs.

  18. Recent computational advances in the identification of allosteric sites in proteins.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Zhang, Jian

    2014-10-01

    Allosteric modulators have the potential to fine-tune protein functional activity. Therefore, the targeting of allosteric sites, as a strategy in drug design, is gaining increasing attention. Currently, it is not trivial to find and characterize new allosteric sites by experimental approaches. Alternatively, computational approaches are useful in helping researchers analyze and select potential allosteric sites for drug discovery. Here, we review state-of-the-art computational approaches directed at predicting putative allosteric sites in proteins, along with examples of successes in identifying allosteric sites utilizing these methods. We also discuss the challenges in developing reliable methods for predicting allosteric sites and tactics to resolve demanding tasks. PMID:25107670

  19. [Allosteric effect of serotonin and mianserin on the kinetics of specific [3H]-ligand binding to adrenergic and muscarinic receptors in the rat cerebral cortex membranes].

    PubMed

    Manukhin, B N; Nesterova, L A

    2015-01-01

    The effects of serotonin receptor activation (by serotonin) and inhibition (by mianserin) on the properties of the α1-, α2-adrenoreceptors, and muscarinic cholinergic receptors in subcellular membrane fractions from the rat cerebral cortex were studied. Experimental data on the kinetics of specific antagonists binding to adrenergic and muscarinic receptors were analyzed by graphical and mathematical methods. The results suggest the presence of allosteric (cross-talk) interaction. In the control, α1- and α2-adrenoreceptors were represented by a single pool, and muscarinic receptors, by two pools. Two pools of adrenoreceptors with different affinity were detected against the background of serotonin. It was found that mianserin induces the formation of two pools of only (α2-receptors and muscarinic receptors are represented by two pools differing in the main parameters, such as dissociation constants and adrenoreceptor concentrations, in the control and experimental groups. It was shown that the allosteric effect of serotonin and mianserin is manifested in the inhibition of muscarinic receptors. It was assumed that the adrenergic and cholinergic receptors exist as dimers. The interaction between the adrenergic, cholinergic, and serotonergic systems is likely to be implemented at the cell membrane level.

  20. Dopamine transporter ligands: recent developments and therapeutic potential.

    PubMed

    Runyon, Scott P; Carroll, F Ivy

    2006-01-01

    The dopamine transporter (DAT) is a target for the development of pharmacotherapies for a number of central disorders including Parkinson's disease, Alzheimer's disease, schizophrenia, Tourette's syndrome, Lesch-Nyhan disease, attention deficit hyperactivity disorder (ADHD), obesity, depression, and stimulant abuse as well as normal aging. Considerable effort continues to be devoted to the development of new ligands for the DAT. In this review, we present some of the more interesting ligands developed during the last few years from the 3-phenytropane, 1,4-dialkylpiperazine, phenylpiperidine, and benztropine classes of DAT uptake inhibitors as well as a few less studied miscellaneous DAT uptake inhibitors. Studies related to the therapeutic potential of some of the more studied compounds are presented. A few of the compounds have been studied as pharmacotherapies for Parkinson's disease, ADHD, and obesity. However, most of the drug discovery studies have been directed toward pharmacotherapies for stimulant abuse (mainly cocaine). A number of the compounds showed decreased cocaine maintained responding in rhesus monkeys trained to self-administer cocaine. One compound, GBR 12,909, was evaluated in a Phase 1 clinical trial. PMID:17017960

  1. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    PubMed

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  2. CGP7930: a positive allosteric modulator of the GABAB receptor.

    PubMed

    Adams, C L; Lawrence, A J

    2007-01-01

    CGP7930 (3-(3',5'-Di-tert-butyl-4'-hydroxy)phenyl-2,2-dimethylpropanol) is a positive allosteric modulator of the metabotropic GABAB receptor. CGP7930 has been found to modulate the GABAB receptor in the open, or high affinity, state increasing agonist affinity for the receptor and signal transduction efficacy following agonist stimulation. The GABAB heteromeric subunit B2, involved in signal transduction but not ligand binding, seems to be the site of action of CGP7930 and similar allosteric modulators. When administered alone in naïve animals, CGP7930 acts as an anxiolytic in rodents without other overt behavioral effects and has also been demonstrated to reduce self-administration of nicotine, cocaine, or alcohol in rodents, suggesting that "fine tuning" of the GABAB receptor by positive allosteric modulators may be able to regulate abuse of these drugs. Baclofen, the GABAB agonist, is currently finding use in treating addiction and various other disorders, but this can result in off-target effects and tolerance. CGP7930 when co-administered with baclofen enhances its potency, which could in theory minimize deleterious effects. Further study of CGP7930 is required, but this compound, and others like it, holds potential in a clinical setting. PMID:17894647

  3. Metabotropic glutamate receptor subtype 5: molecular pharmacology, allosteric modulation and stimulus bias.

    PubMed

    Sengmany, K; Gregory, K J

    2016-10-01

    The metabotropic glutamate receptor subtype 5 (mGlu5 ) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR-based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine-tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus-bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  4. Cooperative Electronic and Structural Regulation in a Bioinspired Allosteric Photoredox Catalyst.

    PubMed

    Lifschitz, Alejo M; Young, Ryan M; Mendez-Arroyo, Jose; McGuirk, C Michael; Wasielewski, Michael R; Mirkin, Chad A

    2016-09-01

    Herein, we report the first allosteric photoredox catalyst regulated via constructively coupled structural and electronic control. While often synergistically exploited in nature, these two types of control mechanisms have only been applied independently in the vast majority of allosteric enzyme mimics and receptors in the literature. By embedding a model of photosystem II in a supramolecular coordination complex that responds to chloride as an allosteric effector, we show that distance and electronic control of light harvesting can be married to maximize allosteric regulation of catalytic activity. This biomimetic system is composed of a Bodipy photoantenna, which is capable of transferring excited-state energy to a photoredox pair, wherein the excitation energy is used to generate a catalytically active charge-separated state. The structural aspect of allosteric regulation is achieved by toggling the coordination chemistry of an antenna-functionalized hemilabile ligand via partial displacement from a Rh(I) structual node using chloride. In doing so, the distance between the antenna and the central photoredox catalyst is increased, lowering the inherent efficiency of through-space energy transfer. At the same time, coordination of chloride lowers both the charge of the Rh(I) node and the reduction potential of the Rh(II/I) couple, to the extent that electronic quenching of the antenna excited state is possible via photoinduced electron transfer from the metal center. Compared to a previously developed system that operates solely via electronic regulation, the present system demonstrates that coupling electronic and structural approaches to allosteric regulation gives rise to improved switching ratios between catalytically active and inactive states. Contributions from both structural and electronic control mechanisms are probed via nuclear magnetic resonance, X-ray diffraction, electrochemical, spectroelectrochemical, and transient absorption studies. Overall

  5. 50th anniversary of the word "allosteric".

    PubMed

    Changeux, Jean-Pierre

    2011-07-01

    A brief historical account on the origin and meaning of the word "allosteric" is presented. The word was coined in an attempt to qualify the chemical mechanism of the feedback inhibition of bacterial enzymes by regulatory ligands. The data lead to the proposal that, at variance with the classical mechanism of mutual exclusion by steric hindrance, the inhibition takes place through an "allosteric" interaction between "no overlapping", stereospecifically distinct, sites for substrate and feedback inhibitor, mediated by a discrete reversible alteration of the molecular structure of the protein. PMID:21574197

  6. Melanoma cell galectin-1 ligands functionally correlate with malignant potential*

    PubMed Central

    Yazawa, Erika M.; Geddes-Sweeney, Jenna E.; Cedeno-Laurent, Filiberto; Walley, Kempland C.; Barthel, Steven R.; Opperman, Matthew J.; Liang, Jennifer; Lin, Jennifer Y.; Schatton, Tobias; Laga, Alvaro C.; Mihm, Martin C.; Qureshi, Abrar A.; Widlund, Hans R.; Murphy, George F.; Dimitroff, Charles J.

    2015-01-01

    Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening anti-tumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely-dysplastic nevi as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAMKD) or ST6GalNAc2-overexpressing (ST6O/E) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAMKD or ST6O/E melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1 – melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy. PMID:25756799

  7. Supramolecular Allosteric Cofacial Porphyrin Complexes

    SciTech Connect

    Oliveri, Christopher G.; Gianneschi, Nathan C.; Nguyen, Son Binh T.; Mirkin, Chad A.; Stern, Charlotte L.; Wawrzak, Zdzislaw; Pink, Maren

    2008-04-12

    Nature routinely uses cooperative interactions to regulate cellular activity. For years, chemists have designed synthetic systems that aim toward harnessing the reactivity common to natural biological systems. By learning how to control these interactions in situ, one begins to allow for the preparation of man-made biomimetic systems that can efficiently mimic the interactions found in Nature. To this end, we have designed a synthetic protocol for the preparation of flexible metal-directed supramolecular cofacial porphyrin complexes which are readily obtained in greater than 90% yield through the use of new hemilabile porphyrin ligands with bifunctional ether-phosphine or thioether-phosphine substituents at the 5 and 15 positions on the porphyrin ring. The resulting architectures contain two hemilabile ligand-metal domains (Rh{sup I} or Cu{sup I} sites) and two cofacially aligned porphyrins (Zn{sup II} sites), offering orthogonal functionalities and allowing these multimetallic complexes to exist in two states, 'condensed' or 'open'. Combining the ether-phosphine ligand with the appropriate Rh{sup I} or Cu{sup I} transition-metal precursors results in 'open' macrocyclic products. In contrast, reacting the thioether-phosphine ligand with RhI or CuI precursors yields condensed structures that can be converted into their 'open' macrocyclic forms via introduction of additional ancillary ligands. The change in cavity size that occurs allows these structures to function as allosteric catalysts for the acyl transfer reaction between X-pyridylcarbinol (where X = 2, 3, or 4) and 1-acetylimidazole. For 3- and 4-pyridylcarbinol, the 'open' macrocycle accelerates the acyl transfer reaction more than the condensed analogue and significantly more than the porphyrin monomer. In contrast, an allosteric effect was not observed for 2-pyridylcarbinol, which is expected to be a weaker binder and is unfavorably constrained inside the macrocyclic cavity.

  8. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  9. An allosteric photoredox catalyst inspired by photosynthetic machinery

    PubMed Central

    Lifschitz, Alejo M.; Young, Ryan M.; Mendez-Arroyo, Jose; Stern, Charlotte L.; McGuirk, C. Michael; Wasielewski, Michael R.; Mirkin, Chad A.

    2015-01-01

    Biological photosynthetic machinery allosterically regulate light harvesting via conformational and electronic changes at the antenna protein complexes as a response to specific chemical inputs. Fundamental limitations in current approaches to regulating inorganic light-harvesting mimics prevent their use in catalysis. Here we show that a light-harvesting antenna/reaction centre mimic can be regulated by utilizing a coordination framework incorporating antenna hemilabile ligands and assembled via a high-yielding, modular approach. As in nature, allosteric regulation is afforded by coupling the conformational changes to the disruptions in the electrochemical landscape of the framework upon recognition of specific coordinating analytes. The hemilabile ligands enable switching using remarkably mild and redox-inactive inputs, allowing one to regulate the photoredox catalytic activity of the photosynthetic mimic reversibly and in situ. Thus, we demonstrate that bioinspired regulatory mechanisms can be applied to inorganic light-harvesting arrays displaying switchable catalytic properties and with potential uses in solar energy conversion and photonic devices. PMID:25817586

  10. Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor.

    PubMed

    Miao, Yinglong; Goldfeld, Dahlia Anne; Moo, Ee Von; Sexton, Patrick M; Christopoulos, Arthur; McCammon, J Andrew; Valant, Celine

    2016-09-20

    Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies. PMID:27601651

  11. Muscarinic and Nicotinic Acetylcholine Receptor Agonists and Allosteric Modulators for the Treatment of Schizophrenia

    PubMed Central

    Jones, Carrie K; Byun, Nellie; Bubser, Michael

    2012-01-01

    Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M1 and M4) and nAChR (α7 and α2β4) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia. PMID:21956443

  12. Discovery and Development of Small Molecule Allosteric Modulators of Glycoprotein Hormone Receptors

    PubMed Central

    Nataraja, Selvaraj G.; Yu, Henry N.; Palmer, Stephen S.

    2015-01-01

    Glycoprotein hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are heterodimeric proteins with a common α-subunit and hormone-specific β-subunit. These hormones are dominant regulators of reproduction and metabolic processes. Receptors for the glycoprotein hormones belong to the family of G protein-coupled receptors. FSH receptor (FSHR) and LH receptor are primarily expressed in somatic cells in ovary and testis to promote egg and sperm production in women and men, respectively. TSH receptor is expressed in thyroid cells and regulates the secretion of T3 and T4. Glycoprotein hormones bind to the large extracellular domain of the receptor and cause a conformational change in the receptor that leads to activation of more than one intracellular signaling pathway. Several small molecules have been described to activate/inhibit glycoprotein hormone receptors through allosteric sites of the receptor. Small molecule allosteric modulators have the potential to be administered orally to patients, thus improving the convenience of treatment. It has been a challenge to develop a small molecule allosteric agonist for glycoprotein hormones that can mimic the agonistic effects of the large natural ligand to activate similar signaling pathways. However, in the past few years, there have been several promising reports describing distinct chemical series with improved potency in preclinical models. In parallel, proposal of new structural model for FSHR and in silico docking studies of small molecule ligands to glycoprotein hormone receptors provide a giant leap on the understanding of the mechanism of action of the natural ligands and new chemical entities on the receptors. This review will focus on the current status of small molecule allosteric modulators of glycoprotein hormone receptors, their effects on common signaling pathways in cells, their utility for clinical application as demonstrated in preclinical models

  13. Novel Allosteric Modulators of G Protein-coupled Receptors*

    PubMed Central

    Gentry, Patrick R.; Sexton, Patrick M.; Christopoulos, Arthur

    2015-01-01

    G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization. PMID:26100627

  14. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  15. Allosteric role of the large-scale domain opening in biological catch-binding

    NASA Astrophysics Data System (ADS)

    Pereverzev, Yuriy V.; Prezhdo, Oleg V.; Sokurenko, Evgeni V.

    2009-05-01

    The proposed model demonstrates the allosteric role of the two-domain region of the receptor protein in the increased lifetimes of biological receptor/ligand bonds subjected to an external force. The interaction between the domains is represented by a bounded potential, containing two minima corresponding to the attached and separated conformations of the two protein domains. The dissociative potential with a single minimum describing receptor/ligand binding fluctuates between deep and shallow states, depending on whether the domains are attached or separated. A number of valuable analytic expressions are derived and are used to interpret experimental data for two catch bonds. The P-selectin/P-selectin-glycoprotein-ligand-1 (PSGL-1) bond is controlled by the interface between the epidermal growth factor (EGF) and lectin domains of P-selectin, and the type 1 fimbrial adhesive protein (FimH)/mannose bond is governed by the interface between the lectin and pilin domains of FimH. Catch-binding occurs in these systems when the external force stretches the receptor proteins and increases the interdomain distance. The allosteric effect is supported by independent measurements, in which the domains are kept separated by attachment of another ligand. The proposed model accurately describes the experimentally observed anomalous behavior of the lifetimes of the P-selectin/PSGL-1 and FimH/mannose complexes as a function of applied force and provides valuable insights into the mechanism of catch-binding.

  16. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    NASA Technical Reports Server (NTRS)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  17. New multifunctional ligands for potential use in the design therapeutic or diagnostic radiopharmaceutical imaging agents

    DOEpatents

    Katti, Kattesh V.; Volkert, Wynn A.; Ketring, Alan R.; Singh, Prahlad R.

    1997-01-01

    A class of diagnostic and therapeutic compounds derived from phosphinimines that include ligands containing either a single phosphinimine functionality or both a phosphinimine group and a phosphine or arsine group, or an aminato group, or a second phosphinimine moiety. These phosphinimine ligands are complexed to early transition metal radionuclides (e.g. .sup.99m Tc or .sup.186 Re/.sup.188 Re) or late transition metals (e.g., .sup.105 Rh or .sup.109 Pd). The complexes with these metals .sup.186 Re/.sup.188 Re, .sup.99m Tc and .sup.109 Pd exhibit a high in vitro and high in vivo stability. The complexes are formed in high yields and can be neutral or charged. These ligands can also be used to form stable compounds with paramagnetic transition metals (e.g. Fe and Mn) for potential use as MRI contrast agents. Applications for the use of ligands and making the ligands are also disclosed.

  18. A Random Forest Model for Predicting Allosteric and Functional Sites on Proteins.

    PubMed

    Chen, Ava S-Y; Westwood, Nicholas J; Brear, Paul; Rogers, Graeme W; Mavridis, Lazaros; Mitchell, John B O

    2016-04-01

    We created a computational method to identify allosteric sites using a machine learning method trained and tested on protein structures containing bound ligand molecules. The Random Forest machine learning approach was adopted to build our three-way predictive model. Based on descriptors collated for each ligand and binding site, the classification model allows us to assign protein cavities as allosteric, regular or orthosteric, and hence to identify allosteric sites. 43 structural descriptors per complex were derived and were used to characterize individual protein-ligand binding sites belonging to the three classes, allosteric, regular and orthosteric. We carried out a separate validation on a further unseen set of protein structures containing the ligand 2-(N-cyclohexylamino) ethane sulfonic acid (CHES). PMID:27491922

  19. Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions

    SciTech Connect

    Liu, Wei; Chun, Eugene; Thompson, Aaron A.; Chubukov, Pavel; Xu, Fei; Katritch, Vsevolod; Han, Gye Won; Roth, Christopher B.; Heitman, Laura H.; IJzerman, Adriaan P.; Cherezov, Vadim; Stevens, Raymond C.

    2012-08-31

    Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A{sub 2A} adenosine receptor by replacing its third intracellular loop with apocytochrome b{sub 562}RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp{sup 2.50}. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

  20. An Allosteric Circuit in Caspase-1

    SciTech Connect

    Datta, D.; Scheer, J.M.; Romanowski, M.J.; Wells, J.A.

    2009-05-14

    Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface approximately 15 A from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them-Arg286 and Glu390, which form a salt bridge-have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k(cat)/K(m)). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k(cat)/K(m). X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or 'hot wire' that promotes site-to-site coupling.

  1. Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content.

    PubMed

    Arias, Hugo R; Ravazzini, Federica; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Feuerbach, Dominik; Boffi, Juan C; Draczkowski, Piotr; Montag, Dirk; Brown, Brandon M; Elgoyhen, Ana Belén; Jozwiak, Krzysztof; Puia, Giulia

    2016-07-01

    The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

  2. Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content.

    PubMed

    Arias, Hugo R; Ravazzini, Federica; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Feuerbach, Dominik; Boffi, Juan C; Draczkowski, Piotr; Montag, Dirk; Brown, Brandon M; Elgoyhen, Ana Belén; Jozwiak, Krzysztof; Puia, Giulia

    2016-07-01

    The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets. PMID:27129924

  3. Synthesis of Phthalimide Derivatives as Potential PPAR-γ Ligands.

    PubMed

    Eom, So Hyeon; Liu, Sen; Su, Mingzhi; Noh, Tae Hwan; Hong, Jongki; Kim, Nam Deuk; Chung, Hae Young; Yang, Min Hye; Jung, Jee H

    2016-01-01

    Paecilocin A, a phthalide derivative isolated from the jellyfish-derived fungus Paecilomyces variotii, activates PPAR-γ (Peroxisome proliferator-activated receptor gamma) in rat liver Ac2F cells. Based on a SAR (Structure-activity relationships) study and in silico analysis of paecilocin A-mimetic derivatives, additional N-substituted phthalimide derivatives were synthesized and evaluated for PPAR-γ agonistic activity in both murine liver Ac2F cells and in human liver HepG2 cells by luciferase assay, and for adipogenic activity in 3T3-L1 cells. Docking simulation indicated PD6 was likely to bind most strongly to the ligand binding domain of PPAR-γ by establishing crucial H-bonds with key amino acid residues. However, in in vitro assays, PD1 and PD2 consistently displayed significant PPAR-γ activation in Ac2F and HepG2 cells, and adipogenic activity in 3T3-L1 preadipocytes. PMID:27338418

  4. Synthesis of Phthalimide Derivatives as Potential PPAR-γ Ligands

    PubMed Central

    Eom, So Hyeon; Liu, Sen; Su, Mingzhi; Noh, Tae Hwan; Hong, Jongki; Kim, Nam Deuk; Chung, Hae Young; Yang, Min Hye; Jung, Jee H.

    2016-01-01

    Paecilocin A, a phthalide derivative isolated from the jellyfish-derived fungus Paecilomyces variotii, activates PPAR-γ (Peroxisome proliferator-activated receptor gamma) in rat liver Ac2F cells. Based on a SAR (Structure-activity relationships) study and in silico analysis of paecilocin A-mimetic derivatives, additional N-substituted phthalimide derivatives were synthesized and evaluated for PPAR-γ agonistic activity in both murine liver Ac2F cells and in human liver HepG2 cells by luciferase assay, and for adipogenic activity in 3T3-L1 cells. Docking simulation indicated PD6 was likely to bind most strongly to the ligand binding domain of PPAR-γ by establishing crucial H-bonds with key amino acid residues. However, in in vitro assays, PD1 and PD2 consistently displayed significant PPAR-γ activation in Ac2F and HepG2 cells, and adipogenic activity in 3T3-L1 preadipocytes. PMID:27338418

  5. Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics Approach

    PubMed Central

    Ivetac, Anthony; Andrew McCammon, J

    2010-01-01

    To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the β1 (β1AR) and β2 (β2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify ‘hot spots’ with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 μs. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies. PMID:20626410

  6. Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.

    PubMed

    Laprairie, Robert B; Kulkarni, Abhijit R; Kulkarni, Pushkar M; Hurst, Dow P; Lynch, Diane; Reggio, Patricia H; Janero, David R; Pertwee, Roger G; Stevenson, Lesley A; Kelly, Melanie E M; Denovan-Wright, Eileen M; Thakur, Ganesh A

    2016-06-15

    One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse

  7. Allosteric Coupling in the Bacterial Adhesive Protein FimH*

    PubMed Central

    Rodriguez, Victoria B.; Kidd, Brian A.; Interlandi, Gianluca; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2013-01-01

    The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH. PMID:23821547

  8. Allosteric dominance in carbamoyl phosphate synthetase.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1999-02-01

    A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 degreesC. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed. PMID:9931004

  9. Potential Toxicity of Up-Converting Nanoparticles Encapsulated with a Bilayer Formed by Ligand Attraction

    PubMed Central

    2015-01-01

    The cellular toxicity of nanoparticles that were capped with a bilayered ligand was studied using an up-converting (UC) phosphor material as a representative nanoparticle (NP). The results indicate that although UC NPs are known to be nontoxic, the toxicity of the NPs depends strongly on ligand coordination conditions, in addition to the other commonly known parameters such as size, structure, surface charge etc. Oleate-capped hydrophobic NaYF4:Yb,Er NPs were surface modified to yield three extreme conditions: bare particles that were stripped of the oleate ligands; particles with covalently bound poly(ethylene glycol) (PEG) ligands; and particles with an bilayer of PEG-oleate ligands using the oleate surface group that was remained after synthesis. It was found that the bare particles and the covalent PEG NPs induced little toxicity. However, particles that were rendered biocompatible by forming a bilayer with an amphiphilic ligand (i.e., PEG-oleate) resulted in significant cell toxicity. These findings strongly suggest that the PEG-oleate group dissociated from the bilayered oleate-capped NPs, resulting in significant toxicity by exposing the hydrophobic oleate-capped NPs to the cell. Based on results with bare particles, the NaLnF4:Yb,Er (Ln = Y, Gd) up-converting phosphors are essentially less-toxic. Capping and functionalizing these particles with ligand intercalation may, however, not be a suitable method for rendering the NPs suitable for bioapplication as the ligand can potentially dissociate upon cellular interaction, leading to significant toxicity. PMID:24971524

  10. Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor

    PubMed Central

    Guo, Dong; Venhorst, Suzanne N; Massink, Arnault; van Veldhoven, Jacobus P D; Vauquelin, Georges; IJzerman, Adriaan P; Heitman, Laura H

    2014-01-01

    Background and Purpose Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well. PMID:25040887

  11. ASD v3.0: unraveling allosteric regulation with structural mechanisms and biological networks

    PubMed Central

    Shen, Qiancheng; Wang, Guanqiao; Li, Shuai; Liu, Xinyi; Lu, Shaoyong; Chen, Zhongjie; Song, Kun; Yan, Junhao; Geng, Lv; Huang, Zhimin; Huang, Wenkang; Chen, Guoqiang; Zhang, Jian

    2016-01-01

    Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries. PMID:26365237

  12. Phthalocyanines: a new class of G-quadruplex-ligands with many potential applications.

    PubMed

    Yaku, Hidenobu; Fujimoto, Takeshi; Murashima, Takashi; Miyoshi, Daisuke; Sugimoto, Naoki

    2012-06-25

    A G-quadruplex is a four-stranded DNA structure featuring stacked guanine tetrads, G-quartets. Formation of a G-quadruplex in telomere DNA can inhibit telomerase activity; therefore, development of G-quadruplex-ligands, which induce and/or stabilize G-quadruplexes, has become an area of great interest. Phthalocyanine derivatives have substantial potential as high-affinity G-quadruplex-ligands because these planar chromophores are similar in size and shape to the G-quartets. Here, we focus on the latest findings on phthalocyanine derivatives as G-quadruplex-ligands, and discuss the mechanisms by which phthalocyanines bind to G-quadruplexes with high affinity and selectivity. We also discuss potential biomedical and organic electronic applications of phthalocyanines that are dependent on their photophysical properties.

  13. Phthalocyanines: a new class of G-quadruplex-ligands with many potential applications.

    PubMed

    Yaku, Hidenobu; Fujimoto, Takeshi; Murashima, Takashi; Miyoshi, Daisuke; Sugimoto, Naoki

    2012-06-25

    A G-quadruplex is a four-stranded DNA structure featuring stacked guanine tetrads, G-quartets. Formation of a G-quadruplex in telomere DNA can inhibit telomerase activity; therefore, development of G-quadruplex-ligands, which induce and/or stabilize G-quadruplexes, has become an area of great interest. Phthalocyanine derivatives have substantial potential as high-affinity G-quadruplex-ligands because these planar chromophores are similar in size and shape to the G-quartets. Here, we focus on the latest findings on phthalocyanine derivatives as G-quadruplex-ligands, and discuss the mechanisms by which phthalocyanines bind to G-quadruplexes with high affinity and selectivity. We also discuss potential biomedical and organic electronic applications of phthalocyanines that are dependent on their photophysical properties. PMID:22590705

  14. Studying the allosteric energy cycle by isothermal titration calorimetry.

    PubMed

    Martinez-Julvez, Marta; Abian, Olga; Vega, Sonia; Medina, Milagros; Velazquez-Campoy, Adrian

    2012-01-01

    Isothermal titration calorimetry (ITC) is a powerful biophysical technique which allows a complete thermodynamic characterization of protein interactions with other molecules. The possibility of dissecting the Gibbs energy of interaction into its enthalpic and entropic contributions, as well as the detailed additional information experimentally accessible on the intermolecular interactions (stoichiometry, cooperativity, heat capacity changes, and coupled equilibria), make ITC a suitable technique for studying allosteric interactions in proteins. Two experimental methodologies for the characterization of allosteric heterotropic ligand interactions by ITC are described in this chapter, illustrated with two proteins with markedly different structural and functional features: a photosynthetic electron transfer protein and a drug target viral protease.

  15. Controlling allosteric networks in proteins

    NASA Astrophysics Data System (ADS)

    Dokholyan, Nikolay

    2013-03-01

    We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

  16. Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

    PubMed

    Yilmaz, Oya Gursoy; Olmez, Elif Ozkirimli; Ulgen, Kutlu O

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1.

  17. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  18. Novel properties and potential applications of functional ligand-modified polyoxotitanate cages.

    PubMed

    Li, Ning; Matthews, Peter D; Luo, He-Kuan; Wright, Dominic S

    2016-09-28

    Functional ligand-modified polyoxotitanate (L-POT) cages of the general type [TixOy(OR)z(L)m] (OR = alkoxide, L = functional ligand) can be regarded as molecular fragments of surface-sensitized solid-state TiO2, and are of value as models for studying the interfacial charge and energy transfer between the bound functional ligands and a bulk semiconductor surface. These L-POTs have also had a marked impact in many other research fields, such as single-source precursors for TiO2 deposition, inorganic-organic hybrid material construction, photocatalysis, photoluminescence, asymmetric catalysis and gas adsorption. Their atomically well-defined structures provide the basis for the understanding of structure/property relationships and ultimately for the rational design of new cages targeting specific uses. This highlight focuses on recent advances in L-POTs research, with emphasis on their novel properties and potential applications. PMID:27332621

  19. Use of natural AhR ligands as potential therapeutic modalities against inflammatory disorders

    PubMed Central

    Busbee, Philip B; Rouse, Michael; Nagarkatti, Mitzi; Nagarkatti, Prakash S

    2014-01-01

    The aim of this review is to discuss research involving ligands for the aryl hydrocarbon receptor (AhR) and their role in immunomodulation. While activation of the AhR is well known for its ability to regulate the biochemical and toxic effects of environmental chemicals, more recently an exciting discovery has been made indicating that AhR ligation can also regulate T-cell differentiation, specifically through activation of Foxp3+ regulatory T cells (Tregs) and downregulation of the proinflammatory Th17 cells. Such findings have opened new avenues of research on the possibility of targeting the AhR to treat inflammatory and autoimmune diseases. Specifically, this review will discuss the current research involving natural and dietary AhR ligands. In addition, evidence indicating the potential use of these ligands in regulating inflammation in various diseases will be highlighted. The importance of the AhR in immunological processes can be illustrated by expression of this receptor on a majority of immune cell types. In addition, AhR signaling pathways have been reported to influence a number of genes responsible for mediating inflammation and other immune responses. As interest in the AhR and its ligands increases, it seems prudent to consolidate current research on the contributions of these ligands to immune regulation during the course of inflammatory diseases. PMID:23731446

  20. Potential uses of silica-bonded macrocyclic ligands for separation of metal ions from nuclear waste

    SciTech Connect

    Bruening, R.L.; Camaioni, D.M.; Colton, N.G.; Morrey, J.R.

    1991-11-01

    This paper explores the potential of a relatively new separation material that is obtained by covalently binding macrocyclic ligands to silica gel. Fortunately, neutral macrocyclic ligands can be bound to silica gel such that metal binding constants do not differ significantly from the binding constants of the free ligands so that selectivities of free macrocyclic ligands can be used in designing silica-bound materials with appropriate selectivities. Accordingly, macrocyclic ligands known to have selectivities for Pd{sup +2}, Ag{sup +}, Ru{sup +3}, Sr{sup +2}, and Cs{sup +} were covalently bound to silica gel. These materials were then tested for their ability to separate these ions from a synthetic test solution representative of a nuclear process waste stream. Cs{sup +} and Sr{sup +2} are of interest because their radioactive isotopes are major radioactive constituents of defense nuclear wastes accumulated at the Hanford site. Removal of precious metals such as Pd{sup +2}, Ag{sup +} and Ru{sup +3} present in nuclear defense waste are of interest not just because of their obvious economic value, but also because these metals may hinder the waste vitrification process for confining radionuclides.

  1. New multifunctional ligands for potential use in the design therapeutic or diagnostic radiopharmaceutical imaging agents

    DOEpatents

    Katti, K.V.; Volkert, W.A.; Ketring, A.R.; Singh, P.R.

    1997-02-11

    A class of diagnostic and therapeutic compounds are derived from phosphinimines that include ligands containing either a single phosphinimine functionality or both a phosphinimine group and a phosphine or arsine group, or an aminato group, or a second phosphinimine moiety. These phosphinimine ligands are complexed to early transition metal radionuclides (e.g., {sup 99m}Tc or {sup 186}Re/{sup 188}Re) or late transition metals (e.g., {sup 105}Rh or {sup 109}Pd). The complexes with these metals {sup 186}Re/{sup 188}Re, {sup 99m}Tc and {sup 109}Pd exhibit a high in vitro and high in vivo stability. The complexes are formed in high yields and can be neutral or charged. These ligands can also be used to form stable compounds with paramagnetic transition metals (e.g., Fe and Mn) for potential use as MRI contrast agents. Applications for the use of ligands and making the ligands are also disclosed.

  2. Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

    PubMed Central

    Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

    2015-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

  3. Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor.

    PubMed

    Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U Helena; Tresadern, Gary; Ulens, Chris

    2015-05-12

    The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

  4. Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.

    PubMed

    Williams, Dustin K; Wang, Jingyi; Papke, Roger L

    2011-10-15

    Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.

  5. Allosteric effects of carbamoyl phosphate synthetase from Escherichia coli are entropy-driven.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1996-09-10

    When catalyzing the formation of MgATP and carbamate from MgADP and carbamoyl phosphate, Escherichia coli carbamoyl phosphate synthetase (CPS) binds MgADP with a large negative change in heat capacity. The magnitude of this heat capacity change is not appreciably altered by the presence of a saturating concentration of either the allosteric activator ornithine or the inhibitor UMP despite the substantial and opposing effects these ligands have on the binding affinity for MgADP. By contrast, no detectable change in heat capacity is associated with the thermodynamic coupling between MgADP and either ornithine or UMP. The sign of the apparently constant enthalpic and entropic contributions to the coupling free energy for each of these ligands is opposite that of the coupling free energy, indicating that the observed allosteric phenomenology is in net opposed by the enthalpy of the interaction and instead arises from a change in entropy of the system. IMP produces only a very small allosteric effect as indicated by a near-zero value for the MgADP-IMP coupling free energy. However, the enthalpic and entropic contributions are individually larger in absolute value for the IMP coupling than for those pertaining to the other allosteric ligands, and entropy dominates the coupling free energy above 36 degrees C, causing IMP to become an activator at high temperature. In addition, the sign of the coupling enthalpy and entropy for IMP has the same sign as the coupling enthalpy and entropy produced by ornithine, suggesting that IMP and ornithine may similarly influence the enzyme at a molecular level despite binding to different allosteric sites on the enzyme. The data are consistent with a model in which the actions of the allosteric ligands arise primarily from changes in the conformational degeneracy introduced by each ligand. With this model, one can also rationalize the failure of these allosteric ligands to substantially influence kcat. PMID:8794775

  6. Effectors of hemoglobin. Separation of allosteric and affinity factors.

    PubMed Central

    Marden, M C; Bohn, B; Kister, J; Poyart, C

    1990-01-01

    The relative contributions of the allosteric and affinity factors toward the change in p50 have been calculated for a series of effectors of hemoglobin (Hb). Shifts in the ligand affinity of deoxy Hb and the values for 50% ligand saturation (p50) were obtained from oxygen equilibrium data. Because the high-affinity parameters (liganded conformation) are poorly determined from the equilibrium curves, they were determined from kinetic measurements of the association and dissociation rates with CO as ligand. The CO on-rates were obtained by flash photolysis measurements. The off-rates were determined from the rate of oxidation of HbCO by ferricyanide, or by replacement of CO with NO. The partition function of fully liganded hemoglobin for oxygen and CO is only slightly changed by the effectors. Measurements were made in the presence of the effectors 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), and two recently synthesized derivatives of Bzf (LR16 and L35). Values of p50 change by over a factor of 60; the on-rates decrease by nearly a factor of 8, with little change in the off-rates for the liganded conformation. The data indicate that both allosteric and affinity parameters are changed by the effectors; the changes in ligand affinity represent the larger contribution toward shifts in p50. PMID:2306490

  7. Molecular dynamics simulations and modelling of the residue interaction networks in the BRAF kinase complexes with small molecule inhibitors: probing the allosteric effects of ligand-induced kinase dimerization and paradoxical activation.

    PubMed

    Verkhivker, G M

    2016-10-20

    Protein kinases are central to proper functioning of cellular networks and are an integral part of many signal transduction pathways. The family of protein kinases represents by far the largest and most important class of therapeutic targets in oncology. Dimerization-induced activation has emerged as a common mechanism of allosteric regulation in BRAF kinases, which play an important role in growth factor signalling and human diseases. Recent studies have revealed that most of the BRAF inhibitors can induce dimerization and paradoxically stimulate enzyme transactivation by conferring an active conformation in the second monomer of the kinase dimer. The emerging connections between inhibitor binding and BRAF kinase domain dimerization have suggested a molecular basis of the activation mechanism in which BRAF inhibitors may allosterically modulate the stability of the dimerization interface and affect the organization of residue interaction networks in BRAF kinase dimers. In this work, we integrated structural bioinformatics analysis, molecular dynamics and binding free energy simulations with the protein structure network analysis of the BRAF crystal structures to determine dynamic signatures of BRAF conformations in complexes with different types of inhibitors and probe the mechanisms of the inhibitor-induced dimerization and paradoxical activation. The results of this study highlight previously unexplored relationships between types of BRAF inhibitors, inhibitor-induced changes in the residue interaction networks and allosteric modulation of the kinase activity. This study suggests a mechanism by which BRAF inhibitors could promote or interfere with the paradoxical activation of BRAF kinases, which may be useful in informing discovery efforts to minimize the unanticipated adverse biological consequences of these therapeutic agents.

  8. Mapping the Binding of GluN2B-Selective N-Methyl-d-aspartate Receptor Negative Allosteric Modulators

    PubMed Central

    Yuan, Hongjie; Karakas, Erkan; Geballe, Matthew; Furukawa, Hiro; Liotta, Dennis C.; Snyder, James P.; Traynelis, Stephen F.

    2012-01-01

    We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists. PMID:22596351

  9. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

    Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.

  10. Induced Long-Range Attractive Potentials of Human Serum Albumin by Ligand Binding

    SciTech Connect

    Sato, Takaaki; Komatsu, Teruyuki; Nakagawa, Akito; Tsuchida, Eishun

    2007-05-18

    Small-angle x-ray scattering and dielectric spectroscopy investigation on the solutions of recombinant human serum albumin and its heme hybrid revealed that heme incorporation induces a specific long-range attractive potential between protein molecules. This is evidenced by the enhanced forward intensity upon heme binding, despite no hindrance to rotatory Brownian motion, unbiased colloid osmotic pressure, and discontiguous nearest-neighbor distance, confirming monodispersity of the proteins. The heme-induced potential may play a trigger role in recognition of the ligand-filled human serum albumins in the circulatory system.

  11. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

  12. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  13. Chemical potential of bound ligand, an important parameter for free energy transduction

    PubMed Central

    Tanford, Charles

    1981-01-01

    The chemical potential (μL,b) of a ligand L bound to a protein or enzyme can be rigorously defined, and this paper describes some of its properties in relation to other thermodynamic parameters, with emphasis on thermodynamic parameters that may be used in the elucidation of the mechanism of biological free energy transduction. Free energy transduction involves the transfer of free energy from one molecule to another, and the actual transfer may often occur while both molecules are bound to the transducer enzyme, which means that μL,b for one bound ligand increases at the expense of μL,b for the other. The free energy change for the overall reaction may be very small, and it is not possible to express the phenomenon of transfer, in thermodynamic terms, without the explicit use of μL,b as a parameter. PMID:16592948

  14. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

    PubMed

    Kahsai, Alem W; Wisler, James W; Lee, Jungmin; Ahn, Seungkirl; Cahill Iii, Thomas J; Dennison, S Moses; Staus, Dean P; Thomsen, Alex R B; Anasti, Kara M; Pani, Biswaranjan; Wingler, Laura M; Desai, Hemant; Bompiani, Kristin M; Strachan, Ryan T; Qin, Xiaoxia; Alam, S Munir; Sullenger, Bruce A; Lefkowitz, Robert J

    2016-09-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs. PMID:27398998

  15. Functional anatomy of an allosteric protein

    NASA Astrophysics Data System (ADS)

    Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

    2013-12-01

    Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2-αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

  16. Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2

    PubMed Central

    Rastelli, Giulio; Anighoro, Andrew; Chripkova, Martina; Carrassa, Laura; Broggini, Massimo

    2014-01-01

    Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75–1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells. PMID:24911186

  17. Allosteric proteins as logarithmic sensors

    PubMed Central

    Olsman, Noah; Goentoro, Lea

    2016-01-01

    Many sensory systems, from vision and hearing in animals to signal transduction in cells, respond to fold changes in signal relative to background. Responding to fold change requires that the system senses signal on a logarithmic scale, responding identically to a change in signal level from 1 to 3, or from 10 to 30. It is an ongoing search in the field to understand the ways in which a logarithmic sensor can be implemented at the molecular level. In this work, we present evidence that logarithmic sensing can be implemented with a single protein, by means of allosteric regulation. Specifically, we find that mathematical models show that allosteric proteins can respond to stimuli on a logarithmic scale. Next, we present evidence from measurements in the literature that some allosteric proteins do operate in a parameter regime that permits logarithmic sensing. Finally, we present examples suggesting that allosteric proteins are indeed used in this capacity: allosteric proteins play a prominent role in systems where fold-change detection has been proposed. This finding suggests a role as logarithmic sensors for the many allosteric proteins across diverse biological processes. PMID:27410043

  18. Allosteric modulation in monomers and oligomers of a G protein-coupled receptor

    PubMed Central

    Shivnaraine, Rabindra V; Kelly, Brendan; Sankar, Krishana S; Redka, Dar'ya S; Han, Yi Rang; Huang, Fei; Elmslie, Gwendolynne; Pinto, Daniel; Li, Yuchong; Rocheleau, Jonathan V; Gradinaru, Claudiu C; Ellis, John; Wells, James W

    2016-01-01

    The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations. DOI: http://dx.doi.org/10.7554/eLife.11685.001 PMID:27151542

  19. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  20. Synthesis and biological evaluation of negative allosteric modulators of the Kv11.1(hERG) channel.

    PubMed

    Yu, Zhiyi; van Veldhoven, Jacobus P D; 't Hart, Ingrid M E; Kopf, Adrian H; Heitman, Laura H; IJzerman, Adriaan P

    2015-12-01

    We synthesized and evaluated a series of compounds for their allosteric modulation at the Kv11.1 (hERG) channel. Most compounds were negative allosteric modulators of [(3)H]dofetilide binding to the channel, in particular 7f, 7h-j and 7p. Compounds 7f and 7p were the most potent negative allosteric modulators amongst all ligands, significantly increasing the dissociation rate of dofetilide in the radioligand kinetic binding assay, while remarkably reducing the affinities of dofetilide and astemizole in a competitive displacement assay. Additionally, both 7f and 7p displayed peculiar displacement characteristics with Hill coefficients significantly distinct from unity as shown by e.g., dofetilide, further indicative of their allosteric effects on dofetilide binding. Our findings in this investigation yielded several promising negative allosteric modulators for future functional and clinical research with respect to their antiarrhythmic propensities, either alone or in combination with known Kv11.1 blockers. PMID:26519929

  1. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  2. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  3. Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators.

    PubMed

    Lisi, George P; Manley, Gregory A; Hendrickson, Heidi; Rivalta, Ivan; Batista, Victor S; Loria, J Patrick

    2016-07-01

    The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26- to 4,900-fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Furthermore, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity. PMID:27238967

  4. Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease.

    PubMed

    Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

    2016-01-01

    At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD.

  5. Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson's Disease? The Potential Role of Zolpidem in the Treatment of Parkinson's Disease

    PubMed Central

    Daniele, Antonio; Panza, Francesco; Greco, Antonio; Logroscino, Giancarlo; Seripa, Davide

    2016-01-01

    At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD. PMID:27293955

  6. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    PubMed Central

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  7. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  8. Nicotinic acetylcholine receptor ligands; a patent review (2006-2011)

    PubMed Central

    Gündisch, Daniela; Eibl, Christoph

    2012-01-01

    Introduction Nicotinic acetylcholine receptors (nAChRs), pentameric ligand-gated cation channels, are potential targets for the development of therapeutics for a variety of disease states. Areas covered This article is reviewing recent advances in the development of small molecule ligands for diverse nAChR subtypes and is a continuation of an earlier review in this journal. Expert opinion The development of nAChR ligands with preference for α4β2 or α7 subtypes for the treatment of CNS disorders are in the most advanced developmental stage. In addition, there is a fast growing interest to generate so-called PAMs, positive allosteric modulators, to influence the channels’ functionalities. PMID:22098319

  9. Characterization of a Ligand Binding Site in the Human Transient Receptor Potential Ankyrin 1 Pore

    PubMed Central

    Klement, Göran; Eisele, Lina; Malinowsky, David; Nolting, Andreas; Svensson, Mats; Terp, Gitte; Weigelt, Dirk; Dabrowski, Michael

    2013-01-01

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule. PMID:23442958

  10. Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore.

    PubMed

    Klement, Göran; Eisele, Lina; Malinowsky, David; Nolting, Andreas; Svensson, Mats; Terp, Gitte; Weigelt, Dirk; Dabrowski, Michael

    2013-02-19

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

  11. Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

    NASA Astrophysics Data System (ADS)

    Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

    2016-01-01

    Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

  12. The qEEG Signature of Selective NMDA NR2B Negative Allosteric Modulators; A Potential Translational Biomarker for Drug Development

    PubMed Central

    Keavy, Deborah; Bristow, Linda J.; Sivarao, Digavalli V.; Batchelder, Margaret; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E.; Weed, Michael R.

    2016-01-01

    The antidepressant activity of the N-methyl-D-aspartate (NMDA) receptor channel blocker, ketamine, has led to the investigation of negative allosteric modulators (NAMs) selective for the NR2B receptor subtype. The clinical development of NR2B NAMs would benefit from a translational pharmacodynamic biomarker that demonstrates brain penetration and functional inhibition of NR2B receptors in preclinical species and humans. Quantitative electroencephalography (qEEG) is a translational measure that can be used to demonstrate pharmacodynamic effects across species. NMDA receptor channel blockers, such as ketamine and phencyclidine, increase the EEG gamma power band, which has been used as a pharmacodynamic biomarker in the development of NMDA receptor antagonists. However, detailed qEEG studies with ketamine or NR2B NAMs are lacking in nonhuman primates. The aim of the present study was to determine the effects on the qEEG power spectra of the NR2B NAMs traxoprodil (CP-101,606) and BMT-108908 in nonhuman primates, and to compare them to the NMDA receptor channel blockers, ketamine and lanicemine. Cynomolgus monkeys were surgically implanted with EEG radio-telemetry transmitters, and qEEG was measured after vehicle or drug administration. The relative power for a number of frequency bands was determined. Ketamine and lanicemine increased relative gamma power, whereas the NR2B NAMs traxoprodil and BMT-108908 had no effect. Robust decreases in beta power were elicited by ketamine, traxoprodil and BMT-108908; and these agents also produced decreases in alpha power and increases in delta power at the doses tested. These results suggest that measurement of power spectra in the beta and delta bands may represent a translational pharmacodynamic biomarker to demonstrate functional effects of NR2B NAMs. The results of these studies may help guide the selection of qEEG measures that can be incorporated into early clinical evaluation of NR2B NAMs in healthy humans. PMID:27035340

  13. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

    NASA Astrophysics Data System (ADS)

    Novinec, Marko; Korenč, Matevž; Caflisch, Amedeo; Ranganathan, Rama; Lenarčič, Brigita; Baici, Antonio

    2014-02-01

    Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.

  14. Doing justice to allosteric regulation.

    PubMed

    Keller, Evelyn Fox

    2015-06-01

    Jacques Monod gave us not only our first regulatory system, but also our first smart molecules - i.e., he gave us allosteric proteins. But both of these contributions hung in a certain tension with his primary commitments. In particular, I focus here on the ways in which his ontological commitments constrained his thinking about the power of allostery. Although he wrote that "so far as regulation through allosteric interaction is concerned, everything is possible", for him, not everything was conceivable. In particular, what was not conceivable was a challenge to the primacy of DNA.

  15. Doing justice to allosteric regulation.

    PubMed

    Keller, Evelyn Fox

    2015-06-01

    Jacques Monod gave us not only our first regulatory system, but also our first smart molecules - i.e., he gave us allosteric proteins. But both of these contributions hung in a certain tension with his primary commitments. In particular, I focus here on the ways in which his ontological commitments constrained his thinking about the power of allostery. Although he wrote that "so far as regulation through allosteric interaction is concerned, everything is possible", for him, not everything was conceivable. In particular, what was not conceivable was a challenge to the primacy of DNA. PMID:25908117

  16. Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery.

    PubMed

    Patching, Simon G

    2014-01-01

    Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

  17. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes. PMID:17168764

  18. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

  19. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    SciTech Connect

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. )

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  20. Organometallic Palladium Complexes with a Water-Soluble Iminophosphorane Ligand as Potential Anticancer Agents

    PubMed Central

    Carreira, Monica; Calvo-Sanjuán, Rubén; Sanaú, Mercedes; Marzo, Isabel; Contel, María

    2012-01-01

    The synthesis and characterization of a new water-soluble iminophosphorane ligand TPA=N-C(O)-2BrC6H4 (C,N-IM; TPA = 1,3,5-triaza-7-phosphaadamantane) 1 is reported. Oxidative addition of 1 to Pd2(dba)3 affords the orthopalladated dimer [Pd(μ-Br){C6H4(C(O)N=TPA-kC,N)-2}]2 (2) as a mixture of cis and trans isomers (1:1 molar ratio) where the iminophosphorane moeity behaves as a C,N-pincer ligand. By addition of different neutral or monoanionic ligands to 2, the bridging bromide can be cleaved and a variety of hydrophilic or water-soluble mononuclear organometallic palladium(II) complexes of the type [Pd{C6H4(C(O)N=TPA-kC,N)-2}(L-L)] (L-L = acac (3); S2CNMe2 (4); 4,7-Diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt C12H6N2(C6H4SO3Na)2 (5)); [Pd{C6H4(C(O)N=TPA-kC,N)-2}(L)Br] (L = P(mC6H4SO3Na)3 (6); P(3-Pyridyl)3 (7)) and, [Pd(C6H4(C(O)N=TPA)-2}(TPA)2Br] (8) are obtained as single isomers. All new complexes were tested as potential anticancer agents and their cytotoxicity properties were evaluated in vitro against human Jurkat-T acute lymphoblastic leukemia cells, normal T-lymphocytes (PBMC) and DU-145 human prostate cancer cells. Compounds [Pd(μ-Br){C6H4(C(O)N=TPA-kC,N)-2}]2 (2) and [Pd{C6H4(C(O)N=TPA-kC,N)-2}(acac)] 3 (which has been crystallographically characterized) display the higher cytotoxicity against the above mentioned cancer cell lines while being less toxic to normal T-lymphocytes (peripheral blood mononuclear cells: PBMC). In addition, 3 is very toxic to cisplatin resistant Jurkat shBak indicating a cell death pathway that may be different to that of cisplatin. The interaction of 2 and 3 with plasmid (pBR322) DNA is much weaker than that of cisplatin pointing to an alternative biomolecular target for these cytotoxic compounds. All the compounds show an interaction with human serum albumin (HSA) faster than that of cisplatin. PMID:23066172

  1. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling

    PubMed Central

    Bock, Andreas; Merten, Nicole; Schrage, Ramona; Dallanoce, Clelia; Bätz, Julia; Klöckner, Jessica; Schmitz, Jens; Matera, Carlo; Simon, Katharina; Kebig, Anna; Peters, Lucas; Müller, Anke; Schrobang-Ley, Jasmin; Tränkle, Christian; Hoffmann, Carsten; De Amici, Marco; Holzgrabe, Ulrike; Kostenis, Evi; Mohr, Klaus

    2012-01-01

    Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M2 acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling. PMID:22948826

  2. Macrophage Membrane Potential Changes Associated with γ 2b/γ 1 Fc Receptor-Ligand Binding

    NASA Astrophysics Data System (ADS)

    Young, John Ding-E; Unkeless, Jay C.; Kaback, H. Ronald; Cohn, Zanvil A.

    1983-03-01

    We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (Δ Psi ) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in Δ Psi , but a sustained depolarization lasting ≈ 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+, K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

  3. Baclofen and Other GABAB Receptor Agents Are Allosteric Modulators of the CXCL12 Chemokine Receptor CXCR4

    PubMed Central

    Kussrow, Amanda; Olmsted, Ian Roys; Sandoz, Guillaume; Bornhop, Darryl J.; Nahon, Jean-Louis

    2013-01-01

    CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca2+ channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K+ (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases. PMID:23843532

  4. Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli.

    PubMed

    Kim, J; Raushel, F M

    2001-09-18

    , whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another. PMID:11551199

  5. Identification of the Allosteric Regulatory Site of Insulysin

    SciTech Connect

    Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W.; Gerrard, Juliet Ann

    2011-06-24

    Background Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. Principal Findings The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Conclusions/Significance Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

  6. Identification of the Allosteric Regulatory Site of Insulysin

    SciTech Connect

    Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W.

    2012-05-25

    Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the A{beta} peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

  7. Allosteric Modulators for mGlu Receptors

    PubMed Central

    Gasparini, F; Spooren, W

    2007-01-01

    The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions. The potential use of mGluRs as drug targets for various nervous system pathologies such as anxiety, depression, schizophrenia, pain or Parkinson’s disease has triggered an intense search for subtype selective modulators and resulted in the identification of numerous novel pharmacological agents capable to modulate the receptor activity through an interaction at an allosteric site located in the transmembrane domain. The present review presents the most recent developments in the identification and the characterization of allosteric modulators for the mGlu receptors. PMID:19305801

  8. Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders

    PubMed Central

    Conn, P. Jeffrey; Lindsley, Craig W.; Meiler, Jens; Niswender, Colleen M.

    2014-01-01

    Novel allosteric modulators of G protein-coupled receptors (GPCRs) are providing fundamental advances in the development of GPCR ligands with high subtype selectivity and novel modes of efficacy that have not been possible with traditional approaches. As new allosteric modulators are advancing as drug candidates, we are developing an increased understanding of the major advantages and broad range of activities that can be achieved with these agents through selective modulation of specific signalling pathways, differential effects on GPCR homodimers versus heterodimers, and other properties. This understanding creates exciting opportunities, as well as unique challenges, in the optimization of novel therapeutic agents for disorders of the central nervous system. PMID:25176435

  9. In vitro selection of allosteric ribozymes that sense the bacterial second messenger c-di-GMP.

    PubMed

    Furukawa, Kazuhiro; Gu, Hongzhou; Breaker, Ronald R

    2014-01-01

    Recently, a number of study have shown the ligand-dependent allosteric ribozymes can be harnessed as biosensors, high-throughput screening, and agents for the control of gene expression in vivo, called artificial riboswitches. In this chapter, we describe how in vitro selection can be used to create an allosteric ribozyme that senses bacterial second messenger cyclic-di-GMP (c-di-GMP). A hammerhead ribozyme was joined to a natural c-di-GMP class I riboswitch aptamer via communication modules. Both c-di-GMP-activating and -inhibiting ribozyme can be obtained by this approach. PMID:24549622

  10. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    PubMed

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  11. Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles.

    PubMed

    Bueno, Ana B; Showalter, Aaron D; Wainscott, David B; Stutsman, Cynthia; Marín, Aranzazu; Ficorilli, James; Cabrera, Over; Willard, Francis S; Sloop, Kyle W

    2016-05-13

    Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and

  12. Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles*

    PubMed Central

    Showalter, Aaron D.; Wainscott, David B.; Stutsman, Cynthia; Marín, Aranzazu; Ficorilli, James; Cabrera, Over

    2016-01-01

    Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5′-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9–36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [3H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and

  13. Latest advances in novel cannabinoid CB2 ligands for drug abuse and their therapeutic potential

    PubMed Central

    Yang, Peng; Wang, Lirong; Xie, Xiang-Qun

    2012-01-01

    The field of cannabinoid (CB) drug research is experiencing a challenge as the CB1 antagonist Rimonabant, launched in 2006 as an anorectic/anti-obesity drug, was withdrawn from the European market due to the complications of suicide and depression as side effects. There is interest in developing CB2 drugs without CB1 psychotropic side effects for drug-abuse treatment and therapeutic medication. The CB1 receptor was discovered predominantly in the brain, whereas the CB2 is mainly expressed in peripheral cells and tissues, and is involved in immune signal transduction. Conversely, the CB2 receptor was recently detected in the CNS, for example, in the microglial cells and the neurons. While the CB2 neurons activity remains controversial, the CB2 receptor is an attractive therapeutic target for neuropathic pain, immune system, cancer and osteoporosis without psychoactivity. This review addresses CB drug abuse and therapeutic potential with a focus on the most recent advances on new CB2 ligands from the literature as well as patents. PMID:22300098

  14. Structurally Similar Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors Exhibit Five Distinct Pharmacological Effects*

    PubMed Central

    Gill-Thind, JasKiran K.; Dhankher, Persis; D'Oyley, Jarryl M.; Sheppard, Tom D.; Millar, Neil S.

    2015-01-01

    Activation of nicotinic acetylcholine receptors (nAChRs) is associated with the binding of agonists such as acetylcholine to an extracellular site that is located at the interface between two adjacent receptor subunits. More recently, there has been considerable interest in compounds, such as positive and negative allosteric modulators (PAMs and NAMs), that are able to modulate nAChR function by binding to distinct allosteric sites. Here we examined a series of compounds differing only in methyl substitution of a single aromatic ring. This series of compounds includes a previously described α7-selective allosteric agonist, cis-cis-4-p-tolyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4MP-TQS), together with all other possible combinations of methyl substitution at a phenyl ring (18 additional compounds). Studies conducted with this series of compounds have revealed five distinct pharmacological effects on α7 nAChRs. These five effects can be summarized as: 1) nondesensitizing activation (allosteric agonists), 2) potentiation associated with minimal effects on receptor desensitization (type I PAMs), 3) potentiation associated with reduced desensitization (type II PAMs), 4) noncompetitive antagonism (NAMs), and 5) compounds that have no effect on orthosteric agonist responses but block allosteric modulation (silent allosteric modulators (SAMs)). Several lines of experimental evidence are consistent with all of these compounds acting at a common, transmembrane allosteric site. Notably, all of these chemically similar compounds that have been classified as nondesensitizing allosteric agonists or as nondesensitizing (type II) PAMs are cis-cis-diastereoisomers, whereas all of the NAMs, SAMs, and type I PAMs are cis-trans-diastereoisomers. Our data illustrate the remarkable pharmacological diversity of allosteric modulators acting on nAChRs. PMID:25516597

  15. Structures of pyruvate kinases display evolutionarily divergent allosteric strategies.

    PubMed

    Morgan, Hugh P; Zhong, Wenhe; McNae, Iain W; Michels, Paul A M; Fothergill-Gilmore, Linda A; Walkinshaw, Malcolm D

    2014-09-01

    The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k cat/S 0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k cat/S 0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.

  16. Allosteric conformational spread: Exact results using a simple transfer matrix method

    NASA Astrophysics Data System (ADS)

    Mochrie, S. G. J.; Mack, A. H.; Regan, L.

    2010-09-01

    A transfer matrix method is described for the conformational spread (CS) model of allosteric cooperativity within a one-dimensional arrangement of four-state binding sites. Each such binding site can realize one of two possible conformational states. Each of these states can either bind ligand or not bind ligand. Thus, analytical expressions that are exact within the context of the CS model are derived for the grand partition function, for the mean fraction of binding sites occupied by ligand versus ligand concentration, and for the mean fraction of binding sites in a given allosteric state versus ligand concentration. The utility of our analytical results is demonstrated by least-mean-square fitting of prior experimental results obtained on the bacterial flagellar motor for the fraction of FliM/FliG/FliN complexes with CheY-P bound [V. Sourjik and H. C. Berg, Proc. Natl. Acad. Sci. U.S.A. 99, 12669 (2002)10.1073/pnas.192463199] and for the cw bias [P. Cluzel , Science 287, 1652 (2000)10.1126/science.287.5458.1652], which plausibly may be identified as the fraction of protomers realizing state 2. Finally, the relationships between our analytical results and the classical Monod-Wyman-Changeaux, Koshland-Nemethy-Filmer, and McGhee-Von Hippel treatments of allosteric cooperativity are elucidated, as is the connection to an earlier approximate analytical treatment of the CS model.

  17. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    NASA Astrophysics Data System (ADS)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  18. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells.

    PubMed

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway. PMID:27657873

  19. Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

    PubMed

    Makarov, G I; Golovin, A V; Sumbatyan, N V; Bogdanov, A A

    2015-08-01

    The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed. PMID:26547073

  20. Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

    PubMed

    Makarov, G I; Golovin, A V; Sumbatyan, N V; Bogdanov, A A

    2015-08-01

    The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed.

  1. Rational design of dualsteric GPCR ligands: quests and promise

    PubMed Central

    Mohr, Klaus; Tränkle, Christian; Kostenis, Evi; Barocelli, Elisabetta; De Amici, Marco; Holzgrabe, Ulrike

    2010-01-01

    Dualsteric ligands represent a novel mode of targeting G protein-coupled receptors (GPCRs). These compounds attach simultaneously to both, the orthosteric transmitter binding site and an additional allosteric binding area of a receptor protein. This approach allows the exploitation of favourable characteristics of the orthosteric and the allosteric site by a single ligand molecule. The orthosteric interaction provides high affinity binding and activation of receptors. The allosteric interaction yields receptor subtype-selectivity and, in addition, may modulate both, efficacy and intracellular signalling pathway activation. Insight into the spatial arrangement of the orthosteric and the allosteric site is far advanced in the muscarinic acetylcholine receptor, and the design of dualsteric muscarinic agonists has now been accomplished. Using the muscarinic receptor as a paradigm, this review summarizes the way from suggestive evidence for an orthosteric/allosteric overlap binding to the rational design and experimental validation of dualsteric ligands. As allosteric interactions are increasingly described for GPCRs and as insight into the spatial geometry of ligand/GPCR-complexes is growing impressively, the rational design of dualsteric drugs is a promising new approach to achieve fine-tuned GPCR-modulation. This article is part of a themed section on Molecular Pharmacology of GPCR. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00695.x PMID:20136835

  2. Allosteric Modulation of the Calcium-Sensing Receptor

    PubMed Central

    Jensen, Anders A; Bräuner-Osborne, Hans

    2007-01-01

    The calcium (Ca2+)-sensing receptor (CaR) belongs to family C of the G-protein coupled receptors (GPCRs). The receptor is activated by physiological levels of Ca2+ (and Mg2+) and positively modulated by a range of proteinogenic L-α-amino acids. Recently, several synthetic allosteric modulators of the receptor have been developed, which either act as positive modulators (termed calcimimetics) or negative modulators (termed calcilytics). These ligands do not activate the wild-type receptor directly, but rather shift the concentration-response curves of Ca2+ to the left or right, respectively. Like other family C GPCRs, the CaR contains a large amino-terminal domain and a 7-transmembrane domain. Whereas the endogenous ligands for the receptor, Ca2+, Mg2+ and the L-α-amino acids, bind to the amino-terminal domain, most if not all of the synthetic modulators published so far bind to the 7-transmembrane domain. The most prominent physiological function of the CaR is to maintain the extracellular Ca2+ level in a very tight range via control of secretion of parathyroid hormone (PTH). Influence on e.g. secretion of calcitonin from thyroid C-cells and direct action on the tubule of the kidney also contribute to the control of the extracellular Ca2+ level. This control over PTH and Ca2+ levels is partially lost in patients suffering from primary and secondary hyperparathyroidism. The perspectives in CaR as a therapeutic target have been underlined by the recent approval of the calcimimetic cinacalcet for the treatment of certain forms of primary and secondary hyperparathyroidism. Cinacalcet is the first clinically administered allosteric modulator acting on a GPCR, and thus the compound constitutes an important proof-of-concept for future development of allosteric modulators on other GPCR drug targets. PMID:19305800

  3. Potential Application of Alchemical Free Energy Simulations to Discriminate GPCR Ligand Efficacy.

    PubMed

    Lee, Hui Sun; Seok, Chaok; Im, Wonpil

    2015-03-10

    G protein-coupled receptors (GPCRs) play fundamental roles in physiological processes by modulating diverse signaling pathways and thus have been one of the most important drug targets. Based on the fact that GPCR-mediated signaling is modulated in a ligand-specific manner such as agonist, inverse agonist, and neutral antagonist (termed ligand efficacy), quantitative characterization of the ligand efficacy is essential for rational design of selective modulators for GPCR targets. As experimental approaches for this purpose are time-, cost-, and labor-intensive, computational tools that can systematically predict GPCR ligand efficacy can have a big impact on GPCR drug design. Here, we have performed free energy perturbation molecular dynamics simulations to calculate absolute binding free energy of an inverse agonist, a neutral antagonist, and an agonist to β2-adrenergic receptor (β2-AR) active and inactive states, respectively, in explicit lipid bilayers. Relatively short alchemical free energy calculations reveal that both the time-series of the total binding free energy and decomposed energy contributions can be used as relevant physical properties to discriminate β2-AR ligand efficacy. This study illustrates a merit of the current approach over simple, fast docking calculations or highly expensive millisecond-time scale simulations. PMID:26579772

  4. Bovine Norovirus: Carbohydrate Ligand, Environmental Contamination, and Potential Cross-Species Transmission via Oysters ▿ †

    PubMed Central

    Zakhour, Maha; Maalouf, Haifa; Di Bartolo, Ilaria; Haugarreau, Larissa; Le Guyader, Françoise S.; Ruvoën-Clouet, Nathalie; Le Saux, Jean-Claude; Ruggeri, Franco Maria; Pommepuy, Monique; Le Pendu, Jacques

    2010-01-01

    Noroviruses (NoV) are major agents of acute gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Previous studies showed that some human strains bind to oyster tissues through carbohydrate ligands that are similar to their human receptors. Thus, based on presentation of shared norovirus carbohydrate ligands, oysters could selectively concentrate animal strains with increased ability to overcome species barriers. In comparison with human GI and GII strains, bovine GIII NoV strains, although frequently detected in bovine feces and waters of two estuaries of Brittany, were seldom detected in oysters grown in these estuaries. Characterization of the carbohydrate ligand from a new GIII strain indicated recognition of the alpha-galactosidase (α-Gal) epitope not expressed by humans, similar to the GIII.2 Newbury2 strain. This ligand was not detectable on oyster tissues, suggesting that oysters may not be able to accumulate substantial amounts of GIII strains due to the lack of shared carbohydrate ligand and that they should be unable to contribute to select GIII strains with an increased ability to recognize humans. PMID:20709837

  5. Allosteric sodium in class A GPCR signaling

    PubMed Central

    Katritch, Vsevolod; Fenalti, Gustavo; Abola, Enrique E.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

    2014-01-01

    Despite their functional and structural diversity, G protein-coupled receptors (GPCRs) share a common mechanism of signal transduction via conformational changes in the seven-transmembrane (7TM) helical domain. New major insights into this mechanism come from the recent crystallographic discoveries of a partially hydrated sodium ion that is specifically bound in the middle of the 7TM bundle of multiple class A GPCRs. This review discusses the remarkable structural conservation and distinct features of the Na+ pocket in this most populous GPCR class, as well as the conformational collapse of the pocket on receptor activation. New insights help to explain allosteric effects of sodium on GPCR agonist binding and activation, and sodium’s role as a potential co-factor in class A GPCR function. PMID:24767681

  6. Classification and comparison of ligand-binding sites derived from grid-mapped knowledge-based potentials.

    PubMed

    Hoppe, Christian; Steinbeck, Christoph; Wohlfahrt, Gerd

    2006-03-01

    We describe the application of knowledge-based potentials implemented in the MOE program to compare the ligand-binding sites of several proteins. The binding probabilities for a polar and a hydrophobic probe are calculated on a grid to allow easy comparison of binding sites of superimposed related proteins. The method is fast and simple enough to simultaneously use structural information of multiple proteins of a target family. The method can be used to rapidly cluster proteins into subfamilies according to the similarity of hydrophobic and polar fields of their ligand-binding sites. Regions of the binding site which are common within a protein family can be identified and analysed for the design of family-targeted libraries or those which differ for improvement of ligand selectivity. The field-based hierarchical clustering is demonstrated for three protein families: the ligand-binding domains of nuclear receptors, the ATP-binding sites of protein kinases and the substrate binding sites of proteases. More detailed comparisons are presented for serine proteases of the chymotrypsin family, for the peroxisome proliferator-activated receptor subfamily of nuclear receptors and for progesterone and androgen receptor. The results are in good accordance with structure-based analysis and highlight important differences of the binding sites, which have been also described in the literature.

  7. Allosteric Optical Control of a Class B G‐Protein‐Coupled Receptor

    PubMed Central

    Broichhagen, Johannes; Johnston, Natalie R.; von Ohlen, Yorrick; Meyer‐Berg, Helena; Jones, Ben J.; Bloom, Stephen R.; Rutter, Guy A.

    2016-01-01

    Abstract Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca2+, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R. PMID:27059784

  8. The allosteric transition of GroEL induced by metal fluoride-ADP complexes.

    PubMed

    Inobe, Tomonao; Kikushima, Kenji; Makio, Tadashi; Arai, Munehito; Kuwajima, Kunihiro

    2003-05-23

    structurally equivalent to the allosteric state induced by ATP. These results suggested that both the size and coordination geometry of gamma-phosphate (and its analogs) are related to the allosteric transition of GroEL. It was therefore concluded that the tetrahedral geometry of gamma-phosphate (or its analogs) and the inter-atomic distance ( approximately 1.6A) between phosphorus (vanadium, or metal atom) and oxygen (or fluorine) are both important for inducing the allosteric transition of GroEL, leading to the high selectivity of GroEL for ATP about ligand adenine nucleotides, which function as the preferred allosteric ligand. PMID:12742022

  9. Unraveling structural mechanisms of allosteric drug action.

    PubMed

    Nussinov, Ruth; Tsai, Chung-Jung

    2014-05-01

    Orthosteric drugs block the active site to obstruct function; allosteric drugs modify the population of the active state, to modulate function. Available data lead us to propose that allosteric drugs can constitute anchors and drivers. The anchor docks into an allosteric pocket. The conformation with which it interacts is unchanged during the transition between the inactive and active states. The anchor provides the foundation that allows the driver to exert a 'pull' and/or 'push' action that shifts the receptor population from the inactive to the active state. The presence or absence of driver atom in an allosteric drug can exert opposite agonism. We map a strategy for driver identification and expect the allosteric trigger concept to transform agonist/antagonist drug discovery.

  10. Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

    PubMed Central

    Sung, Yun-Min; Wilkins, Angela D.; Rodriguez, Gustavo J.; Wensel, Theodore G.; Lichtarge, Olivier

    2016-01-01

    The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues. PMID:26979958

  11. Potential Application of Kläui Ligands in Actinide Separations

    SciTech Connect

    Lumetta, Gregg J.; McNamara, Bruce K.; Hubler, Timothy L.; Wester, Dennis W.; Li, Jun; Latesky, Stanley L.

    2006-07-31

    We have undertaken a systematic study of the complexation of Cp*Co[P(O)(OR)2]3- (Cp* = pentamethylcyclopentadienyl) ligands with f-block metal ions (i.e., lanthanides and actinides). As part of this work, the complexation of La3+ ion with Cp*Co[P(O)(OR)2]3- ligands has been studied as the alkyl group was systematically varied from methyl to n-propyl (R = -CH3, -CH2CH3, and -CH2 CH2CH3). For ligands in which R = -CH3 or -CH2CH3, complexes with ligand-to-La stoichiometries of 1:1 and 2:1 were formed. In contrast, only the 1:1 complex was isolated when R = CH2CH2CH3. A prototypical extraction chromatography resin containing Cp*Co[P(O)(OEt)2]3- (1b) has been prepared. The resin consists of 0.75 wt% 1b on Amberlite? XAD-7. This resin strongly sorbs Am3+ and Pu4+. The sorption of these ions decreases with increasing nitric acid concentration, but this effect is more pronounced for Am3+. This allows for convenient separation of Am3+ from Pu4+ by simple adjustments in the HNO3 concentration. The tripodal geometry of 1b disfavors the complexation of UO22+, so sorption of U(VI) by the 1b-containing resin is weak.

  12. Biological redundancy of endogenous GPCR ligands in the gut and the potential for endogenous functional selectivity

    PubMed Central

    Thompson, Georgina L.; Canals, Meritxell; Poole, Daniel P.

    2014-01-01

    This review focuses on the existence and function of multiple endogenous agonists of the somatostatin and opioid receptors with an emphasis on their expression in the gastrointestinal tract. These agonists generally arise from the proteolytic cleavage of prepropeptides during peptide maturation or from degradation of peptides by extracellular or intracellular endopeptidases. In other examples, endogenous peptide agonists for the same G protein-coupled receptors can be products of distinct genes but contain high sequence homology. This apparent biological redundancy has recently been challenged by the realization that different ligands may engender distinct receptor conformations linked to different intracellular signaling profiles and, as such the existence of distinct ligands may underlie mechanisms to finely tune physiological responses. We propose that further characterization of signaling pathways activated by these endogenous ligands will provide invaluable insight into the mechanisms governing biased agonism. Moreover, these ligands may prove useful in the design of novel therapeutic tools to target distinct signaling pathways, thereby favoring desirable effects and limiting detrimental on-target effects. Finally we will discuss the limitations of this area of research and we will highlight the difficulties that need to be addressed when examining endogenous bias in tissues and in animals. PMID:25506328

  13. Assessment of the genotoxic potential of the antipsychotic sigma receptor ligand E-5842.

    PubMed

    Guzmán, Antonio; García, Concepción; Fernández de Henestrosa, Antonio R; Riley, Sue; Ruiz, Maria Teresa; Marín, Ana-Paz; Tortajada, Araceli

    2006-06-16

    The genotoxic potential of E-5842, a sigma ligand compound being developed as an antipsychotic drug, was evaluated by means of an extensive battery of in vitro and in vivo assays. Negative results were obtained in an Ames test (up to 5000 μg/plate), a mouse lymphoma assay (up to 535.1 μg/ml (-S9) and 891.8 μg/ml (+S9)), an in vivo rat hepatocyte micronucleus assay (up to 100 mg/kg/day on 2 days), and a two-dose mouse micronucleus assay (up to 40 mg/kg/day on 2 days). In a single-dose mouse bone-marrow micronucleus assay (up to 400 mg/kg; 24, 48 and 72 h sampling) a slight and non-statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was observed 48 h after administration of a 200 mg/kg dose, in the absence of bone-marrow toxicity. This minor increase in MNPCE frequency was considered of questionable biological relevance, because it was observed under conditions of marked animal toxicity including mortality. In addition, it occurred in association with a strong hypothermic effect produced by administration of E-5842. A clear increase in the frequency of structural chromosomal aberrations was observed in human lymphocytes at concentrations ≥350.6 and 1685.4 μg/ml in the presence and absence of S9, respectively. Mitotic accumulation was observed at those concentrations at which clastogenic effects were observed, a condition that may have masked toxicity. Concentrations lacking clastogenic effects in this chromosome aberration assay (300.7 and 173.2 μg/ml in the presence and absence of S9, respectively) were well in excess of maximum human plasma concentrations attained in clinical studies at the maximum tolerated dose (19.1 ng/ml). A weight-of-evidence analysis, taking into consideration the results obtained in the different in vitro and in vivo assays and the conditions of clinical use, suggest that E-5842 would not pose a genotoxic risk under clinical conditions.

  14. Herbo-mineral based Schiff base ligand and its metal complexes: Synthesis, characterization, catalytic potential and biological applications.

    PubMed

    Kareem, Abdul; Laxmi; Arshad, Mohammad; Nami, Shahab A A; Nishat, Nahid

    2016-07-01

    Schiff base ligand, (L), derived from condensation reaction of 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, (curcumin), with pyridine-3-carboxamide, (nicotinamide), and its complexes of Co(II), Ni(II) and Cu(II) ions, containing 1,10-phenanthroline as auxiliary ligand were synthesized and characterized by various physico-chemical techniques. From the micro analytical data, the stoichiometry of the complexes 1:1 (metal: ligand) was ascertained. The Co(II) and Cu(II) forms octahedral complexes, while the geometric structure around Ni(II) atom can be described as square planar. The catalytic potential of the metal complexes have been evaluated by recording the rate of decomposition of hydrogen peroxide. The results reveal that the percent decomposition of H2O2increases with time and the highest value (50.50%) was recorded for Co(II) complex. The ligand and its complexes were also screened for their in vitro antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pyogenes and Pseudomonas aeruginosa. The relative order of antibacterial activity against S. Pyogenes, S. aureus and E. coli is Cu(II)>Ni(II)>Co(II)>(L); while with P. aeruginosa, K. pneumoniae the order of activity is Cu(II)>Co(II)>Ni(II)>(L). The anthelmintic screening was performed using Pheretima posthuma. The order of anthelmintic activity of ligand and its complexes is [(Phen)CuLCl2]>[(Phen)CoLCl2]>[(Phen)NiL]Cl2>(L).

  15. Synthesis, characterisation, spectral, thermal, XRD, molecular modelling and potential antibacterial study of metal complexes containing octadentate azodye ligands

    NASA Astrophysics Data System (ADS)

    Mahapatra, Bipin Bihari; Chaulia, Satyanarayan; Sarangi, Ashish Kumar; Dehury, Satyanarayan; Panda, Jnyanaranjan

    2015-05-01

    Twelve tetrametallic complexes of Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Hg(II) with two new octadentate azodye ligands, 4,4‧-bis(2‧,4‧-dihydroxy-5‧carboxyphenylazo) diphenylether (LH6) and 4,4‧-bis(2‧,4‧-dihydroxy-5‧-acylphenylazo) diphenylether (L‧H4) have been synthesised. The structural elucidation of the complexes was made basing upon analytical, conductance, magnetic susceptibility, IR, electronic spectra, ESR, NMR, ESI-MS, TG, DTG, DTA and X-ray diffraction (powder pattern) data. The cobalt (II) and nickel (II) complexes are found to be octahedral, copper (II) complexes are distorted octahedral and a tetrahedral stereochemistry has been suggested to zinc (II), cadmium (II) and mercury (II) complexes. The thermal analysis data provided the kinetic parameters as order of decomposition reaction, activation energy and frequency factor. The geometry of the ligands and their Co(II), Ni(II), Cu(II) and Zn(II) complexes were optimised and their physicochemical properties were calculated by using molecular modelling procedure. The ESI-MS determination supports the molecular formula and molecular weight of the ligands and the complexes. The Ni(II) complex is found to have a triclinic crystal system. The potential antibacterial study of the two ligands and eight metal complexes was made by cup-plate method against one gram positive and one gram negative bacteria. The results showed increase in the activity of some metal complexes as compare with azodye ligands.

  16. Herbo-mineral based Schiff base ligand and its metal complexes: Synthesis, characterization, catalytic potential and biological applications.

    PubMed

    Kareem, Abdul; Laxmi; Arshad, Mohammad; Nami, Shahab A A; Nishat, Nahid

    2016-07-01

    Schiff base ligand, (L), derived from condensation reaction of 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, (curcumin), with pyridine-3-carboxamide, (nicotinamide), and its complexes of Co(II), Ni(II) and Cu(II) ions, containing 1,10-phenanthroline as auxiliary ligand were synthesized and characterized by various physico-chemical techniques. From the micro analytical data, the stoichiometry of the complexes 1:1 (metal: ligand) was ascertained. The Co(II) and Cu(II) forms octahedral complexes, while the geometric structure around Ni(II) atom can be described as square planar. The catalytic potential of the metal complexes have been evaluated by recording the rate of decomposition of hydrogen peroxide. The results reveal that the percent decomposition of H2O2increases with time and the highest value (50.50%) was recorded for Co(II) complex. The ligand and its complexes were also screened for their in vitro antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pyogenes and Pseudomonas aeruginosa. The relative order of antibacterial activity against S. Pyogenes, S. aureus and E. coli is Cu(II)>Ni(II)>Co(II)>(L); while with P. aeruginosa, K. pneumoniae the order of activity is Cu(II)>Co(II)>Ni(II)>(L). The anthelmintic screening was performed using Pheretima posthuma. The order of anthelmintic activity of ligand and its complexes is [(Phen)CuLCl2]>[(Phen)CoLCl2]>[(Phen)NiL]Cl2>(L). PMID:27107703

  17. Electrophilic PPARγ ligands inhibit corneal fibroblast to myofibroblast differentiation in vitro: a potentially novel therapy for corneal scarring.

    PubMed

    Kuriyan, A E; Lehmann, G M; Kulkarni, A A; Woeller, C F; Feldon, S E; Hindman, H B; Sime, P J; Huxlin, K R; Phipps, R P

    2012-01-01

    A critical component of corneal scarring is the TGFβ-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-methyl ester (CDDO-Me) and 15-deoxy-Δ(-12,14)-prostaglandin J(2) (15d-PGJ(2)) for inhibiting TGFβ-induced myofibroblast differentiation in vitro. TGFβ was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ(2) were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFβ-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ(2)) potently inhibited TGFβ-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ(2) are strong inhibitors of TGFβ-induced corneal fibroblast to myofibroblast differentiation in vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.

  18. Opioid ligands having delayed long-term antagonist activity: potential pharmacotherapies for opioid abuse.

    PubMed

    Husbands, Stephen M; Lewis, John W

    2003-03-01

    Buprenorphine is a partial agonist at the micro -opioid receptor with long duration of action and also exhibits delayed antagonist activity. Buprenorphine is finding increasing use as a treatment agent for opioid abuse, though its low efficacy is not well tolerated by all addicts. There is interest in developing a higher efficacy version of buprenorphine and in this mini-review some of the ligands recently discovered, that share with buprenorphine a profile of agonism followed by delayed antagonism, are discussed.

  19. Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Snoeberger, Ning-Shiuan Nicole

    Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

  20. First Demonstration of Positive Allosteric-like Modulation at the Human Wild Type Translocator Protein (TSPO).

    PubMed

    Narlawar, Rajeshwar; Werry, Eryn L; Scarf, Alana M; Hanani, Raphy; Chua, Sook Wern; King, Victoria A; Barron, Melissa L; Martins, Ralph N; Ittner, Lars M; Rendina, Louis M; Kassiou, Michael

    2015-11-12

    We show that changing the number and position of nitrogen atoms in the heteroatomic core of a pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild type human TSPO.

  1. Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

    SciTech Connect

    Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.; Lovell, Scott; Karanicolas, John

    2012-08-07

    Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492G in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.

  2. Identification of an allosteric binding site for RORγt inhibition

    PubMed Central

    Scheepstra, Marcel; Leysen, Seppe; van Almen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors. PMID:26640126

  3. The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.

    PubMed

    Cecchini, Marco; Changeux, Jean-Pierre

    2015-09-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  4. Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

    PubMed

    Cressman, William J; Beckett, Dorothy

    2016-01-19

    Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

  5. Potential New Ligand Systems for Binding Uranyl Ions in Seawater Environments

    SciTech Connect

    Arnold, John

    2014-12-13

    Work began this quarter on a new project involving a combined computational and biosynthetic approach to selective recognition of uranyl ion in aqueous solution. This project exploits the results of computational studies to discover new ligand classes. Synthetic studies will follow to generate target systems for uranyl binding and determination of binding constants. The process will be iterative, with results from computation informing synthesis, and vice versa. The theme of the ligand classes to be examined initially will be biologically based. New phosphonate-containing α-amino acid N-carboxyanhydride (NCA) monomers were used recently to prepare well-defined phosphonate-containing poly-peptides and block copolypeptides. Our first approach is to utilize these phosphate- and phosphonate-containing NCAs for the coordination of uranyl. The work includes the laboratory-scale preparation of a series of NCAs and the full thermodynamic and spectroscopic characterization of the resulting uranyl complexes. We are also evaluating the sequestering activity in different physiological and environmental conditions of these copolymers as well as their biodegradability.

  6. Recent developments in adenosine receptor ligands and their potential as novel drugs☆

    PubMed Central

    Müller, Christa E.; Jacobson, Kenneth A.

    2012-01-01

    Medicinal chemical approaches have been applied to all four of the adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3) to create selective agonists and antagonists for each. The most recent class of selective AR ligands to be reported is the class of A2BAR agonists. The availability of these selective ligands has facilitated research on therapeutic applications of modulating the ARs and in some cases has provided clinical candidates. Prodrug approaches have been developed which improve the bioavailability of the drugs, reduce side-effects, and/or may lead to site-selective effects. The A2A agonist regadenoson (Lexiscan®), a diagnostic drug for myocardial perfusion imaging, is the first selective AR agonist to be approved. Other selective agonists and antagonists are or were undergoing clinical trials for a broad range of indications, including capadenoson and tecadenoson (A1 agonists) for atrial fibrillation, or paroxysmal supraventricular tachycardia, respectively, apadenoson and binodenoson (A2A agonists) for myocardial perfusion imaging, preladenant (A2A antagonist) for the treatment of Parkinson’s disease, and CF101 and CF102 (A3 agonists) for inflammatory diseases and cancer, respectively. This article is part of a Special Issue entitled: “Adenosine Receptors”. PMID:21185259

  7. Allosteric function and dysfunction of the prion protein.

    PubMed

    Linden, Rafael; Cordeiro, Yraima; Lima, Luis Mauricio T R

    2012-04-01

    Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP(C)), its conformational conversion, aggregation and precipitation. We recently proposed that PrP(C) serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP(C), which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP(C) and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP(C), and that allosteric dysfunction of PrP(C) has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP(C), as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.

  8. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

  9. An alternative Fc gamma-receptor ligand: potential role in T-cell development.

    PubMed Central

    Sandor, M; Galon, J; Takacs, L; Tatsumi, Y; Mueller, A L; Sautes, C; Lynch, R G

    1994-01-01

    Fetal pre-T cells express low-affinity receptors for IgG (Fc gamma R) at a developmental stage prior to the rearrangement and expression of immunoglobulin genes. The present studies investigated the possible functional significance of Fc gamma R on fetal pre-T cells. Between 13 and 17 days of fetal development a subpopulation of T-cell receptor-, Thy-1+ thymocytes express for gamma R. The same cells contain mRNA for several forms of Fc gamma R (Fc gamma RII beta 1, beta 2, and Fc gamma RIII). Concurrently, a Pgp-1-, Thy-1-, surface-immunoglobulin- fetal thymic cell binds recombinant soluble Fc gamma R. In principle this cell can interact with the pre-T cells through this counter-receptor. To test this possibility anti-Fc gamma RII/III antibody (2.4G2) was injected into pregnant mice and then into their offspring for 6 wk postpartum. The injected antibody induced a slight increase in the proportion of CD4 or CD8 single-positive, alpha/beta T cells in the thymus. However, in fetal thymic cultures in the presence of 2.4G2 or the recombinant soluble Fc gamma R there was an accelerated differentiation of thymocytes to single-positive, CD3-bright, heat-stable antigen-dull, alpha/beta T cells. These experiments show that Fc gamma Rs are present on pre-T cells during early fetal thymic development, and that a non-IgG ligand of the Fc gamma R is expressed concurrently on Thy- fetal thymocytes. Furthermore, the presumed interaction of Fc gamma R and the alternative ligand(s) influences T-cell development. IgG binding could be an adapted function of Fc gamma Rs, and, as shown for many members of the Ig super family, these receptors may have originally served as cell-cell recognition/interaction molecules required for hematopoietic development. Images Fig. 2 PMID:7809135

  10. Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines.

    PubMed

    Mony, Laetitia; Zhu, Shujia; Carvalho, Stéphanie; Paoletti, Pierre

    2011-06-17

    NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.

  11. Disentangling the web of allosteric communication in a homotetramer: heterotropic activation in phosphofructokinase from Escherichia coli.

    PubMed

    Fenton, Aron W; Paricharttanakul, N Monique; Reinhart, Gregory D

    2004-11-01

    Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites and four allosteric sites. Understanding the allosteric activation of EcPFK by MgADP has been complicated by the complex web of possible interactions, including active site homotropic interactions, allosteric site homotropic interactions, and heterotropic interactions between active and allosteric sites. The current work has simplified this web of possible interactions to a series of single heterotropic interactions by forming and isolating hybrid tetramers. Each of the four unique heterotropic interactions have independently been isolated and compared to a control that has all four of the unique heterotropic interactions. If the interactions are labeled with the distances between interacting ligands, the 45-A interaction contributes 20% +/- 1%, the 33-A interaction contributes 34% +/- 1%, the 30-A interaction contributes 21% +/- 1%, and the 23-A interaction contributes 25% +/- 1% with respect to the total free energy of MgADP/fructose 6-phosphate (Fru-6-P) activation in the control. The free energies of the isolated interactions sum to 100% +/- 2% of the total. Therefore, the four unique interactions are all contributors to activation, are nonequivalent, and are additive.

  12. Global Low Frequency Protein Motions in Long-Range Allosteric Signaling

    NASA Astrophysics Data System (ADS)

    McLeish, Tom; Rogers, Thomas; Townsend, Philip; Burnell, David; Pohl, Ehmke; Wilson, Mark; Cann, Martin; Richards, Shane; Jones, Matthew

    2015-03-01

    We present a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ``signalling at a distance.'' Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes we demonstrate negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ``control sites.'' The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ``design space'' that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analyzing naturally occurring CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

  13. Modulation of Global Low-Frequency Motions Underlies Allosteric Regulation: Demonstration in CRP/FNR Family Transcription Factors

    PubMed Central

    Burnell, David; Jones, Matthew L.; Richards, Shane A.; McLeish, Tom C. B.; Pohl, Ehmke; Wilson, Mark R.; Cann, Martin J.

    2013-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein “design space” that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to

  14. Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

    PubMed

    Rodgers, Thomas L; Townsend, Philip D; Burnell, David; Jones, Matthew L; Richards, Shane A; McLeish, Tom C B; Pohl, Ehmke; Wilson, Mark R; Cann, Martin J

    2013-09-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein "design space" that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to

  15. The Dipole Potential Modifies the Clustering and Ligand Binding Affinity of ErbB Proteins and Their Signaling Efficiency

    PubMed Central

    Kovács, Tamás; Batta, Gyula; Hajdu, Tímea; Szabó, Ágnes; Váradi, Tímea; Zákány, Florina; Csomós, István; Szöllősi, János; Nagy, Peter

    2016-01-01

    Although activation of the ErbB family of receptor tyrosine kinases (ErbB1-4) is driven by oligomerization mediated by intermolecular interactions between the extracellular, the kinase and the transmembrane domains, the transmembrane domain has been largely neglected in this regard. The largest contributor to the intramembrane electric field, the dipole potential, alters the conformation of transmembrane peptides, but its effect on ErbB proteins is unknown. Here, we show by Förster resonance energy transfer (FRET) and number and brightness (N&B) experiments that the epidermal growth factor (EGF)-induced increase in the homoassociation of ErbB1 and ErbB2 and their heteroassociation are augmented by increasing the dipole potential. These effects were even more pronounced for ErbB2 harboring an activating Val → Glu mutation in the transmembrane domain (NeuT). The signaling capacity of ErbB1 and ErbB2 was also correlated with the dipole potential. Since the dipole potential decreased the affinity of EGF to ErbB1, the augmented growth factor-induced effects at an elevated dipole potential were actually induced at lower receptor occupancy. We conclude that the dipole potential plays a permissive role in the clustering of ErbB receptors and that the effects of lipid rafts on ligand binding and receptor signaling can be partially attributed to the dipole potential. PMID:27775011

  16. Galantamine ameliorates the impairment of recognition memory in mice repeatedly treated with methamphetamine: involvement of allosteric potentiation of nicotinic acetylcholine receptors and dopaminergic-ERK1/2 systems.

    PubMed

    Noda, Yukihiro; Mouri, Akihiro; Ando, Yu; Waki, Yukari; Yamada, Shin-Nosuke; Yoshimi, Akira; Yamada, Kiyofumi; Ozaki, Norio; Wang, Dayong; Nabeshima, Toshitaka

    2010-11-01

    Galantamine, a drug used to treat Alzheimer's disease, inhibits acetylcholinesterase (AChE) and allosterically modulates nicotinic acetylcholine receptors (nAChRs) resulting in stimulation of catecholamine neurotransmission. In this study, we investigated whether galantamine exerts cognitive-improving effects through the allosteric modulation of nAChRs in an animal model of methamphetamine (Meth) psychosis. The mice treated with Meth (1 mg/kg.d) for 7 d showed memory impairment in a novel object recognition test. Galantamine (3 mg/kg) ameliorated the memory impairment, and it increased the extracellular dopamine release in the prefrontal cortex (PFC) of Meth-treated mice. Donepezil, an AChE inhibitor (1 mg/kg) increased the extracellular ACh release in the PFC, whereas it had no effect on the memory impairment in Meth-treated mice. The nAChR antagonist, mecamylamine, and dopamine D1 receptor antagonist, SCH 23390, blocked the ameliorating effect of galantamine on Meth-induced memory impairment, whereas the muscarinic AChR antagonist, scopolamine, had no effect. The effects of galantamine on extracellular dopamine release were also antagonized by mecamylamine. Galantamine attenuated the defect of the novelty-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). The ameliorating effect of galantamine on recognition memory in Meth-treated mice was negated by microinjection of an ERK inhibitor, PD98059, into the PFC. These results suggest that the ameliorating effect of galantamine on Meth-induced memory impairment is associated with indirect activation of dopamine D1 receptor-ERK1/2 following augmentation with dopaminergic neurotransmission in the PFC through the allosteric activation of nAChRs. Galantamine could be a useful therapeutic agent for treating cognitive deficits in schizophrenia/Meth psychosis, as well as Alzheimer's disease.

  17. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

    PubMed Central

    Pantel, Jacques; Williams, Savannah Y.; Mi, Dehui; Sebag, Julien; Corbin, Jackie D.; Weaver, C. David; Cone, Roger D.

    2011-01-01

    The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z’=0.50). A pilot screen run on the Microsource Spectrum compound library (n= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists –the β2AR being endogenously expressed in HEK293 cells-, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of an HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  18. Consideration of allosterism and interacting proteins in the physiological functions of the serotonin transporter.

    PubMed

    Zhong, Huailing; Sánchez, Connie; Caron, Marc G

    2012-02-15

    The serotonin transporter (SERT) functions to transport serotonin (5-HT) from the extracellular space into neurons to maintain homeostatic control of 5-HT. It is the molecular target for selective serotonin reuptake inhibitor (SSRI) antidepressants. Preclinical research has shown that some SERT inhibitors can bind to two distinct binding sites on the SERT, a primary high affinity binding site and a low affinity allosteric binding site. Mutational studies of the SERT and computational modeling methods with escitalopram resulted in the identification of key amino acid residues important for the function of the allosteric binding site. While this allosteric binding site appears to influence the clinical efficacy of escitalopram under physiological conditions, the molecular mechanism of this effect is still poorly understood and may involve a large network of protein-protein interactions with the SERT. Dynamic interfaces between the SERT and the SERT interacting proteins (SIPs) potentially influence not only the SERT on its uptake function, its regulation, and trafficking, but also on known as well as yet to be identified non-canonical signaling pathways through SIPs. In this commentary, we outline approaches in the areas of selective small-molecule allosteric compound discovery, biochemistry, in vivo genetic knock-in mouse models, as well as computational and structural biology. These studies of the intra-molecular allosteric modulation of the SERT in the context of the myriad of potential inter-molecular signaling interactions with SIPs may help uncover unknown physiological functions of the SERT.

  19. A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death

    PubMed Central

    Furness, SGB; Hare, DL; Kourakis, A; Turnley, AM; Wookey, PJ

    2016-01-01

    We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis. PMID:27777788

  20. Kinetics of protein-ligand unbinding via smoothed potential molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Mollica, Luca; Decherchi, Sergio; Zia, Syeda Rehana; Gaspari, Roberto; Cavalli, Andrea; Rocchia, Walter

    2015-06-01

    Drug discovery is expensive and high-risk. Its main reasons of failure are lack of efficacy and toxicity of a drug candidate. Binding affinity for the biological target has been usually considered one of the most relevant figures of merit to judge a drug candidate along with bioavailability, selectivity and metabolic properties, which could depend on off-target interactions. Nevertheless, affinity does not always satisfactorily correlate with in vivo drug efficacy. It is indeed becoming increasingly evident that the time a drug spends in contact with its target (aka residence time) can be a more reliable figure of merit. Experimental kinetic measurements are operatively limited by the cost and the time needed to synthesize compounds to be tested, to express and purify the target, and to setup the assays. We present here a simple and efficient molecular-dynamics-based computational approach to prioritize compounds according to their residence time. We devised a multiple-replica scaled molecular dynamics protocol with suitably defined harmonic restraints to accelerate the unbinding events while preserving the native fold. Ligands are ranked according to the mean observed scaled unbinding time. The approach, trivially parallel and easily implementable, was validated against experimental information available on biological systems of pharmacological relevance.

  1. Required allosteric effector site for N-acetylglutamate on carbamoyl-phosphate synthetase I.

    PubMed

    McCudden, C R; Powers-Lee, S G

    1996-07-26

    Carbamoyl-phosphate synthetase I (CPSase I) catalyzes the entry and rate-limiting step in the urea cycle, the pathway by which mammals detoxify ammonia. One facet of CPSase I regulation is a requirement for N-acetylglutamate (AGA), which induces an active enzyme conformation and does not participate directly in the chemical reaction. We have utilized labeling with carbodiimide-activated [14C]AGA to identify peptides 120-127, 234-237, 625-630, and 1351-1356 as potentially being near the binding site for AGA. Identification of peptide 1351-1356 confirms the previous demonstration (Rodriquez-Aparicio, L. B., Guadalajara, A. M., and Rubio, V.(1989) Biochemistry 28, 3070-3074) that the C-terminal region is involved in binding AGA. Identification of peptides 120-127 and 234-237 constitutes the first evidence that the N-terminal region of the synthetase is involved in ligand binding. Since peptides 631-638 and 1327-1348 have been identified near the ATP site of CPSase I (Potter, M. D., and Powers-Lee, S. G.(1992) J. Biol. Chem. 267, 2023-2031), the present finding of involvement of peptides 625-630 and 1351-1356 at an "allosteric" activator site was unexpected. The idea that portions of the AGA effector site might be derived from an ancestral glutamine substrate site via a gene duplication and diversification event was considered. PMID:8663466

  2. Iterative In situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

    PubMed Central

    Millward, Steven W.; Henning, Ryan K.; Kwong, Gabriel A.; Pitram, Suresh; Agnew, Heather D.; Deyle, Kaycie M.; Nag, Arundhati; Hein, Jason; Lee, Su Seong; Lim, Jaehong; Pfeilsticker, Jessica A.; Sharpless, K. Barry; Heath, James R.

    2011-01-01

    We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to pre-inhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties PMID:21962254

  3. Molecular Motions as a Drug Target: Mechanistic Simulations of Anthrax Toxin Edema Factor Function Led to the Discovery of Novel Allosteric Inhibitors

    PubMed Central

    Laine, Élodie; Martínez, Leandro; Ladant, Daniel; Malliavin, Thérèse; Blondel, Arnaud

    2012-01-01

    Edema Factor (EF) is a component of Bacillus anthracis toxin essential for virulence. Its adenylyl cyclase activity is induced by complexation with the ubiquitous eukaryotic cellular protein, calmodulin (CaM). EF and its complexes with CaM, nucleotides and/or ions, have been extensively characterized by X-ray crystallography. Those structural data allowed molecular simulations analysis of various aspects of EF action mechanism, including the delineation of EF and CaM domains through their association energetics, the impact of calcium binding on CaM, and the role of catalytic site ions. Furthermore, a transition path connecting the free inactive form to the CaM-complexed active form of EF was built to model the activation mechanism in an attempt to define an inhibition strategy. The cavities at the surface of EF were determined for each path intermediate to identify potential sites where the binding of a ligand could block activation. A non-catalytic cavity (allosteric) was found to shrink rapidly at early stages of the path and was chosen to perform virtual screening. Amongst 18 compounds selected in silico and tested in an enzymatic assay, 6 thiophen ureidoacid derivatives formed a new family of EF allosteric inhibitors with IC50 as low as 2 micromolars. PMID:23012649

  4. Allosteric modulation of glycine receptors

    PubMed Central

    Yevenes, Gonzalo E; Zeilhofer, Hanns Ulrich

    2011-01-01

    Inhibitory (or strychnine sensitive) glycine receptors (GlyRs) are anion-selective transmitter-gated ion channels of the cys-loop superfamily, which includes among others also the inhibitory γ-aminobutyric acid receptors (GABAA receptors). While GABA mediates fast inhibitory neurotransmission throughout the CNS, the action of glycine as a fast inhibitory neurotransmitter is more restricted. This probably explains why GABAA receptors constitute a group of extremely successful drug targets in the treatment of a wide variety of CNS diseases, including anxiety, sleep disorders and epilepsy, while drugs specifically targeting GlyRs are virtually lacking. However, the spatially more restricted distribution of glycinergic inhibition may be advantageous in situations when a more localized enhancement of inhibition is sought. Inhibitory GlyRs are particularly relevant for the control of excitability in the mammalian spinal cord, brain stem and a few selected brain areas, such as the cerebellum and the retina. At these sites, GlyRs regulate important physiological functions, including respiratory rhythms, motor control, muscle tone and sensory as well as pain processing. In the hippocampus, RNA-edited high affinity extrasynaptic GlyRs may contribute to the pathology of temporal lobe epilepsy. Although specific modulators have not yet been identified, GlyRs still possess sites for allosteric modulation by a number of structurally diverse molecules, including alcohols, neurosteroids, cannabinoids, tropeines, general anaesthetics, certain neurotransmitters and cations. This review summarizes the present knowledge about this modulation and the molecular bases of the interactions involved. PMID:21557733

  5. Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol.

    PubMed

    Olsen, Richard W; Li, Guo-Dong; Wallner, Martin; Trudell, James R; Bertaccini, Edward J; Lindahl, Erik; Miller, Keith W; Alkana, Ronald L; Davies, Daryl L

    2014-03-01

    The molecular mechanism(s) of action of anesthetic, and especially, intoxicating doses of alcohol (ethanol [EtOH]) have been of interest even before the advent of the Research Society on Alcoholism. Recent physiological, genetic, and biochemical studies have pin-pointed molecular targets for anesthetics and EtOH in the brain as ligand-gated ion channel (LGIC) membrane proteins, especially the pentameric (5 subunit) Cys-loop superfamily of neurotransmitter receptors including nicotinic acetylcholine (nAChRs), GABAA (GABAA Rs), and glycine receptors (GlyRs). The ability to demonstrate molecular and structural elements of these proteins critical for the behavioral effects of these drugs on animals and humans provides convincing evidence for their role in the drugs' actions. Amino acid residues necessary for pharmacologically relevant allosteric modulation of LGIC function by anesthetics and EtOH have been identified in these channel proteins. Site-directed mutagenesis revealed potential allosteric modulatory sites in both the trans-membrane domain (TMD) and extracellular domain (ECD). Potential sites of action and binding have been deduced from homology modeling of other LGICs with structures known from crystallography and cryo-electron microscopy studies. Direct information about ligand binding in the TMD has been obtained by photoaffinity labeling, especially in GABAA Rs. Recent structural information from crystallized procaryotic (ELIC and GLIC) and eukaryotic (GluCl) LGICs allows refinement of the structural models including evaluation of possible sites of EtOH action.

  6. Organism-adapted specificity of the allosteric regulation of pyruvate kinase in lactic acid bacteria.

    PubMed

    Veith, Nadine; Feldman-Salit, Anna; Cojocaru, Vlad; Henrich, Stefan; Kummer, Ursula; Wade, Rebecca C

    2013-01-01

    Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may

  7. A Common Molecular Motif Characterizes Extracellular Allosteric Enhancers of GPCR Aminergic Receptors and Suggests Enhancer Mechanism of Action

    PubMed Central

    Bernstein, Robert Root; Dillon, Patrick F

    2014-01-01

    Several classes of compounds that have no intrinsic activity on aminergic systems nonetheless enhance the potency of aminergic receptor ligands three-fold or more while significantly increasing their duration of activity, preventing tachyphylaxis and reversing fade. Enhancer compounds include ascorbic acid, ethylenediaminetetraacetic acid, cortico-steroids, opioid peptides, opiates and opiate antagonists. This paper provides the first review of aminergic enhancement, demonstrating that all enhancers have a common, inobvious molecular motif and work through a common mechanism that is manifested by three common characteristics. First, aminergic enhancers bind directly to the amines they enhance, suggesting that the common structural motif is reflected in common binding targets. Second, one common target is the first extracellular loop of aminergic receptors. Third, at least some enhancers are antiphosphodiesterases. These observations suggest that aminergic enhancers act on the extracellular surface of aminergic receptors to keep the receptor in its high affinity state, trapping the ligand inside the receptor. Enhancer binding produces allosteric modifications of the receptor structure that interfere with phosphorylation of the receptor, thereby inhibiting down-regulation of the receptor. The mechanism explains how enhancers potentiate aminergic activity and increase duration of activity and makes testable predictions about additional compounds that should act as aminergic enhancers. PMID:25174918

  8. Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 beta-lactamase.

    PubMed

    Mathieu, Valéry; Fastrez, Jacques; Soumillion, Patrice

    2010-09-01

    In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) beta-lactamase. The first one, cpTEM-1-His(3) was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn(2+). The second one, cpTEM-1-3M-His(2), was selected by a directed evolution strategy. It is allosterically down-regulated by Zn(2+), Ni(2+) and Co(2+) with binding affinities around 300 microM.

  9. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875.

    PubMed

    Srivastava, Ankita; Yano, Jason; Hirozane, Yoshihiko; Kefala, Georgia; Gruswitz, Franz; Snell, Gyorgy; Lane, Weston; Ivetac, Anthony; Aertgeerts, Kathleen; Nguyen, Jasmine; Jennings, Andy; Okada, Kengo

    2014-09-01

    Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3 Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.

  10. Exploration of Allosteric Agonism Structure-Activity Relationships within an Acetylene Series of Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254)

    PubMed Central

    Turlington, Mark; Noetzel, Meredith J.; Chun, Aspen; Zhou, Ya; Gogliotti, Rocco D.; Nguyen, Elizabeth D.; Gregory, Karen J.; Vinson, Paige N.; Rook, Jerri M.; Gogi, Kiran K.; Xiang, Zixiu; Bridges, Thomas M.; Daniels, J. Scott; Jones, Carrie; Niswender, Colleen M.; Meiler, Jens; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs. PMID:24050755

  11. Targeting the thyroid-stimulating hormone receptor with small molecule ligands and antibodies

    PubMed Central

    Davies, Terry F; Latif, Rauf

    2015-01-01

    Introduction The thyroid-stimulating hormone receptor (TSHR) is the essential molecule for thyroid growth and thyroid hormone production. Since it is also a key autoantigen in Graves’ disease and is involved in thyroid cancer pathophysiology, the targeting of the TSHR offers a logical model for disease control. Areas covered We review the structure and function of the TSHR and the progress in both small molecule ligands and TSHR antibodies for their therapeutic potential. Expert opinion Stabilization of a preferential conformation for the TSHR by allosteric ligands and TSHR antibodies with selective modulation of the signaling pathways is now possible. These tools may be the next generation of therapeutics for controlling the pathophysiological consequences mediated by the effects of the TSHR in the thyroid and other extrathyroidal tissues. PMID:25768836

  12. Radiosynthesis of [18F]ATPFU: a potential PET ligand for mTOR.

    PubMed

    Majo, Vattoly J; Simpson, Norman R; Prabhakaran, Jaya; Mann, J John; Kumar, J S Dileep

    2014-11-01

    Mammalian target of rapamycin (mTOR) plays a pivotal role in many aspects of cellular proliferation, and recent evidence suggests that an altered mTOR signaling pathway plays a central role in the pathogenesis of aging, tumor progression, neuropsychiatric, and major depressive disorder. Availability of a mTOR-specific PET tracer will facilitate monitoring early response to treatment with mTOR inhibitors that are under clinical development. Towards this we have developed the radiosynthesis of [(18)F]1-(4-(4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-1-(2,2,2-trifluoroethyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenyl)-3-(2-fluoroethyl)urea [(18)F]ATPFU ([(18)F]1) as an mTOR PET ligand. Synthesis of reference 1 and the precursor for radiolabeling, 4-(4-8-oxa-3-azabicyclo[3.2.1]-octan-3yl)-1-(2,2,2-trifluoroethyl)-1H-pyrazolo[3,4-d]pyrimidin-6yl)aniline (10), were achieved from beta-chloroaldehyde 3 in 4 and 5 steps, respectively, with an overall yield of 25-28%. [(18)F]Fluoroethylamine was prepared by heating N-[2-(toluene-4-sulfonyloxy)ethyl]phthalimide with [(18)F]fluoride ion in acetonitrile. [(18)F]1 was obtained by slow distillation under argon of [(18) F]FCH2CH2NH2 into amine 10 that was pre-treated with triphosgene at 0-5 °C. The total time required for the two-step radiosynthesis including semi-preparative HPLC purification was 90 min, and the overall radiochemical yield of [(18)F]1 for the process was 15 ± 5% based on [(18)F]fluoride ion (decay corrected). At the end of synthesis (EOS), the specific activity was 37-74 GBq/µmol (N = 6). PMID:25359578

  13. Enzyme-substrate complexes of allosteric citrate synthase: evidence for a novel intermediate in substrate binding.

    PubMed

    Duckworth, Harry W; Nguyen, Nham T; Gao, Yin; Donald, Lynda J; Maurus, Robert; Ayed, Ayeda; Bruneau, Brigitte; Brayer, Gary D

    2013-12-01

    The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.

  14. Structural flexibility, an essential component of the allosteric activation in Escherichia coli glucosamine-6-phosphate deaminase.

    PubMed

    Rudiño-Piñera, E; Morales-Arrieta, S; Rojas-Trejo, S P; Horjales, E

    2002-01-01

    A new crystallographic structure of the free active-site R conformer of the allosteric enzyme glucosamine-6-phosphate deaminase from Escherichia coli, coupled with previously reported structures of the T and R conformers, generates a detailed description of the heterotropic allosteric transition in which structural flexibility plays a central role. The T conformer's external zone [Horjales et al. (1999), Structure, 7, 527-536] presents higher B values than in the R conformers. The ligand-free enzyme (T conformer) undergoes an allosteric transition to the free active-site R conformer upon binding of the allosteric activator. This structure shows three alternate conformations of the mobile section of the active-site lid (residues 163-182), in comparison to the high B values for the unique conformation of the T conformer. One of these alternate R conformations corresponds to the active-site lid found when the substrate is bound. The disorder associated with the three alternate conformations can be related to the biological regulation of the K(m) of the enzyme for the reaction, which is metabolically required to maintain adequate concentrations of the activator, which holds the enzyme in its R state. Seven alternate conformations for the active-site lid and three for the C-terminus were refined for the T structure using isotropic B factors. Some of these conformers approach that of the R conformer in geometry. Furthermore, the direction of the atomic vibrations obtained with anisotropic B refinement supports the hypothesis of an oscillating rather than a tense T state. The concerted character of the allosteric transition is also analysed in view of the apparent dynamics of the conformers.

  15. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

    PubMed Central

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-01-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  16. Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

    PubMed Central

    Bridgham, Jamie T.; Keay, June; Ortlund, Eric A.; Thornton, Joseph W.

    2014-01-01

    An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become “stuck” in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations

  17. Vestigialization of an allosteric switch: genetic and structural mechanisms for the evolution of constitutive activity in a steroid hormone receptor.

    PubMed

    Bridgham, Jamie T; Keay, June; Ortlund, Eric A; Thornton, Joseph W

    2014-01-01

    An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become "stuck" in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations to

  18. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  19. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

    NASA Astrophysics Data System (ADS)

    Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

    2015-01-01

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  20. An allosteric receptor by simultaneous "casting" and "molding" in a dynamic combinatorial library.

    PubMed

    Li, Jianwei; Nowak, Piotr; Otto, Sijbren

    2015-01-12

    Allosteric synthetic receptors are difficult to access by design. Herein we report a dynamic combinatorial strategy towards such systems based on the simultaneous use of two different templates. Through a process of simultaneous casting (the assembly of a library member around a template) and molding (the assembly of a library member inside the binding pocket of a template), a Russian-doll-like termolecular complex was obtained with remarkable selectivity. Analysis of the stepwise formation of the complex indicates that binding of the two partners by the central macrocycle exhibits significant positive cooperativity. Such allosteric systems represent hubs that may have considerable potential in systems chemistry.

  1. A 3-D QSAR-BASED IDENTIFICATION ALGORITHM FOR POTENTIAL ESTROGEN RECEPTOR LIGANDS

    EPA Science Inventory

    Recent reports concerning the lethal effects of solar ultraviolet-B (UV-B) radiation on amphibians suggest that this stressor has the potential to impact some amphibian populations. In this study embryos and larvae of three anuran species, Rana pipiens, R. clamitans, and R. septe...

  2. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1.

    PubMed

    Fay, Jonathan F; Farrens, David L

    2015-07-01

    G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

  3. An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

    PubMed Central

    J Gingell, Joseph; Simms, John; Barwell, James; Poyner, David R; Watkins, Harriet A; Pioszak, Augen A; Sexton, Patrick M; Hay, Debbie L

    2016-01-01

    G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor

  4. Moving Beyond Active-Site Detection: MixMD Applied to Allosteric Systems.

    PubMed

    Ghanakota, Phani; Carlson, Heather A

    2016-08-25

    Mixed-solvent molecular dynamics (MixMD) is a hotspot-mapping technique that relies on molecular dynamics simulations of proteins in binary solvent mixtures. Previous work on MixMD has established the technique's effectiveness in capturing binding sites of small organic compounds. In this work, we show that MixMD can identify both competitive and allosteric sites on proteins. The MixMD approach embraces full protein flexibility and allows competition between solvent probes and water. Sites preferentially mapped by probe molecules are more likely to be binding hotspots. There are two important requirements for the identification of ligand-binding hotspots: (1) hotspots must be mapped at very high signal-to-noise ratio and (2) the hotspots must be mapped by multiple probe types. We have developed our mapping protocol around acetonitrile, isopropanol, and pyrimidine as probe solvents because they allowed us to capture hydrophilic, hydrophobic, hydrogen-bonding, and aromatic interactions. Charged probes were needed for mapping one target, and we introduce them in this work. In order to demonstrate the robust nature and wide applicability of the technique, a combined total of 5 μs of MixMD was applied across several protein targets known to exhibit allosteric modulation. Most notably, all the protein crystal structures used to initiate our simulations had no allosteric ligands bound, so there was no preorganization of the sites to predispose the simulations to find the allosteric hotspots. The protein test cases were ABL Kinase, Androgen Receptor, CHK1 Kinase, Glucokinase, PDK1 Kinase, Farnesyl Pyrophosphate Synthase, and Protein-Tyrosine Phosphatase 1B. The success of the technique is demonstrated by the fact that the top-four sites solely map the competitive and allosteric sites. Lower-ranked sites consistently map other biologically relevant sites, multimerization interfaces, or crystal-packing interfaces. Lastly, we highlight the importance of including protein

  5. Computational fragment-based drug design to explore the hydrophobic sub-pocket of the mitotic kinesin Eg5 allosteric binding site.

    PubMed

    Oguievetskaia, Ksenia; Martin-Chanas, Laetitia; Vorotyntsev, Artem; Doppelt-Azeroual, Olivia; Brotel, Xavier; Adcock, Stewart A; de Brevern, Alexandre G; Delfaud, Francois; Moriaud, Fabrice

    2009-08-01

    Eg5, a mitotic kinesin exclusively involved in the formation and function of the mitotic spindle has attracted interest as an anticancer drug target. Eg5 is co-crystallized with several inhibitors bound to its allosteric binding pocket. Each of these occupies a pocket formed by loop 5/helix alpha2 (L5/alpha2). Recently designed inhibitors additionally occupy a hydrophobic pocket of this site. The goal of the present study was to explore this hydrophobic pocket with our MED-SuMo fragment-based protocol, and thus discover novel chemical structures that might bind as inhibitors. The MED-SuMo software is able to compare and superimpose similar interaction surfaces upon the whole protein data bank (PDB). In a fragment-based protocol, MED-SuMo retrieves MED-Portions that encode protein-fragment binding sites and are derived from cross-mining protein-ligand structures with libraries of small molecules. Furthermore we have excluded intra-family MED-Portions derived from Eg5 ligands that occupy the hydrophobic pocket and predicted new potential ligands by hybridization that would fill simultaneously both pockets. Some of the latter having original scaffolds and substituents in the hydrophobic pocket are identified in libraries of synthetically accessible molecules by the MED-Search software.

  6. Estrogen Receptors Alpha (ERα) and Beta (ERβ): Subtype-Selective Ligands and Clinical Potential

    PubMed Central

    Paterni, Ilaria; Granchi, Carlotta; Katzenellenbogen, John A.; Minutolo, Filippo

    2014-01-01

    Estrogen receptors alpha (ERα) and beta (ERβ) are nuclear transcription factors that are involved in the regulation of many complex physiological processes in humans. Modulation of these receptors by prospective therapeutic agents is currently being considered for prevention and treatment of a wide variety of pathological conditions, such as, cancer, metabolic and cardiovascular diseases, neurodegeneration, inflammation, and osteoporosis. This review provides an overview and update of compounds that have been recently reported as modulators of ERs, with a particular focus on their potential clinical applications. PMID:24971815

  7. Monovalent cation and amiloride analog modulation of adrenergic ligand binding to the unglycosylated alpha 2B-adrenergic receptor subtype

    SciTech Connect

    Wilson, A.L.; Seibert, K.; Brandon, S.; Cragoe, E.J. Jr.; Limbird, L.E. )

    1991-04-01

    The unglycosylated alpha 2B subtype of the alpha 2-adrenergic receptor found in NG-108-15 cells possesses allosteric regulation of adrenergic ligand binding by monovalent cations and 5-amino-substituted amiloride analogs. These findings demonstrate that allosteric modulation of adrenergic ligand binding is not a property unique to the alpha 2A subtype. The observation that amiloride analogs as well as monovalent cations can modulate adrenergic ligand binding to the nonglycosylated alpha 2B subtype indicates that charge shielding due to carbohydrate moieties does not play a role in this allosteric modulation but, rather, these regulatory effects result from interactions of cations and amiloride analogs with the protein moiety of the receptor. Furthermore, the observation that both alpha 2A and alpha 2B receptor subtypes are modulated by amiloride analogs suggests that structural domains that are conserved between the two are likely to be involved in this allosteric modulation.

  8. Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment.

    PubMed

    García-Echeverría, Carlos

    2010-08-01

    Over the past few years a number of components of the PI3K/mTOR pathway have been the subject of intense drug discovery activities both in pharmaceutical companies and in academia. This review article summarizes progress made in the identification and development of allosteric and ATP-competitive kinase inhibitors of mTOR and their potential therapeutic use in oncology.

  9. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  10. Early-Late Heterobimetallic Complexes Linked by Phosphinoamide Ligands. Tuning Redox Potentials and Small Molecule Activation

    SciTech Connect

    Thomas, Christine M.

    2015-08-01

    Recent attention in the chemical community has been focused on the energy efficient and environmentally benign conversion of abundant small molecules (CO2, H2O, etc.) to useful liquid fuels. This project addresses these goals by examining fundamental aspects of catalyst design to ultimately access small molecule activation processes under mild conditions. Specifically, Thomas and coworkers have targetted heterobimetallic complexes that feature metal centers with vastly different electronic properties, dictated both by their respective positions on the periodic table and their coordination environment. Unlike homobimetallic complexes featuring identical or similar metals, the bonds between metals in early/late heterobimetallics are more polarized, with the more electron-rich late metal center donating electron density to the more electron-deficient early metal center. While metal-metal bonds pose an interesting strategy for storing redox equivalents and stabilizing reactive metal fragments, the polar character of metal-metal bonds in heterobimetallic complexes renders these molecules ideally poised to react with small molecule substrates via cleavage of energy-rich single and double bonds. In addition, metal-metal interactions have been shown to dramatically affect redox potentials and promote multielectron redox activity, suggesting that metal-metal interactions may provide a mechanism to tune redox potentials and access substrate reduction/activation at mild overpotentials. This research project has provided a better fundamental understanding of how interactions between transition metals can be used as a strategy to promote and/or control chemical transformations related to the clean production of fuels. While this project focused on the study of homogeneous systems, it is anticipated that the broad conclusions drawn from these investigations will be applicable to heterogeneous catalysis as well, particularly on heterogeneous processes that occur at interfaces in

  11. Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus.

    PubMed

    Ortigosa, Allison D; Kimmel, Jennifer L; Reinhart, Gregory D

    2004-01-20

    A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.

  12. 13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase*

    PubMed Central

    Kudalkar, Shalley N.; Nikas, Spyros P.; Kingsley, Philip J.; Xu, Shu; Galligan, James J.; Rouzer, Carol A.; Banerjee, Surajit; Ji, Lipin; Eno, Marsha R.; Makriyannis, Alexandros; Marnett, Lawrence J.

    2015-01-01

    Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism. PMID:25648895

  13. Detecting Allosteric Networks Using Molecular Dynamics Simulation.

    PubMed

    Bowerman, S; Wereszczynski, J

    2016-01-01

    Allosteric networks allow enzymes to transmit information and regulate their catalytic activities over vast distances. In principle, molecular dynamics (MD) simulations can be used to reveal the mechanisms that underlie this phenomenon; in practice, it can be difficult to discern allosteric signals from MD trajectories. Here, we describe how MD simulations can be analyzed to reveal correlated motions and allosteric networks, and provide an example of their use on the coagulation enzyme thrombin. Methods are discussed for calculating residue-pair correlations from atomic fluctuations and mutual information, which can be combined with contact information to identify allosteric networks and to dynamically cluster a system into highly correlated communities. In the case of thrombin, these methods show that binding of the antagonist hirugen significantly alters the enzyme's correlation landscape through a series of pathways between Exosite I and the catalytic core. Results suggest that hirugen binding curtails dynamic diversity and enforces stricter venues of influence, thus reducing the accessibility of thrombin to other molecules. PMID:27497176

  14. Progress in developing cholecystokinin (CCK)/gastrin receptor ligands which have therapeutic potential

    PubMed Central

    Berna, Marc J.; Tapia, Jose A.; Sancho, Veronica; Jensen, Robert T.

    2007-01-01

    Summary Gastrin and CCK are two of the oldest hormones and within the last 15 years there has been an exponential increase in knowledge of their pharmacology, cell biology, receptors (CCK1R, CCK2R) and roles in physiology and pathological conditions. Despite these advances there is no approved disease indication for CCK receptor antagonists and only minor use of agonists. In this review the important factors determining this slow therapeutic development are reviewed. To assess this it is necessary to briefly review what is known about the roles of CCK receptors (CCK1R, CCK2R) in normal human physiology, their role in pathologic conditions, the selectivity of available potent CCKR agonists/antagonists as well as review their use in human conditions to date and the results. Despite extensive studies in animals and some in humans, recent studies suggest that monotherapy with CCK1R agonists will not be effective in obesity, nor CCK2R antagonists in panic disorders or CCK2R antagonists to inhibit growth of pancreatic cancer. Areas that require more study include the use of CCK2R agonists for imaging tumors and radiotherapy, CCK2R antagonists in hypergastrinemic states especially with long term PPI use and for potentiation of analgesia as well as use of CCK1R antagonists for a number of gastrointestinal disorders [motility disorders (irritable bowel syndrome, dyspepsia, constipation) and pancreatitis (acute, chronic)]. PMID:17997137

  15. Discovery of very late antigen-4 (VLA-4, alpha4beta1 integrin) allosteric antagonists.

    PubMed

    Chigaev, Alexandre; Wu, Yang; Williams, D Bart; Smagley, Yelena; Sklar, Larry A

    2011-02-18

    Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ∼2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies. PMID:21131351

  16. Regulatory changes in the control of carbamoyl phosphate synthetase induced by truncation and mutagenesis of the allosteric binding domain.

    PubMed

    Czerwinski, R M; Mareya, S M; Raushel, F M

    1995-10-24

    Carbamoyl phosphate synthetase from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases in all of the enzymatic activities catalyzed by the enzyme. A 40-fold decrease in the glutamine-dependent ATPase activity was observed for the delta 14 truncation. Similar losses in activity were also observed for the delta 50, delta 65, delta 91, and delta 119 mutant proteins. However, formation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the delta 91 or delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7577987

  17. Design, Synthesis and Biological Evaluation of Aminoalkylindole Derivatives as Cannabinoid Receptor Ligands with Potential for Treatment of Alcohol Abuse

    PubMed Central

    Vasiljevik, Tamara; Franks, Lirit N.; Ford, Benjamin M.; Douglas, Justin T.; Prather, Paul L.; Fantegrossi, William E.; Prisinzano, Thomas E.

    2013-01-01

    Attenuation of increased endocannabinoid signaling with a CB1R neutral antagonist might offer a new therapeutic direction for treatment of alcohol abuse. We have recently reported that a mono-hydroxylated metabolite of the synthetic aminoalkylindole cannabinoid JHW-073 (3) exhibits neutral antagonist activity at CB1Rs and thus may serve as a promising lead for the development of novel alcohol abuse therapies. In the current study, we show that systematic modification of an aminoalkylindole scaffold identified two new compounds with dual CB1R antagonist/CB2R agonist activity. Similar to the CB1R antagonist/inverse agonist rimonabant, analogues 27 and 30 decrease oral alcohol self-administration, without affecting total fluid intake and block the development of alcohol-conditioned place preference. Collectively, these initial findings suggest that design and systematic modification of aminoalkylindoles such as 3 may lead to development of novel cannabinoid ligands with dual CB1R antagonist/CB2R agonist activity with potential for use as treatments of alcohol abuse. PMID:23631463

  18. System with potential dual modes of metal-ligand cooperation: highly catalytically active pyridine-based PNNH-Ru pincer complexes.

    PubMed

    Fogler, Eran; Garg, Jai Anand; Hu, Peng; Leitus, Gregory; Shimon, Linda J W; Milstein, David

    2014-11-24

    Metal-ligand cooperation (MLC) plays an important role in catalysis. Systems reported so far are generally based on a single mode of MLC. We report here a system with potential for MLC by both amine-amide and aromatization-dearomatization ligand transformations, based on a new class of phosphino-pyridyl ruthenium pincer complexes, bearing sec-amine coordination. These pincer complexes are effective catalysts under unprecedented mild conditions for acceptorless dehydrogenative coupling of alcohols to esters at 35 °C and hydrogenation of esters at room temperature and 5 atm H2. The likely actual catalyst, a novel, crystallographically characterized monoanionic de-aromatized enamido-Ru(II) complex, was obtained by deprotonation of both the N-H and the methylene proton of the N-arm of the pincer ligand. PMID:25331061

  19. Structural Basis for Negative Allosteric Modulation of GluN2A-Containing NMDA Receptors.

    PubMed

    Yi, Feng; Mou, Tung-Chung; Dorsett, Katherine N; Volkmann, Robert A; Menniti, Frank S; Sprang, Stephen R; Hansen, Kasper B

    2016-09-21

    NMDA receptors mediate excitatory synaptic transmission and regulate synaptic plasticity in the central nervous system, but their dysregulation is also implicated in numerous brain disorders. Here, we describe GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo state of the GluN1 ligand-binding domain (LBD), which is incapable of triggering channel gating. We describe structural determinants of NAM binding in crystal structures of the GluN1/2A LBD heterodimer, and analyses of NAM-bound LBD structures corresponding to active and inhibited receptor states reveal a molecular switch in the modulatory binding site that mediate the allosteric inhibition. NAM binding causes displacement of a valine in GluN2A and the resulting steric effects can be mitigated by the transition from glycine bound to apo state of the GluN1 LBD. This work provides mechanistic insight to allosteric NMDA receptor inhibition, thereby facilitating the development of novel classes NMDA receptor modulators as therapeutic agents. PMID:27618671

  20. TNF-related apoptosis-inducing ligand (TRAIL): a potential candidate for combined treatment of hematological malignancies.

    PubMed

    Secchiero, Paola; Vaccarezza, Mauro; Gonelli, Arianna; Zauli, Giorgio

    2004-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF gene superfamily, which induces apoptosis through engagement of death receptors. TRAIL is unusual as compared to the other cytokines of this family, as it interacts with a complex system of receptors consisting of two pro-apoptotic death receptors (TRAIL-R1 and TRAIL-R2) and three decoy receptors (TRAIL-R3, TRAIL-R4 and osteoprotegerin). Moreover, with respect to other members of the TNF superfamily, such as CD95L and TNF-alpha, TRAIL has generated great interest as a potential tumor-specific cancer therapeutic because as a stable soluble trimer it selectively induces apoptosis in many transformed cells but not in normal cells. Of note, TRAIL cytotoxicity is at least partially independent of the major systems involved in resistance to chemotherapy, such as p53 wild-type function and multidrug resistance (MDR) genes. Since one fundamental problem of most cancers is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of conventional chemotherapy, new therapeutic approaches that either restore the pro-apoptotic activity of chemotherapeutic drugs or by-pass the mechanisms of resistance are highly desirable. This review will focus on the potential of TRAIL for its application in the therapy of hematological malignancies, used either alone or in combination with chemotherapy. The scenario emerging from the literature is that the treatment and management of hematological malignancies will require the rational combination of TRAIL plus conventional or new drugs in a regimen that would optimize the anti-neoplastic activity in malignant cells resistant to chemotherapy through restoration of the pro-apoptotic activity of TRAIL. PMID:15579063

  1. Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.

    PubMed

    McGresham, Maria S; Reinhart, Gregory D

    2015-01-27

    The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol(-1) (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol(-1) increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild

  2. Assessment of subunit-dependent direct gating and allosteric modulatory effects of carisoprodol at GABA(A) receptors.

    PubMed

    Kumar, Manoj; González, Lorie A; Dillon, Glenn H

    2015-10-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1-6 and z = 1-3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects.

  3. The global allostery model of hemoglobin: an allosteric mechanism involving homotropic and heterotropic interactions.

    PubMed

    Yonetani, Takashi; Tsuneshige, Antonio

    2003-06-01

    Studies of the allosteric mechanism of hemoglobin (Hb) have evolved from phenomenological descriptions to structure-based molecular mechanisms, as the molecular structures of Hb in deoxy and ligated states have been elucidated. The MWC two-state concerted model has been the widely accepted as the most plausible of the allosteric mechanisms of Hb. It assumes that the O2-affinity of Hb is regulated/controlled primarily by the T/R quaternary structural transition and that heterotropic effectors bind preferentially to T (deoxy) Hb to shift the T/R allosteric equilibrium toward the T state. However, recent more comprehensive O2-binding measurements of Hb have revealed a new mechanism, the Global Allostery model. It describes that the O2-affinity and the cooperativity are modulated in greater extents and the Bohr effect is generated primarily by the tertiary structural changes in both T (deoxy) and R (ligated) states of Hb. Differential interactions of heterotropic allosteric effectors with both T (deoxy) and R (ligated) states of Hb induce these tertiary structural changes. The X-ray structure of a complex of R (ligated) Hb with BZF, a potent heterotropic effector, has revealed the stereo-chemical influence of these effectors on the structure of R (ligated) Hb, resulting in the reduction of the ligand affinity in R (ligated) Hb. This model stresses the fundamental importance of the heterotropic interactions in regulation/control of the functionality of Hb. They alter the tertiary structures of both T (deoxy) and R (oxy) Hb, leading to large-scale modulations of the O2 affinity (KT and KR), and consequently the cooperativity (KR/KT) and the Bohr effect (delta P50/delta pH) from a global viewpoint of allostery in Hb.

  4. Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

    PubMed

    Alvarez-Añorve, Laura I; Alonzo, Diego A; Mora-Lugo, Rodrigo; Lara-González, Samuel; Bustos-Jaimes, Ismael; Plumbridge, Jacqueline; Calcagno, Mario L

    2011-12-01

    The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

  5. Determination of ζ-potential, charge, and number of organic ligands on the surface of water soluble quantum dots by capillary electrophoresis.

    PubMed

    Voráčová, Ivona; Klepárník, Karel; Lišková, Marcela; Foret, František

    2015-03-01

    The number of charges and/or organic ligands covalently attached to the surface of CdTe quantum dot nanoparticles has been determined from their electrophoretic mobilities measured in capillaries filled with free electrolyte buffers. Three sizes of water soluble CdTe quantum dots with 3-mercaptopropionic and thioglycolic acids as surface ligands were prepared. Their electrophoretic mobilities in different pH and ionic strength values of separation buffers were measured by capillary electrophoresis with laser induced fluorescence detection. The ζ-potentials determined from electrophoretic mobilities using analytical solution of Henry function proposed by Ohshima were in the range from -30 to -100 mV. Charges of QDs were calculated from ζ-potentials. As a result, numbers of organic ligands bonded to QDs surface were determined to be 13, 14, and 15 for the sizes of 3.1, 3.5, and 3.9 nm, respectively. The dissociation constants of organic ligands bonded on QDs surfaces estimated from the dependence of QDs charge on pH of the separation buffer were 7.8 and 7.9 for 3-mercaptopropionic acid and 6.9 for thioglycolic acid. PMID:25521532

  6. Syntheses of 2-amino and 2-halothiazole derivatives as high-affinity metabotropic glutamate receptor subtype 5 ligands and potential radioligands for in vivo imaging.

    PubMed

    Siméon, Fabrice G; Wendahl, Matthew T; Pike, Victor W

    2011-02-10

    The structure of the potent selective mGlu(5) ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu(5) ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed subnanomolar mGlu(5) affinity. 10b also displayed >10000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [(18)F]fluoride ion (t(1/2) = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu(5) with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu(5) and may also serve as a lead to a potential (123)I-labeled ligand for imaging brain mGlu(5) with single photon emission computed tomography. PMID:21207959

  7. Syntheses of 2-Amino and 2-Halothiazole Derivatives as High-Affinity Metabotropic Glutamate Receptor Subtype 5 Ligands and Potential Radioligands for In Vivo Imaging

    PubMed Central

    Siméon, Fabrice G; Wendahl, Matthew T.; Pike, Victor W.

    2011-01-01

    The structure of the potent selective mGlu5 ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu5 ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed sub-nanomolar mGlu5 affinity. 10b also displayed >10,000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [18F]fluoride ion (t1/2 = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu5 with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu5 and may also serve as a lead to a potential 123I-labeled ligand for imaging brain mGlu5 with single photon emission computed tomography. PMID:21207959

  8. Design of an allosterically regulated retroaldolase

    PubMed Central

    Raymond, Elizabeth A; Mack, Korrie L; Yoon, Jennifer H; Moroz, Olesia V; Moroz, Yurii S; Korendovych, Ivan V

    2015-01-01

    We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca2+ ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue. PMID:25516403

  9. 5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

    PubMed

    Massink, Arnault; Louvel, Julien; Adlere, Ilze; van Veen, Corine; Huisman, Berend J H; Dijksteel, Gabrielle S; Guo, Dong; Lenselink, Eelke B; Buckley, Benjamin J; Matthews, Hayden; Ranson, Marie; Kelso, Michael; IJzerman, Adriaan P

    2016-05-26

    The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site. PMID:27124340

  10. Molecular Basis of Allosteric Transitions: GroEL

    NASA Astrophysics Data System (ADS)

    Horovitz, Amnon

    Chaperonins such as GroEL from Escherichia coli are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. Here, we describe some of the various functional (allosteric) states of GroEL, the pathways by which they inter-convert and the coupling between allosteric transitions and protein folding reactions.

  11. 2013 Philip S. Portoghese Medicinal Chemistry Lectureship: Drug Discovery Targeting Allosteric Sites†

    PubMed Central

    2015-01-01

    The identification of sites on receptors topographically distinct from the orthosteric sites, so-called allosteric sites, has heralded novel approaches and modes of pharmacology for target modulation. Over the past 20 years, our understanding of allosteric modulation has grown significantly, and numerous advantages, as well as caveats (e.g., flat structure–activity relationships, species differences, “molecular switches”), have been identified. For multiple receptors and proteins, numerous examples have been described where unprecedented levels of selectivity are achieved along with improved physiochemical properties. While not a panacea, these novel approaches represent exciting opportunities for tool compound development to probe the pharmacology and therapeutic potential of discrete molecular targets, as well as new medicines. In this Perspective, in commemoration of the 2013 Philip S. Portoghese Medicinal Chemistry Lectureship (LindsleyC. W.Adventures in allosteric drug discovery. Presented at the 246th National Meeting of the American Chemical Society, Indianapolis, IN, September 10, 2013; The 2013 Portoghese Lectureship), several vignettes of drug discovery campaigns targeting novel allosteric mechanisms will be recounted, along with lessons learned and guidelines that have emerged for successful lead optimization. PMID:25180768

  12. Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding.

    PubMed

    Casino, Patricia; Niks, Dimitri; Ngo, Huu; Pan, Peng; Brzovic, Peter; Blumenstein, Lars; Barends, Thomas Reinier; Schlichting, Ilme; Dunn, Michael F

    2007-07-01

    Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.

  13. Tuning redox potentials of bis(imino)pyridine cobalt complexes: an experimental and theoretical study involving solvent and ligand

    SciTech Connect

    Moyses Araujo, C.; Doherty, Mark D.; Konezny, Steven J.; Luca, Oana R.; Usyatinsky, Alex; Grade, Hans; Lobkovsky, Emil; Soloveichik, Grigorii L.; Crabtree, Robert H.; Batista, Victor S.

    2012-01-01

    The structure and electrochemical properties of a series of bis(imino)pyridine CoII complexes (NNN)CoX₂ and [(NNN)₂Co][PF₆]₂ (NNN = 2,6-bis[1-(4-R-phenylimino)ethyl]pyridine, with R = CN, CF₃, H, CH₃, OCH₃, N(CH₃)₂; NNN = 2,6-bis[1-(2,6-(iPr)₂-phenylimino)ethyl]pyridine and X = Cl, Br) were studied using a combination of electrochemical and theoretical methods. Cyclic voltammetry measurements and DFT/B3LYP calculations suggest that in solution (NNN)CoCl₂ complexes exist in equilibrium with disproportionation products [(NNN)₂Co]²⁺ [CoCl₄]²⁻ with the position of the equilibrium heavily influenced by both the solvent polarity and the steric and electronic properties of the bis(imino)pyridine ligands. In strong polar solvents (e.g., CH₃CN or H₂O) or with electron donating substituents (R = OCH₃ or N(CH₃)₂) the equilibrium is shifted and only oxidation of the charged products [(NNN)₂Co]²⁺ and [CoCl₄]²⁻ is observed. Conversely, in nonpolar organic solvents such as CH₂Cl₂ or with electron withdrawing substituents (R = CN or CF₃), disproportionation is suppressed and oxidation of the (NNN)CoCl₂ complexes leads to 18e⁻ CoIII complexes stabilized by coordination of a solvent moiety. In addition, the [(NNN)₂Co][PF₆]₂ complexes exhibit reversible CoII/III oxidation potentials that are strongly dependent on the electron withdrawing/donating nature of the N-aryl substituents, spanning nearly 750 mV in acetonitrile. The resulting insight on the regulation of redox properties of a series of bis(imino)pyridine cobalt(II) complexes should be particularly valuable to tune suitable conditions for reactivity.

  14. Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

    PubMed Central

    Chio, Cynthia M.; Yu, Xiaoling; Bishop, Anthony C.

    2015-01-01

    Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are critical regulators of metazoan cell signaling and have emerged as potential drug targets for a range of human diseases. Strategies for chemically targeting the function of individual PTPs selectively could serve to elucidate the signaling roles of these enzymes and would potentially expedite validation of the therapeutic promise of PTP inhibitors. Here we report a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs; these sites, which can be introduced into target PTPs through protein engineering, serve to sensitize target PTPs to potent and selective inhibition by a biarsenical small molecule. Building on the recent discovery of a naturally occurring cryptic allosteric site in wild-type Src-homology-2 domain containing PTP (Shp2) that can be targeted by biarsenical compounds, we hypothesized that Shp2’s unusual sensitivity to biarsenicals could be strengthened through rational design and that the Shp2-specific site could serve as a blueprint for the introduction of non-natural inhibitor sensitivity in other PTPs. Indeed, we show here that the strategic introduction of a cysteine residue at a position removed from the Shp2 active site can serve to increase the potency and selectivity of the interaction between Shp2’s allosteric site and the biarsenical inhibitor. Moreover, we find that “Shp2-like” allosteric sites can be installed de novo in PTP enzymes that do not possess naturally occurring sensitivity to biarsenical compounds. Using primary-sequence alignments to guide our enzyme engineering, we have successfully introduced allosteric-inhibition sites in four classical PTPs—PTP1B, PTPH-1, FAP-1, and HePTP—from four different PTP subfamilies, suggesting that our sensitization approach can likely be applied widely across the classical PTP family to generate biarsenical-responsive PTPs. PMID:25828055

  15. Universal allosteric mechanism for Gα activation by GPCRs

    PubMed Central

    Flock, Tilman; Venkatakrishnan, A. J.; Kayikci, Melis; Tate, Christopher G.; Veprintsev, Dmitry B.; Babu, M. Madan

    2016-01-01

    G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ~800 human GPCRs and 16 different Gα proteins, does a universal allosteric mechanism govern Gα activation? Here we show that different GPCRs interact and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that can undergo disorder-order transitions decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, whilst conserving the allosteric activation mechanism. PMID:26147082

  16. Universal allosteric mechanism for Gα activation by GPCRs.

    PubMed

    Flock, Tilman; Ravarani, Charles N J; Sun, Dawei; Venkatakrishnan, A J; Kayikci, Melis; Tate, Christopher G; Veprintsev, Dmitry B; Babu, M Madan

    2015-08-13

    G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism. PMID:26147082

  17. Investigation of allosteric modulation mechanism of metabotropic glutamate receptor 1 by molecular dynamics simulations, free energy and weak interaction analysis

    NASA Astrophysics Data System (ADS)

    Bai, Qifeng; Yao, Xiaojun

    2016-02-01

    Metabotropic glutamate receptor 1 (mGlu1), which belongs to class C G protein-coupled receptors (GPCRs), can be coupled with G protein to transfer extracellular signal by dimerization and allosteric regulation. Unraveling the dimer packing and allosteric mechanism can be of great help for understanding specific regulatory mechanism and designing more potential negative allosteric modulator (NAM). Here, we report molecular dynamics simulation studies of the modulation mechanism of FITM on the wild type, T815M and Y805A mutants of mGlu1 through weak interaction analysis and free energy calculation. The weak interaction analysis demonstrates that van der Waals (vdW) and hydrogen bonding play an important role on the dimer packing between six cholesterol molecules and mGlu1 as well as the interaction between allosteric sites T815, Y805 and FITM in wild type, T815M and Y805A mutants of mGlu1. Besides, the results of free energy calculations indicate that secondary binding pocket is mainly formed by the residues Thr748, Cys746, Lys811 and Ser735 except for FITM-bound pocket in crystal structure. Our results can not only reveal the dimer packing and allosteric regulation mechanism, but also can supply useful information for the design of potential NAM of mGlu1.

  18. Modeling the Contribution of Allosteric Regulation for Flux Control in the Central Carbon Metabolism of E. coli

    PubMed Central

    Machado, Daniel; Herrgård, Markus J.; Rocha, Isabel

    2015-01-01

    Modeling cellular metabolism is fundamental for many biotechnological applications, including drug discovery and rational cell factory design. Central carbon metabolism (CCM) is particularly important as it provides the energy and precursors for other biological processes. However, the complex regulation of CCM pathways has still not been fully unraveled and recent studies have shown that CCM is mostly regulated at post-transcriptional levels. In order to better understand the role of allosteric regulation in controlling the metabolic phenotype, we expand the reconstruction of CCM in Escherichia coli with allosteric interactions obtained from relevant databases. This model is used to integrate multi-omics datasets and analyze the coordinated changes in enzyme, metabolite, and flux levels between multiple experimental conditions. We observe cases where allosteric interactions have a major contribution to the metabolic flux changes. Inspired by these results, we develop a constraint-based method (arFBA) for simulation of metabolic flux distributions that accounts for allosteric interactions. This method can be used for systematic prediction of potential allosteric regulation under the given experimental conditions based on experimental data. We show that arFBA allows predicting coordinated flux changes that would not be predicted without considering allosteric regulation. The results reveal the importance of key regulatory metabolites, such as fructose-1,6-bisphosphate, in controlling the metabolic flux. Accounting for allosteric interactions in metabolic reconstructions reveals a hidden topology in metabolic networks, improving our understanding of cellular metabolism and fostering the development of novel simulation methods that account for this type of regulation. PMID:26501058

  19. Identification of diaryl ether-based ligands for estrogen-related receptor α as potential antidiabetic agents.

    PubMed

    Patch, Raymond J; Searle, Lily L; Kim, Alexander J; De, Debyendu; Zhu, Xizhen; Askari, Hossein B; O'Neill, John C; Abad, Marta C; Rentzeperis, Dionisios; Liu, Jianying; Kemmerer, Michael; Lin, Ling; Kasturi, Jyotsna; Geisler, John G; Lenhard, James M; Player, Mark R; Gaul, Micheal D

    2011-02-10

    Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has been functionally implicated in the regulation of energy homeostasis. Herein is described the development of diaryl ether based thiazolidenediones, which function as selective ligands against this receptor. Series optimization provided several potent analogues that inhibit the recruitment of a coactivator peptide fragment in in vitro biochemical assays (IC(50) < 150 nM) and cellular two-hybrid reporter assays against the ligand binding domain (IC(50) = 1-5 μM). A cocrystal structure of the ligand-binding domain of ERRα with lead compound 29 revealed the presence of a covalent interaction between the protein and ligand, which has been shown to be reversible. In diet-induced murine models of obesity and in an overt diabetic rat model, oral administration of 29 normalized insulin and circulating triglyceride levels, improved insulin sensitivity, and was body weight neutral. This provides the first demonstration of functional activities of an ERRα ligand in metabolic animal models.

  20. Bis(methylpyridine)-EDTA derivative as a potential ligand for PET imaging: synthesis, complexation, and biological evaluation.

    PubMed

    Singh, Pooja; Aggarwal, Swati; Tiwari, Anjani K; Kumar, Vikas; Pratap, Ramendra; Chuttani, Krishna; Mishra, Anil K

    2014-12-01

    A novel transitional metal ligand derivatized from EDTA-conjugated 2-amino-4-methyl pyridine, an acyclic vehicle (EDTA-Mepy2 ) was designed, synthesized, and characterized for PET imaging with ⁶⁸Ga. The drug likeliness and appropriate lipophilicity were first analyzed by molecular docking studies which shows interactive property of ligand with serum albumin protein (HSA: PDB 1E78), at Lys199, Arg257, and His242 residues, which make it more appropriate in transportation as a specific ligand for PET imaging. As a confirmation, binding constant of the ligand with human serum albumin was calculated at λex = 350 nm which was found to be 4.9 × 10³ m⁻¹. The pharmacokinetics of (68) Ga-EDTA-Mepy2 was analyzed by blood kinetics (t(1/2) slow: 3 h 56 min and t(1/2) fast: 32 min) and biodistribution (maximum % ID/g was found in kidney at 1 h). Further the capability of this ligand was analyzed as optical marker also, by recording λex = 380 nm, RFU = 8000; 710 nm, RFU = 1000 units at fixed λem = 280 nm. Additionally, in physiological conditions where its stability was calculated, suggests 15-20 times selectivity over the endogenously present metal ions (KG aL /KZ nL = 14.3, KG aL /KC uL = 18.1).

  1. An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

    PubMed

    Motamedi-Shad, Neda; Jagger, Alistair M; Liedtke, Maximilian; Faull, Sarah V; Nanda, Arjun Scott; Salvadori, Enrico; Wort, Joshua L; Kay, Christopher W M; Heyer-Chauhan, Narinder; Miranda, Elena; Perez, Juan; Ordóñez, Adriana; Haq, Imran; Irving, James A; Lomas, David A

    2016-10-01

    Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

  2. An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

    PubMed

    Motamedi-Shad, Neda; Jagger, Alistair M; Liedtke, Maximilian; Faull, Sarah V; Nanda, Arjun Scott; Salvadori, Enrico; Wort, Joshua L; Kay, Christopher W M; Heyer-Chauhan, Narinder; Miranda, Elena; Perez, Juan; Ordóñez, Adriana; Haq, Imran; Irving, James A; Lomas, David A

    2016-10-01

    Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation. PMID:27407165

  3. Reduced retinoid signaling in the skin after systemic retinoid-X receptor ligand treatment in mice with potential relevance for skin disorders.

    PubMed

    Mihály, Johanna; Gericke, Janine; Aydemir, Gamze; Weiss, Kathrin; Carlsen, Harald; Blomhoff, Rune; Garcia, Javier; Rühl, Ralph

    2012-01-01

    Retinoid-X receptor (RXR)- and retinoic acid receptor (RAR)-mediated signaling is induced by retinoic acids (RA), which are involved in the regulation of skin permeability, differentiation and immune response. Dysregulation of retinoid signaling is present in various skin disorders. Topically and systemically administered synthetic RAR or RXR agonists might influence retinoid-mediated signaling in the skin of RARE reporter animals and gene expression analysis for retinoid, skin homeostasis and skin inflammation marker genes and local retinoid concentrations. Mice were treated orally and topically with synthetic ligands and bioimaging, QRT-PCR and retinoid analysis were performed. Topical application of the synthetic RAR ligand AM580 significantly enhanced retinoid signaling in skin while topical application of the RXR ligand LG268 did not influence retinoic acid receptor response elements (RARE)-mediated signaling. Systemic treatments with LG268 decreased the expression of genes involved in skin homeostasis, RA synthesis and skin RA concentrations, while it increased various markers for skin inflammation and RA degradation, which corresponds to decreased skin RARE signaling. We conclude from these observations that increased systemic concentrations of an RXR -ligand may be one reason for reduced retinoid signaling, -reduced all-trans RA levels in the skin, reduced epidermal homeostasis and increased skin inflammation marker expression with potential relevance for various skin disorders, like atopic dermatitis.

  4. Small-world networks of residue interactions in the Abl kinase complexes with cancer drugs: topology of allosteric communication pathways can determine drug resistance effects.

    PubMed

    Tse, A; Verkhivker, G M

    2015-07-01

    The human protein kinases play a fundamental regulatory role in orchestrating functional processes in complex cellular networks. Understanding how conformational equilibrium between functional kinase states can be modulated by ligand binding or mutations is critical for quantifying molecular basis of allosteric regulation and drug resistance. In this work, molecular dynamics simulations of the Abl kinase complexes with cancer drugs (Imatinib and Dasatinib) were combined with structure-based network modeling to characterize dynamics of the residue interaction networks in these systems. The results have demonstrated that structural architecture of kinase complexes can produce a small-world topology of the interaction networks. Our data have indicated that specific Imatinib binding to a small number of highly connected residues could lead to network-bridging effects and allow for efficient allosteric communication, which is mediated by a dominant pathway sensitive to the unphosphorylated Abl state. In contrast, Dasatinib binding to the active kinase form may activate a broader ensemble of allosteric pathways that are less dependent on the phosphorylation status of Abl and provide a better balance between the efficiency and resilience of signaling routes. Our results have unveiled how differences in the residue interaction networks and allosteric communications of the Abl kinase complexes can be directly related to drug resistance effects. This study offers a plausible perspective on how efficiency and robustness of the residue interaction networks and allosteric pathways in kinase structures may be associated with protein responses to drug binding.

  5. Modification of protein structure and function using photoactivated porphyrin ligands

    NASA Astrophysics Data System (ADS)

    Moreno, Gabriel

    2015-03-01

    The tremendous advances in genomic research have sparked an interest in investigating the possibility to ``manipulate'' the structure of proteins that modify existing functionality. This study makes use of small molecules (e.g., porphyrins) to photosensitize proteins and modify the higher order structure of the polypeptide with the goal of engineering novel functions, or affecting/eliminating native functions. The irradiation of non-covalently bound ligands prompts charge transfer events that have the potential to locally modify the structure of the host protein. The characterization of photoinduced conformational changes in the protein/porphyrin complex is carried out using a combination of electronic spectroscopy and kinetics (e.g., fluorescence spectroscopy, fluorescence decay, circular dichroism). This study is focused primarily on human serum albumin (HSA) as a model. The structure of HSA is well established, the binding sites for an array of ligands are well characterized (including one for protoporphyrins), and HSA provides a series of functions (including some allosteric activity) that can be tested.

  6. Functional prokaryotic-eukaryotic chimera from the pentameric ligand-gated ion channel family.

    PubMed

    Duret, Guillaume; Van Renterghem, Catherine; Weng, Yun; Prevost, Marie; Moraga-Cid, Gustavo; Huon, Christèle; Sonner, James M; Corringer, Pierre-Jean

    2011-07-19

    Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.

  7. Altering residues N125 and D149 impacts sugar effector binding and allosteric parameters in Escherichia coli lactose repressor.

    PubMed

    Xu, Jia; Liu, Shirley; Chen, Mingzhi; Ma, Jianpeng; Matthews, Kathleen S

    2011-10-25

    Lactose repressor protein (LacI), a negative transcriptional regulator in Escherichia coli, relies on an allosteric conformational change for its function. The LacI effector isopropyl-β,D-thiogalactoside (IPTG) promotes this allosteric response and engages the side chains of residues N125 and D149 based on the crystallographic structure of LacI·IPTG. Targeted molecular dynamics (TMD) simulations have indicated involvement of these side chains during the protein structural changes in response to inducer binding. To examine this region further, we applied stochastic boundary molecular dynamics (SBMD) simulation and identified a transient interaction between residues N125 and D149. On the basis of these data, we introduced substitutions for either/both residues and analyzed their impact on protein function. The substitutions utilized were alanine to preclude hydrogen bonding or cysteine to allow disulfide bond formation, which was not observed for N125C/D149C. Minimal impacts were observed on operator affinity for all substitutions, but D149C, N125A/D149A, and N125C/D149C bound to IPTG with 5-8-fold lower affinity than wild-type LacI, and exhibited decreased allosteric amplitude (K(RI/O)/K(R/O)). Of interest, the double mutants did not exhibit an allosteric response to an alternate inducer, 2-phenylethyl-β,D-galactoside (PhEG), despite demonstration of PhEG binding. Further, the presence of the anti-inducer, o-nitrophenyl-β,D-fucoside (ONPF), enhanced operator affinity for wild-type LacI and all other mutant proteins examined, but behaved as an inducer for N125A/D149A, decreasing operator binding affinity. These results confirm the role of residues 125 and 149 in ligand binding and allosteric response and illustrate how readily the function of a regulatory protein can be altered. PMID:21928765

  8. Neurobiological Insights from mGlu Receptor Allosteric Modulation

    PubMed Central

    O’Brien, Daniel E

    2016-01-01

    Allosteric modulation of metabotropic glutamate (mGlu) receptors offers a promising pharmacological approach to normalize neural circuit dysfunction associated with various psychiatric and neurological disorders. As mGlu receptor allosteric modulators progress through discovery and clinical development, both technical advances and novel tool compounds are providing opportunities to better understand mGlu receptor pharmacology and neurobiology. Recent advances in structural biology are elucidating the structural determinants of mGlu receptor–negative allosteric modulation and supplying the means to resolve active, allosteric modulator-bound mGlu receptors. The discovery and characterization of allosteric modulators with novel pharmacological profiles is uncovering the biological significance of their intrinsic agonist activity, biased mGlu receptor modulation, and novel mGlu receptor heterodimers. The development and exploitation of optogenetic and optopharmacological tools is permitting a refined spatial and temporal understanding of both mGlu receptor functions and their allosteric modulation in intact brain circuits. Together, these lines of research promise to provide a more refined understanding of mGlu receptors and their allosteric modulation that will inform the development of mGlu receptor allosteric modulators as neurotherapeutics in the years to come. PMID:26647381

  9. The structural basis of ATP as an allosteric modulator.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-09-01

    Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  10. The Structural Basis of ATP as an Allosteric Modulator

    PubMed Central

    Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-01-01

    Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  11. Engineered allosteric mutants of the integrin alphaMbeta2 I domain: structural and functional studies.

    PubMed Central

    McCleverty, Clare J; Liddington, Robert C

    2003-01-01

    The alpha-I domain, found in the alpha-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity. PMID:12611591

  12. Some metal complexes of three new potentially heptadentate (N4O3) tripodal Schiff base ligands; synthesis, characterizatin and X-ray crystal structure of a novel eight coordinate Gd(III) complex

    NASA Astrophysics Data System (ADS)

    Golbedaghi, Reza; Moradi, Somaeyh; Salehzadeh, Sadegh; Blackman, Allan G.

    2016-03-01

    The symmetrical and asymmetrical potentially heptadentate (N4O3) tripodal Schiff base ligands (H3L1-H3L3) were synthesized from the condensation reaction of three tripodal tetraamine ligands tpt (trpn), tris (3-aminopropyl) amine; ppe (abap), (2-aminoethyl)bis(3-aminopropyl)amine, and tren, tris(2-aminoethyl)amine, with 5-methoxysalicylaldehyde. Then, the reaction of Ln(III) (Ln = Gd, La and Sm), Al(III), and Fe(III) metal ions with the above ligands was investigated. The resulting compounds were characterized by IR, mass spectrometry and elemental analysis in all cases and NMR spectroscopy in the case of the Schiff base ligands. The X-ray crystal structure of the Gd complex of H3L3 ligand showed that in addition to all donor atoms of the ligand one molecule of H2O is also coordinated to the metal ion and a neutral eight-coordinate complex is formed.

  13. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor.

    PubMed

    Isogai, Shin; Deupi, Xavier; Opitz, Christian; Heydenreich, Franziska M; Tsai, Ching-Ju; Brueckner, Florian; Schertler, Gebhard F X; Veprintsev, Dmitry B; Grzesiek, Stephan

    2016-02-11

    G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental

  14. GABAB receptor ligands for the treatment of alcohol use disorder: preclinical and clinical evidence

    PubMed Central

    Agabio, Roberta; Colombo, Giancarlo

    2014-01-01

    The present paper summarizes the preclinical and clinical studies conducted to define the “anti-alcohol” pharmacological profile of the prototypic GABAB receptor agonist, baclofen, and its therapeutic potential for treatment of alcohol use disorder (AUD). Numerous studies have reported baclofen-induced suppression of alcohol drinking (including relapse- and binge-like drinking) and alcohol reinforcing, motivational, stimulating, and rewarding properties in rodents and monkeys. The majority of clinical surveys conducted to date—including case reports, retrospective chart reviews, and randomized placebo-controlled studies—suggest the ability of baclofen to suppress alcohol consumption, craving for alcohol, and alcohol withdrawal symptomatology in alcohol-dependent patients. The recent identification of a positive allosteric modulatory binding site, together with the synthesis of in vivo effective ligands, represents a novel, and likely more favorable, option for pharmacological manipulations of the GABAB receptor. Accordingly, data collected to date suggest that positive allosteric modulators of the GABAB receptor reproduce several “anti-alcohol” effects of baclofen and display a higher therapeutic index (with larger separation—in terms of doses—between “anti-alcohol” effects and sedation). PMID:24936171

  15. Viewpoint: Discriminating between noncompetitive and allosteric interactions.

    PubMed

    Ochs, Raymond S; Ashby, Charles R

    2008-03-01

    Understanding inhibition modes other than competitive is of clear importance in neuropharmacology. However, there appears to be some confusion concerning modes of inhibition that are not of the competitive type. It is critical to make a distinction between "not competitive" and "noncompetitive," as the later is a particular mode of inhibition. Further, there appears to be confusion between noncompetitive and allosteric behavior. Our purpose in this contribution is to explore the basis of these terms so that insight into pharmacological systems has a firmer mechanistic basis.

  16. Ligands Binding and Molecular Simulation: the Potential Investigation of a Biosensor Based on an Insect Odorant Binding Protein

    PubMed Central

    Yi, Xin; Zhang, Yanbo; Wang, Peidan; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua

    2015-01-01

    Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications. PMID:25552932

  17. Ligands binding and molecular simulation: the potential investigation of a biosensor based on an insect odorant binding protein.

    PubMed

    Yi, Xin; Zhang, Yanbo; Wang, Peidan; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua

    2015-01-01

    Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications. PMID:25552932

  18. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation. PMID:26506102

  19. Allosteric control in a metalloprotein dramatically alters function.

    PubMed

    Baxter, Elizabeth Leigh; Zuris, John A; Wang, Charles; Vo, Phu Luong T; Axelrod, Herbert L; Cohen, Aina E; Paddock, Mark L; Nechushtai, Rachel; Onuchic, Jose N; Jennings, Patricia A

    2013-01-15

    Metalloproteins (MPs) comprise one-third of all known protein structures. This diverse set of proteins contain a plethora of unique inorganic moieties capable of performing chemistry that would otherwise be impossible using only the amino acids found in nature. Most of the well-studied MPs are generally viewed as being very rigid in structure, and it is widely thought that the properties of the metal centers are primarily determined by the small fraction of amino acids that make up the local environment. Here we examine both theoretically and experimentally whether distal regions can influence the metal center in the diabetes drug target mitoNEET. We demonstrate that a loop (L2) 20 Å away from the metal center exerts allosteric control over the cluster binding domain and regulates multiple properties of the metal center. Mutagenesis of L2 results in significant shifts in the redox potential of the [2Fe-2S] cluster and orders of magnitude effects on the rate of [2Fe-2S] cluster transfer to an apo-acceptor protein. These surprising effects occur in the absence of any structural changes. An examination of the native basin dynamics of the protein using all-atom simulations shows that twisting in L2 controls scissoring in the cluster binding domain and results in perturbations to one of the cluster-coordinating histidines. These allosteric effects are in agreement with previous folding simulations that predicted L2 could communicate with residues surrounding the metal center. Our findings suggest that long-range dynamical changes in the protein backbone can have a significant effect on the functional properties of MPs.

  20. A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)*

    PubMed Central

    Bai, Fengwei; Asojo, Oluwatoyin A.; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A.; Leng, Lin; Koski, Raymond A.; Powell, Thomas J.; Bucala, Richard; Anthony, Karen G.

    2012-01-01

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases. PMID:22782901

  1. Functional Selectivity in CB2 Cannabinoid Receptor Signaling and Regulation: Implications for the Therapeutic Potential of CB2 Ligands

    PubMed Central

    Atwood, Brady K.; Wager-Miller, James; Haskins, Christopher; Straiker, Alex

    2012-01-01

    Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB1 and CB2 cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB2 to different extents. Because CB2 is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB1 and CB2 trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB2 receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB2 receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB2 internalization. Despite these differences in internalization, both compounds activated CB2 receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of β-arrestin2 to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB2 receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB2 ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB2 receptors and suggest that

  2. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    PubMed

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies. PMID:27335334

  3. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    PubMed

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies.

  4. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  5. Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the δ-opioid receptor.

    PubMed

    Burford, Neil T; Livingston, Kathryn E; Canals, Meritxell; Ryan, Molly R; Budenholzer, Lauren M L; Han, Ying; Shang, Yi; Herbst, John J; O'Connell, Jonathan; Banks, Martyn; Zhang, Litao; Filizola, Marta; Bassoni, Daniel L; Wehrman, Tom S; Christopoulos, Arthur; Traynor, John R; Gerritz, Samuel W; Alt, Andrew

    2015-05-28

    Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

  6. Synthesis and characterisation of new 4-oxo-N-(substituted-thiazol-2-yl)-4H-chromene-2-carboxamides as potential adenosine receptor ligands

    NASA Astrophysics Data System (ADS)

    Cagide, Fernando; Borges, Fernanda; Gomes, Ligia R.; Low, John Nicolson

    2015-06-01

    Chromones are 4H-benzopyran-4-one heterocycles that have been thoroughly studied due to their interesting biological activities. Thiazole based compounds have been used in therapeutics as antimicrobial, antiviral and as antifungal agents for a long time but, in the past decades, they have been identified as potent and selective ligands for adenosine receptor. In continuation of our project related to the syntheses of pharmacologically important heterocycles, a new series of chromone-thiazole hybrids have been designed as potential ligands for human adenosine receptors. In this context, new 4-oxo-N-(substituted-thiazol-2-yl)-4H-chromene-2-carboxamides were synthesized from chromone-2-carboxylic acid by two different amidation methods. The development of dissimilar synthetic approaches provided the possibility of working with diverse reaction conditions, namely with conventional heating and/or microwave irradiation. The structure of the compounds has been established on the basis of NMR and MS spectroscopy and X-ray crystallography. Relevant data related to the molecular geometry and conformation of the chromone-thiazole hybrids has been acquired which can be of the utmost importance to understand ligand-receptor binding.

  7. A selective positive allosteric modulator of metabotropic glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis.

    PubMed

    Benneyworth, Michael A; Xiang, Zixiu; Smith, Randy L; Garcia, Efrain E; Conn, P Jeffrey; Sanders-Bush, Elaine

    2007-08-01

    Recent clinical studies reveal that selective agonists of group II metabotropic glutamate (mGlu) receptors have robust efficacy in treating positive and negative symptoms in patients with schizophrenia. Group II mGlu receptor agonists also modulate the in vivo activity of psychotomimetic drugs and reduce the ability of psychotomimetic hallucinogens to increase glutamatergic transmission. Because increased excitation of the medial prefrontal cortex (mPFC) has been implicated in pathophysiology of schizophrenia, the ability of group II mGlu receptor agonists to reduce hallucinogenic drug action in this region is believed to be directly related to their antipsychotic efficacy. A novel class of ligands, termed positive allosteric modulators, has recently been identified, displaying exceptional mGlu2 receptor selectivity. These compounds do not activate mGlu2 receptors directly but potentiate the ability of glutamate and other agonists to activate this receptor. We now report that the mGlu2 receptor-selective positive allosteric modulator biphenyl-indanone A (BINA) modulates excitatory neurotransmission in the mPFC and attenuates the in vivo actions of the hallucinogenic 5-HT(2A/2C) receptor agonist (-)2,5-dimethoxy-4-bromoamphetamine [(-)DOB]. BINA attenuates serotonin-induced increases in spontaneous excitatory postsynaptic currents in the mPFC, mimicking the effect of the mGlu2/3 receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV). In addition, BINA reduced (-)DOB-induced head twitch behavior and Fos expression in mPFC, effects reversed by pretreatment with the mGlu2/3 receptor antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl) -3 - (xanth-9-yl-)propionic acid (LY341495). These data confirm the relevance of excitatory signaling in the mPFC to the behavioral actions of hallucinogens and further support the targeting of mGlu2 receptors as a novel strategy for treating glutamatergic dysfunction in schizophrenia.

  8. Emerging Computational Methods for the Rational Discovery of Allosteric Drugs

    PubMed Central

    2016-01-01

    Allosteric drug development holds promise for delivering medicines that are more selective and less toxic than those that target orthosteric sites. To date, the discovery of allosteric binding sites and lead compounds has been mostly serendipitous, achieved through high-throughput screening. Over the past decade, structural data has become more readily available for larger protein systems and more membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets. In parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms. As a result of these advances, the field of predictive allosteric drug development is now on the cusp of a new era of rational structure-based computational methods. Here, we review algorithms that predict allosteric sites based on sequence data and molecular dynamics simulations, describe tools that assess the druggability of these pockets, and discuss how Markov state models and topology analyses provide insight into the relationship between protein dynamics and allosteric drug binding. In each section, we first provide an overview of the various method classes before describing relevant algorithms and software packages. PMID:27074285

  9. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Cockrell, Gregory M; Zheng, Yunan; Guo, Wenyue; Peterson, Alexis W; Truong, Jennifer K; Kantrowitz, Evan R

    2013-11-12

    For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

  10. Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

    PubMed

    Lang, Eric J M; Heyes, Logan C; Jameson, Geoffrey B; Parker, Emily J

    2016-02-17

    Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

  11. Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: hit-to-lead optimization.

    PubMed

    Nógrádi, Katalin; Wágner, Gábor; Domány, György; Bobok, Amrita; Magdó, Ildikó; Kiss, Béla; Kolok, Sándor; Fónagy, Katalin; Gyertyán, István; Háda, Viktor; Kóti, János; Gál, Krisztina; Farkas, Sándor; Keserű, György M; Greiner, István; Szombathelyi, Zsolt

    2014-08-15

    An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b]pyridines derivative, which fulfilled the lead criteria.

  12. Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor.

    PubMed

    Grunbeck, Amy; Huber, Thomas; Abrol, Ravinder; Trzaskowski, Bartosz; Goddard, William A; Sakmar, Thomas P

    2012-06-15

    G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce signals. Although GPCRs represent the largest class of therapeutic targets, only a small percentage of their ligand-binding sites are precisely defined. Here we describe the novel application of targeted photo-cross-linking using unnatural amino acids to obtain structural information about the allosteric binding site of a small molecule drug, the CCR5-targeted HIV-1 co-receptor blocker maraviroc. PMID:22455376

  13. An allosteric model for the functional plasticity of olfactory chemoreceptors

    NASA Astrophysics Data System (ADS)

    Colosimo, Alfredo

    2000-12-01

    A simple allosteric model may describe the relatively (a)specific behaviour of olfactory chemoreceptors (OCs) and their functional plasticity with a minimum number of parameters. Allosteric, heterotropic effectors are suggested as a possible cause of variable responses documented, in particular, in frog OCs. As an immediate spinoff of the continuously increasing amount of structural information available on natural OCs, development of appropriate allosteric models is foreseen to provide plausible molecular mechanisms for their complex functional performance. This may also have implications in the design of artificial olfaction systems.

  14. A comparative study of zwitterionic ligands-mediated mineralization and the potential of mineralized zwitterionic matrices for bone tissue engineering

    PubMed Central

    Liu, Pingsheng; Emmons, Erin

    2014-01-01

    Cationic and anionic residues of the extracellular matrices (ECM) of bone play synergistic roles in recruiting precursor ions and templating the nucleation, growth and crystalline transformations of calcium apatite in natural biomineralization. We previously reported that zwitterionic sulfobetaine ligands can template extensive 3-dimensional (3-D) hydroxyapaptite (HA)-mineralization of photo-crosslinked polymethacrylatehydrogels. Here, we compared the potency of two other major zwitterionic ligands, phosphobetaine and carboxybetaine, with that of the sulfobetaine in mediating 3-D mineralization using the crosslinked polymethacrylate hydrogel platform. We confirmed that all three zwitterionic hydrogels were able to effectively template 3-D mineralization, supporting the general ability of zwitterions to mediate templated mineralization. Among them, however, sulfobetaine and phosphobetaine hydrogels templated denser 3-D mineralizationthan the carboxybetaine hydrogel, likely due to their higher free water fractions and better maintenance of zwitterionic nature throughout the pH-changes during the in vitro mineralization process. We further demonstrated that the extensively mineralized zwitterionic hydrogels could be exploited for efficient retention (e.g. 99% retention after 24-h incubation in PBS) of osteogenic growth factor recombinant bone morphogenetic protein-2 (rhBMP-2) and subsequent sustained local release with retained bioactivity. Combined with the excellent cytocompatibility of all three zwitterionic hydrogels and the significantly improved cell adhesive properties of their mineralized matrices, these materials could find promising applications in bone tissue engineering. PMID:25558374

  15. Blockade of Glioma Proliferation Through Allosteric Inhibition of JAK2

    PubMed Central

    He, Kunyan; Qi, Qi; Chan, Chi-Bun; Xiao, Ge; Liu, Xia; Tucker-Burden, Carol; Wang, Liya; Mao, Hui; Lu, Xiang; McDonald, Frank E.; Luo, Hongbo; Fan, Qi-Wen; Weiss, William A.; Sun, Shi-Yong; Brat, Daniel J.; Ye, Keqiang

    2016-01-01

    The gene that encodes the epidermal growth factor receptor (EGFR) is frequently overexpressed or mutated in human cancers, including glioblastoma. However, the efficacy of EGFR-targeted small-molecule inhibitors or monoclonal antibodies in glioblastomas that also have mutation or deletion of the gene encoding phosphatase and tensin homolog (PTEN) has been modest. We found that EGFR signaling was blocked by a small molecule (G5-7) that selectively inhibited Janus kinase 2 (JAK2)–mediated phosphorylation and activation of EGFR and STAT3 (signal transducer and activator of transcription 3) by binding to JAK2, thereby decreasing the activity of downstream signaling by mTOR (mammalian target of rapamycin) and inducing cell cycle arrest. G5-7 inhibited the proliferation of PTEN-deficient glioblastoma cell lines harboring a constitutively active variant of EGFR (U87MG/EGFRvIII) and human glioblastoma explant neurosphere cultures, but the drug only weakly inhibited the proliferation of either glioblastoma cell lines that were wild type for EGFR and stably transfected with PTEN (U87MG/PTEN) or normal neural progenitor cells and astrocytes. Additionally, G5-7 reduced vascular endothelial growth factor (VEGF) secretion and endothelial cell migration and induced apoptosis in glioblastoma xenografts, thereby suppressing glioblastoma growth in vivo. Furthermore, G5-7 was more potent than EGFR or JAK2 inhibitors that interfere with either ligand or adenosine 5′-triphosphate (ATP) binding at impeding glioblastoma cell proliferation, demonstrating that this allosteric JAK2 inhibitor may be an effective clinical strategy. PMID:23838182

  16. Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.

    PubMed

    Roelofs, Kevin G; Wang, Jingxin; Sintim, Herman O; Lee, Vincent T

    2011-09-13

    Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interactions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interactions without the need to purify the protein when performed in cells with low background activity. This method, differential radial capillary action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein-ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through capillary action. We show here that DRaCALA allows detection of specific interactions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli, demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interaction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interactions in a systematic and comprehensive approach.

  17. LigandRNA: computational predictor of RNA-ligand interactions.

    PubMed

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Bujnicki, Janusz M

    2013-12-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

  18. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  19. A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo.

    PubMed

    Henry, E C; Bemis, J C; Henry, O; Kende, A S; Gasiewicz, T A

    2006-06-01

    The aryl hydrocarbon receptor (AhR) is best known as a mediator of toxicity of a diverse family of xenobiotic chemicals such as dioxins and PCBs. However, many naturally occurring compounds also activate AhR. One such compound, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), was isolated from tissue and found to be potent in preliminary tests [J. Song, M. Clagett-Dame, R.E. Peterson, M.E. Hahn, W.M. Westler, R.R. Sicinski, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 99 (2002) 14694-14699]. We have synthesized ITE and [(3)H]ITE and further evaluated its AhR activity in several in vitro and in vivo assays in comparison with the toxic ligand, TCDD. AhR in Hepa1c1c7 cell cytosol bound [(3)H]ITE with high affinity and the AhR.ITE complex formed in vitro bound dioxin response element (DRE) oligonucleotide as potently as TCDD.AhR. In cells treated with ITE, nuclear translocation of AhR, and induction of CYP1A1 protein and of a DRE-dependent luciferase reporter gene were observed. ITE administered to pregnant DRE-LacZ transgenic mice activated fetal AhR, observed as X-gal staining in the same sites as in TCDD-treated mice. However, unlike TCDD, ITE did not induce cleft palate or hydronephrosis. TCDD but not ITE induced thymic atrophy in young adult mice, but both ITE and TCDD caused similar loss of cells and alterations of cell profiles in cultured fetal thymi. These data demonstrate that ITE is a potent AhR agonist in cell extracts, cultured cells, and intact animals, but does not cause the toxicity associated with the more stable xenobiotic ligand, TCDD.

  20. Notes on stochastic (bio)-logic gates: computing with allosteric cooperativity

    NASA Astrophysics Data System (ADS)

    Agliari, Elena; Altavilla, Matteo; Barra, Adriano; Dello Schiavo, Lorenzo; Katz, Evgeny

    2015-05-01

    Recent experimental breakthroughs have finally allowed to implement in-vitro reaction kinetics (the so called enzyme based logic) which code for two-inputs logic gates and mimic the stochastic AND (and NAND) as well as the stochastic OR (and NOR). This accomplishment, together with the already-known single-input gates (performing as YES and NOT), provides a logic base and paves the way to the development of powerful biotechnological devices. However, as biochemical systems are always affected by the presence of noise (e.g. thermal), standard logic is not the correct theoretical reference framework, rather we show that statistical mechanics can work for this scope: here we formulate a complete statistical mechanical description of the Monod-Wyman-Changeaux allosteric model for both single and double ligand systems, with the purpose of exploring their practical capabilities to express noisy logical operators and/or perform stochastic logical operations. Mixing statistical mechanics with logics, and testing quantitatively the resulting findings on the available biochemical data, we successfully revise the concept of cooperativity (and anti-cooperativity) for allosteric systems, with particular emphasis on its computational capabilities, the related ranges and scaling of the involved parameters and its differences with classical cooperativity (and anti-cooperativity).

  1. Notes on stochastic (bio)-logic gates: computing with allosteric cooperativity.

    PubMed

    Agliari, Elena; Altavilla, Matteo; Barra, Adriano; Dello Schiavo, Lorenzo; Katz, Evgeny

    2015-01-01

    Recent experimental breakthroughs have finally allowed to implement in-vitro reaction kinetics (the so called enzyme based logic) which code for two-inputs logic gates and mimic the stochastic AND (and NAND) as well as the stochastic OR (and NOR). This accomplishment, together with the already-known single-input gates (performing as YES and NOT), provides a logic base and paves the way to the development of powerful biotechnological devices. However, as biochemical systems are always affected by the presence of noise (e.g. thermal), standard logic is not the correct theoretical reference framework, rather we show that statistical mechanics can work for this scope: here we formulate a complete statistical mechanical description of the Monod-Wyman-Changeaux allosteric model for both single and double ligand systems, with the purpose of exploring their practical capabilities to express noisy logical operators and/or perform stochastic logical operations. Mixing statistical mechanics with logics, and testing quantitatively the resulting findings on the available biochemical data, we successfully revise the concept of cooperativity (and anti-cooperativity) for allosteric systems, with particular emphasis on its computational capabilities, the related ranges and scaling of the involved parameters and its differences with classical cooperativity (and anti-cooperativity). PMID:25976626

  2. Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

    SciTech Connect

    Li, Ming; Smith, Christopher J.; Walker, Matthew T.; Smith, Thomas J.

    2009-12-01

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  3. The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus.

    PubMed

    Whitaker, Amy M; Reinhart, Gregory D

    2016-10-01

    The allosteric coupling free energy between ligands fructose-6-phosphate (Fru-6-P) and phospho(enol)pyruvate (PEP) for phosphofructokinase-1 (PFK) from the moderate thermophile, Bacillus stearothermophilus (BsPFK), results from compensating enthalpy and entropy components. In BsPFK the positive coupling free energy that defines inhibition is opposite in sign from the negative enthalpy term and is therefore determined by the larger absolute value of the negative entropy term. Variants of BsPFK were made to determine the effect of adding small cavities to the structure on the allosteric function of the enzyme. The BsPFK Ile → Val (cavity containing) mutants have varied values for the coupling free energy between PEP and Fru-6-P, indicating that the modifications altered the effectiveness of PEP as an inhibitor. Notably, the mutation I153V had a substantial positive impact on the magnitude of inhibition by PEP. Van't Hoff analysis determined that this is the result of decreased entropy-enthalpy compensation with a larger change in the enthalpy term compared to the entropy term. PMID:27477958

  4. Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics.

    PubMed

    Kleckner, Ian R; Gollnick, Paul; Foster, Mark P

    2012-01-13

    The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation, we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond-to-millisecond "chemical exchange" time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins. PMID:22115774

  5. Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

    PubMed Central

    Kasbekar, Monica; Fischer, Gerhard; Mott, Bryan T.; Yasgar, Adam; Hyvönen, Marko; Boshoff, Helena I. M.; Abell, Chris; Barry, Clifton E.; Thomas, Craig J.

    2016-01-01

    Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen. PMID:27325754

  6. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    PubMed

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  7. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    PubMed Central

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  8. Metal site occupancy and allosteric switching in bacterial metal sensor proteins

    PubMed Central

    Guerra, Alfredo J.; Giedroc, David P.

    2012-01-01

    All prokaryotes encode a panel of metal sensor or metalloregulatory proteins that govern the expression of genes that allows an organism to quickly adapt to toxicity or deprivation of both biologically essential transition metal ions, e.g., Zn, Cu, Fe, and heavy metal pollutants. As such, metal sensor proteins can be considered arbiters of intracellular transition metal bioavailability and thus potentially control the metallation state of the metalloproteins in the cell. Metal sensor proteins are specialized allosteric proteins that regulate transcription as a result direct binding of one or two cognate metal ions, to the exclusion of all others. In most cases, the binding of the cognate metal ion induces a structural change in a protein oligomer that either activates or inhibits operator DNA binding. A quantitative measure of the degree to which a particular metal drives metalloregulation of operator DNA-binding is the allosteric coupling free energy, ΔGc. In this review, we summarize recent work directed toward understanding metal occupancy and metal selectivity of these allosteric switches in selected families of metal sensor proteins and examine the structural origins of ΔGc in the functional context a thermodynamic “set-point” model of intracellular metal homeostasis. PMID:22178748

  9. Molecular determinants of positive allosteric modulation of the human metabotropic glutamate receptor 2

    PubMed Central

    Farinha, A; Lavreysen, H; Peeters, L; Russo, B; Masure, S; Trabanco, A A; Cid, J; Tresadern, G

    2015-01-01

    Background and Purpose The activation of the metabotropic glutamate receptor 2 (mGlu2) reduces glutamatergic transmission in brain regions where excess excitatory signalling is implicated in disorders such as anxiety and schizophrenia. Positive allosteric modulators (PAMs) can provide a fine-tuned potentiation of these receptors' function and are being investigated as a novel therapeutic approach. An extensive set of mutant human mGlu2 receptors were used to investigate the molecular determinants that are important for positive allosteric modulation at this receptor. Experimental Approach Site-directed mutagenesis, binding and functional assays were employed to identify amino acids important for the activity of nine PAMs. The data from the radioligand binding and mutagenesis studies were used with computational docking to predict a binding mode at an mGlu2 receptor model based on the recent structure of the mGlu1 receptor. Key Results New amino acids in TM3 (R635, L639, F643), TM5 (L732) and TM6 (W773, F776) were identified for the first time as playing an important role in the activity of mGlu2 PAMs. Conclusions and Implications This extensive study furthers our understanding of positive allosteric modulation of the mGlu2 receptor and can contribute to improved future design of mGlu2 PAMs. PMID:25571949

  10. Potentiation of the teratogenic effects induced by coadministration of retinoic acid or phytanic acid/phytol with synthetic retinoid receptor ligands.

    PubMed

    Elmazar, M M A; Nau, H

    2004-11-01

    Previous studies in our laboratory identified retinoid-induced defects that are mediated by RAR-RXR heterodimerization using interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in mice (Elmazar et al. 1997, Toxicol Appl Pharmacol 146:21-28; Elmazar et al. 2001, Toxicol Appl Pharmacol 170:2-9; Nau and Elmazar 1999, Handbook of experimental pharmacology, vol 139, Retinoids, Springer-Verlag, pp 465-487). The present study was designed to investigate whether these RAR-RXR heterodimer-mediated defects can be also induced by interactions of natural and synthetic ligands for retinoid receptors. A non-teratogenic dose of the natural RXR agonist phytanic acid (100 mg/kg orally) or its precursor phytol (500 mg/kg orally) was coadministered with a synthetic RARalpha-agonist (Am580; 5 mg/kg orally) to NMRI mice on day 8.25 of gestation (GD8.25). Furthermore, a non-teratogenic dose of the synthetic RXR agonist LGD1069 (20 mg/kg orally) was also coadministered with the natural RAR agonist, all- trans-retinoic acid (atRA, 20 mg/kg orally) or its precursor retinol (ROH, 50 mg/kg orally) to NMRI mice on GD8.25. The teratogenic outcome was scored in day-18 fetuses. The incidence of Am580-induced resorptions, spina bifida aperta, micrognathia, anotia, kidney hypoplasia, dilated bladder, undescended testis, atresia ani, short and absent tail, fused ribs and fetal weight retardation were potentiated by coadministration of phytanic acid or its precursor phytol. Am580-induced exencephaly and cleft palate, which were not potentiated by coadministration with the synthetic RXR agonists, were also not potentiated by coadministration with either phytanic acid or its precursor phytol. LGD1069 potentiated atRA- and ROH-induced resorption, exencephaly, spina bifida, aperta, ear anotia and microtia, macroglossia, kidney hypoplasia, undescended testis, atresia ani, tail defects and fetal weight retardation, but not cleft palate. These results suggest that synergistic

  11. Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

    SciTech Connect

    Jin,R.; Clark, S.; Weeks, A.; Dudman, J.; Gouaux, E.; Partin, K.

    2005-01-01

    Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1, 3-oxazino benzo-1, 4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the 'hinge' in the ligand-binding core 'clamshell' that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

  12. Ligand-induced Epitope Masking

    PubMed Central

    Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

    2016-01-01

    We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

  13. Preliminary Assessment of Potential for Metal-Ligand Speciation in Aqueous Solution via the Liquid Sampling-Atmospheric Pressure Glow Discharge (LS-APGD) Ionization Source: Uranyl Acetate.

    PubMed

    Zhang, Lynn X; Manard, Benjamin T; Powell, Brian A; Marcus, R Kenneth

    2015-07-21

    The determination of metals, including the generation of metal-ligand speciation information, is essential across a myriad of biochemical, environmental, and industrial systems. Metal speciation is generally affected by the combination of some form of chromatographic separation (reflective of the metal-ligand chemistry) with element-specific detection for the quantification of the metal composing the chromatographic eluent. Thus, the identity of the metal-ligand is assigned by inference. Presented here, the liquid sampling-atmospheric pressure glow discharge (LS-APGD) is assessed as an ionization source for metal speciation, with the uranyl ion-acetate system used as a test system. Molecular mass spectra can be obtained from the same source by simple modification of the sustaining electrolyte solution. Specifically, chemical information pertaining to the degree of acetate complexation of uranyl ion (UO2(2+)) is assessed as a function of pH in the spectral abundance of three metallic species: inorganic (nonligated) uranyl, UO2Ac(H2O)n(MeOH)m(+), and UO2Ac2(H2O)n(MeOH)(m)H(+) (n = 1, 2, 3, ...; m = 1, 2, 3, ...). The product mass spectra are different from what are obtained from electrospray ionization sources that have been applied to this system. The resulting relationships between the speciation and pH values have been compared to calculated concentrations of the corresponding uranyl species: UO2(2+), UO2Ac(+), UO2Ac2. The capacity for the LS-APGD to affect both atomic mass spectra and structurally significant spectra for organometallic complexes is a unique and potentially powerful combination.

  14. How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

    PubMed Central

    Otero, Lisandro H.; Rojas-Altuve, Alzoray; Llarrull, Leticia I.; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W.; Fisher, Jed F.; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A.

    2013-01-01

    The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design. PMID:24085846

  15. How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

    PubMed

    Otero, Lisandro H; Rojas-Altuve, Alzoray; Llarrull, Leticia I; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W; Fisher, Jed F; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A

    2013-10-15

    The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

  16. Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors*

    PubMed Central

    Bauer, Mark; Chicca, Andrea; Tamborrini, Marco; Eisen, David; Lerner, Raissa; Lutz, Beat; Poetz, Oliver; Pluschke, Gerd; Gertsch, Jürg

    2012-01-01

    The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB1). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB1 receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [3H]CP55,940 and [3H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [3H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB1 receptors. Competition binding studies revealed Ki values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [35S]GTPγS binding, and CB1 receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB1 receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling. PMID:22952224

  17. Allosteric Regulation of the Rotational Speed in a Light-Driven Molecular Motor

    PubMed Central

    2016-01-01

    The rotational speed of an overcrowded alkene-based molecular rotary motor, having an integrated 4,5-diazafluorenyl coordination motif, can be regulated allosterically via the binding of metal ions. DFT calculations have been used to predict the relative speed of rotation of three different (i.e., zinc, palladium, and platinum) metal dichloride complexes. The photochemical and thermal isomerization behavior of these complexes has been studied in detail using UV–vis and 1H NMR spectroscopy. Our results confirm that metal coordination induces a contraction of the diazafluorenyl lower half, resulting in a reduction of the steric hindrance in the “fjord” region of the molecule, which causes an increase of the rotational speed. Importantly, metal complexation can be accomplished in situ and is found to be reversible upon the addition of a competing ligand. Consequently, the rotational behavior of these molecular motors can be dynamically controlled with chemical additives. PMID:27669358

  18. Sequence-specific minor groove binding ligands as potential regulators of gene expression in Xenopus laevis oocytes.

    PubMed

    Belikov, S V; Grokhovsky, S L; Isaguliants, M G; Surovaya, A N; Gursky, G V

    2005-10-01

    The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent gene activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the DNA-protein interactions following hormone activation. The strategy of artificial regulating of gene activity by sequence-specific minor groove binding ligands is very attractive. We have synthesized and studied the interaction with DNA of bis-linked netropsin derivatives in which two monomers are attached via short linkers in head-to-head and tail-to-tail manners. We have found that cis-diammine-platinum bridged bis-netropsin added to Xenopus oocytes media penetrates cellular and nuclear membrane and binds selectively to the MMTV promoter at the DNA segment that partly overlaps with the site recognized by glucocorticoid receptor. DNase I footprinting studies demonstrate that there are more stronger binding sites for cis-diammine-platinum bridged bis-netropsin on the naked MMTV DNA which are found to be inaccessible for its binding in oocytes. PMID:16060693

  19. A comparison of novel organoiridium(III) complexes and their ligands as a potential treatment for prostate cancer.

    PubMed

    Hockey, Samantha C; Barbante, Gregory J; Francis, Paul S; Altimari, Jarrad M; Yoganantharajah, Prusothman; Gibert, Yann; Henderson, Luke C

    2016-02-15

    A range of 1,4-substituted 2-pyridyl-N-phenyl triazoles were synthesised and evaluated for their antiproliferative properties against lymph node cancer of the prostate (LNCaP) and bone metastasis of prostate cancer (PC-3) cells. Excellent-to-low IC50 values were determined (5.6-250 μM), and a representative group of 4 ligands were then complexed to iridium(III) giving highly luminescent species. Re-evaluation of these compounds against both cell lines was then undertaken and improved potency (up to 72-fold) was observed, giving IC50 values of 0.36-11 μM for LNCaP and 0.85-5.9 μM for PC-3. Preliminary screens for in vivo toxicity were conducted using a zebrafish model showing a wide range of induced toxicity depending of the compound evaluated. Apoptosis and Caspase-3 levels were also determined and showed no statistical difference between some of the treated specimens and the controls. This study may identify novel therapeutic agents for advanced stage of prostate cancer in humans.

  20. Design, Synthesis and Biological Evaluation of Bivalent Benzoxazolone and Benzothiazolone Ligands as Potential Anti-inflammatory/Analgesic Agents

    PubMed Central

    Abdelazeem, Ahmed H.; Khan, Shabana I.; White, Stephen W.; Sufka, Kenneth J.; McCurdy, Christopher R.

    2015-01-01

    Benzoxazolone and benzothiazolone were used as template blocks to develop two series of dimers as anti-inflammatory and analgesic agents based on the concept of bivalent ligands. The first series (I) involved varying the carbon chain lengths extending from the piperazine core to the nitrogen atom of the dibenzo[d]oxazol-2(3H)-one or dibenzo[d]thiazol-2(3H)-one. The second series (II) was designed by changing the attachment point. All compounds were screened for their in vitro anti-inflammatory activity in terms of the inhibition of inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-κB). Seventeen compounds inhibited both targets. Eleven of them exhibited IC50 values below 3 μM while five compounds showed IC50 values of 1 μM or below. Most of the compounds were found to be devoid of cytotoxicity against mammalian kidney and solid tumors cell lines up to 25μg/mL. In vivo anti-inflammatory and antinociceptive studies revealed that compounds 3j, 5t and 8b have significant anti-inflammatory and analgesic activity comparable to that of indomethacin and ketorolac, respectively. PMID:25975638

  1. Imidazenil, a partial positive allosteric modulator of GABAA receptors, exhibits low tolerance and dependence liabilities in the rat.

    PubMed

    Auta, J; Giusti, P; Guidotti, A; Costa, E

    1994-09-01

    Long-term treatment of rats with full (triazolam) or selective (diazepam) allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors rapidly induced tolerance to the protective effect of these drugs against bicuculline-induced convulsion. In contrast, long-term administration of partial allosteric modulators (imidazenil and bretazenil) of GABAA receptors, in doses equipotent to those of diazepam and triazolam that induce anticonvulsant tolerance, failed to elicit such a tolerance. Furthermore, no cross-tolerance was observed between diazepam and imidazenil. Discontinuation of long-term treatment with diazepam or triazolam, but not of long-term treatment with imidazenil or bretazenil, sensitized rats to behavioral inhibition by punishment (electric shock) in a manner that was potentiated by flumazenil. Administration of a single oral dose of [14C]diazepam or [3H] imidazenil to rats treated repeatedly with the corresponding unlabeled drug or vehicle revealed that the brain concentrations of drugs and their metabolites were similar in both groups of animals. This suggests that tolerance to the full or selective allosteric modulators of GABAA receptors may be associated with changes in the efficacy of the allosteric modulation rather than with changes in drug metabolism. Imidazenil has a longer half-life than an equipotent dose of diazepam and protects rats against bicuculline-induced convulsions for a significantly longer time than diazepam or bretazenil.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Allosteric modulators of the hERG K(+) channel: radioligand binding assays reveal allosteric characteristics of dofetilide analogs.

    PubMed

    Yu, Zhiyi; Klaasse, Elisabeth; Heitman, Laura H; Ijzerman, Adriaan P

    2014-01-01

    Drugs that block the cardiac K(+) channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K(+) channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [(3)H]astemizole and [(3)H]dofetilide to the hERG K(+) channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC50 values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC50 values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K(+) channel, which is discussed in the light of findings on other ion channels. PMID:24200993

  3. The non-ligand binding beta-isoform of the human glucocorticoid receptor (hGR beta): tissue levels, mechanism of action, and potential physiologic role.

    PubMed Central

    de Castro, M.; Elliot, S.; Kino, T.; Bamberger, C.; Karl, M.; Webster, E.; Chrousos, G. P.

    1996-01-01

    BACKGROUND: Alternative splicing of the transcripts of the human glucocorticoid receptor gene results in two mutually exclusive products, the classic, ligand-binding glucocorticoid receptor (hGR alpha), and a dominant negative non-ligand-binding isoform, hGR beta. MATERIALS AND METHODS: We examined the existence of and quantified both hGR alpha and hGR beta isoforms in a panel of human tissues, as well as in intact and fractionated HeLa cells, using specific quantitative Western blots and/or immunocytochemistry. We studied the potential interactions of hGR beta with heat shock protein (hsp) 90 and/or hGR alpha using cross-immunoadsorption/precipitation procedures followed by Western blots. RESULTS: For the first time, we demonstrated the natural existence of the hGR beta protein, which was widely expressed in human tissues. The ratio of immunoreactive hGR alpha to hGR beta varied from 0.2 to 1.0 among different tissues, and was approximately 0.2 in HeLa cells. In the latter, both isoforms were distributed in the cytoplasm and nucleus in the absence of the hormonal ligand, and translocated into the nucleus after addition of dexamethasone. The cytosolic and nuclear hGR alpha-to-hGR beta ratio remained the same before and after dexamethasone exposure, suggesting that upon activation the two isoforms translocated into the nucleus in equal proportions. hGR alpha- and hGR beta-specific antibodies cross-adsorbed and precipitated cytosolic and nuclear glucocorticoid hGR alpha and hGR beta, respectively, as well as hsp90, suggesting that hGR alpha and hGR beta are in complex with hsp90 and/or each other. CONCLUSIONS: The hGR beta protein is widely expressed throughout the human body and present mostly in the cytoplasm of human cells, in complex with hsp90 and other proteins. In the presence of glucocorticoid, hGR beta probably heterodimerizes with ligand-bound hGR alpha and translocates into the nucleus to act as a dominant negative inhibitor of the classic receptor. Images

  4. Immobilizing CC chemokine receptor 4's N-terminal extracellular tail on a capillary to study its potential ligands by capillary electrophoresis.

    PubMed

    Chen, Wenjing; Li, Meina; Yakufu, Pazilaiti; Ling, Xiaomei; Qi, Hui; Xiao, Junhai; Wang, Ying

    2012-04-01

    ML40 is the equivalent peptide derived from the N terminal of CCC4 (CC chemokine receptor 4), which plays a pivotal role in allergic inflammation. A new capillary electrophoresis method was developed to study the interactions between ML40 and its potential ligands in which ML40 was immobilized on the inner wall of capillary as the stationary phase based on the covalent linking technique. The interaction between S009, a known CCR4 antagonist, and the immobilized ML40 was studied to validate the bioactivity of ML40. The electropherogram of S009 showed that the peak height was reduced and the peak width was broadened in the ML40 immobilized capillary. Otherwise, 25 computer-aided design and drafting compounds were screened out using this method. Four compounds' peak widths were broadened and their peak heights were reduced, as with S009. Meanwhile, nonlinear chromatography was used to calculate the constants for the ligand-receptor complex formation. Furthermore, the tertiary amine compounds belonging to the chiral tertiary amines of the type NRR'R″, which are optically inactive resulting from rapid pyramide inversion, were chiral separated by our protein immobilization method for the first time. In general, the methodology presented would be applicable to study compound-ML40 interactions as a reliable and robust screening method for CCR4 antagonist discovery. PMID:22245764

  5. Identification of novel markers of alternative activation and potential endogenous PPARγ ligand production mechanisms in human IL-4 stimulated differentiating macrophages.

    PubMed

    Czimmerer, Zsolt; Varga, Tamas; Poliska, Szilard; Nemet, Istvan; Szanto, Attila; Nagy, Laszlo

    2012-12-01

    We analyzed global gene expression profiles of IL-4 induced alternatively activated as well as IFNγ+TNFα stimulated classically activated human monocyte derived macrophages and identified novel IL-4 regulated alternative activation marker genes including MS4A4A, SLA, CD180, and ENPP2. Transcription factor prediction analysis of IL-4 regulated genes suggested that the regulated genes are involved in a complex regulation of lipid metabolism, defense against cell metabolism derived reactive oxygen species, and basal expression of inflammation linked genes. Both an in silico transcription activation prediction as well as experimental data suggested the presence of alternative macrophage activation specific endogenous PPARγ ligand producing mechanisms. We found the induction of three enzymes whose activity can potentially generate endogenous PPARγ ligands in an IL-4 dependent manner. These are MAOA, ENPP2, and ALOX15 producing 5-methoxy-indole acetate, lysophosphatidic acid (LPA) and 13-hydroxyoctadienoic acid (13-HODE), and/or 15-hydroxyeicosatetraenoic acid (15-HETE), respectively. Our data suggest that global gene expression profiling, combined with computational transcription activity prediction, can lead to identification of transcriptional networks that underpin cellular subtype specification.

  6. Probing the allosteric mechanism in pyrrolysyl-tRNA synthetase using energy-weighted network formalism.

    PubMed

    Bhattacharyya, Moitrayee; Vishveshwara, Saraswathi

    2011-07-19

    Pyrrolysyl-tRNA synthetase (PylRS) is an atypical enzyme responsible for charging tRNA(Pyl) with pyrrolysine, despite lacking precise tRNA anticodon recognition. This dimeric protein exhibits allosteric regulation of function, like any other tRNA synthetases. In this study we examine the paths of allosteric communication at the atomic level, through energy-weighted networks of Desulfitobacterium hafniense PylRS (DhPylRS) and its complexes with tRNA(Pyl) and activated pyrrolysine. We performed molecular dynamics simulations of the structures of these complexes to obtain an ensemble conformation-population perspective. Weighted graph parameters relevant to identifying key players and ties in the context of social networks such as edge/node betweenness, closeness index, and the concept of funneling are explored in identifying key residues and interactions leading to shortest paths of communication in the structure networks of DhPylRS. Further, the changes in the status of important residues and connections and the costs of communication due to ligand induced perturbations are evaluated. The optimal, suboptimal, and preexisting paths are also investigated. Many of these parameters have exhibited an enhanced asymmetry between the two subunits of the dimeric protein, especially in the pretransfer complex, leading us to conclude that encoding of function goes beyond the sequence/structure of proteins. The local and global perturbations mediated by appropriate ligands and their influence on the equilibrium ensemble of conformations also have a significant role to play in the functioning of proteins. Taking a comprehensive view of these observations, we propose that the origin of many functional aspects (allostery and half-sites reactivity in the case of DhPylRS) lies in subtle rearrangements of interactions and dynamics at a global level.

  7. Allosteric regulation in NMDA receptors revealed by the genetically encoded photo-cross-linkers

    PubMed Central

    Tian, Meilin; Ye, Shixin

    2016-01-01

    Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+ for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology. PMID:27713495

  8. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  9. Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

    PubMed Central

    Knapp, James E.; Pahl, Reinhard; Šrajer, Vukica; Royer, William E.

    2006-01-01

    Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 μs after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition. PMID:16684887

  10. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  11. Detection of DNA methyltransferase activity using allosteric molecular beacons.

    PubMed

    Zhang, Weiting; Zu, Xiaolong; Song, Yanling; Zhu, Zhi; Yang, Chaoyong James

    2016-01-21

    Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity. When the specific site in an aMB is methylated by Dam MTase, the probe can be cut by the restriction nuclease DpnI to release a fluorophore labeled aptamer specific for streptavidin (SA) which will bind to SA beads to generate highly fluorescent beads for easy signal readout by a microscope or flow cytometer. However, aMBs maintain a hairpin structure without the binding ability to SA beads in the absence of Dam MTase, leading to weakly fluorescent SA beads. Unlike the existing signal amplified assays, our method is simpler and more convenient. The high performance of the aptamer and the easy bead separation process make this probe superior to other methods for the detection of MTase in complex biological systems. Overall, the proposed method with a detection limit of 0.57 U mL(-1) for Dam MTase shows great potential for further applications in the detection of other MTases, screening of MTase inhibitors, and early diagnosis of cancer.

  12. Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

    PubMed Central

    Bruno, Agostino; Lembo, Francesca; Novellino, Ettore; Stornaiuolo, Mariano; Marinelli, Luciana

    2014-01-01

    Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators. PMID:24441508

  13. Allosteric Inhibition of Human Immunodeficiency Virus Integrase

    PubMed Central

    Gupta, Kushol; Brady, Troy; Dyer, Benjamin M.; Malani, Nirav; Hwang, Young; Male, Frances; Nolte, Robert T.; Wang, Liping; Velthuisen, Emile; Jeffrey, Jerry; Van Duyne, Gregory D.; Bushman, Frederic D.

    2014-01-01

    HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action. PMID:24904063

  14. Allosteric regulation of rhomboid intramembrane proteolysis

    PubMed Central

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-01-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

  15. Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments.

    PubMed

    Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; La Regina, Giuseppe; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

    2015-10-20

    A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

  16. Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments

    NASA Astrophysics Data System (ADS)

    Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; Regina, Giuseppe La; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

    2015-10-01

    A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands.

  17. Endogenous vs Exogenous Allosteric Modulators in GPCRs: A dispute for shuttling CB1 among different membrane microenvironments

    PubMed Central

    Stornaiuolo, Mariano; Bruno, Agostino; Botta, Lorenzo; Regina, Giuseppe La; Cosconati, Sandro; Silvestri, Romano; Marinelli, Luciana; Novellino, Ettore

    2015-01-01

    A Cannabinoid Receptor 1 (CB1) binding site for the selective allosteric modulator ORG27569 is here identified through an integrate approach of consensus pocket prediction, mutagenesis studies and Mass Spectrometry. This unprecedented ORG27569 pocket presents the structural features of a Cholesterol Consensus Motif, a cholesterol interacting region already found in other GPCRs. ORG27569 and cholesterol affects oppositely CB1 affinity for orthosteric ligands. Moreover, the rise in cholesterol intracellular level results in CB1 trafficking to the axonal region of neuronal cells, while, on the contrary, ORG27568 binding induces CB1 enrichment at the soma. This control of receptor migration among functionally different membrane regions of the cell further contributes to downstream signalling and adds a previously unknown mechanism underpinning CB1 modulation by ORG27569 , that goes beyond a mere control of receptor affinity for orthosteric ligands. PMID:26482099

  18. Preclinical pharmacokinetic and toxicological evaluation of MIF-1 peptidomimetic, PAOPA: examining the pharmacology of a selective dopamine D2 receptor allosteric modulator for the treatment of schizophrenia.

    PubMed

    Tan, Mattea L; Basu, Dipannita; Kwiecien, Jacek M; Johnson, Rodney L; Mishra, Ram K

    2013-04-01

    Schizophrenia is a mental illness characterized by a breakdown in cognition and emotion. Over the years, drug treatment for this disorder has mainly been compromised of orthosteric ligands that antagonize the active site of the dopamine D2 receptor. However, these drugs are limited in their use and often lead to the development of adverse movement and metabolic side effects. Allosteric modulators are an emerging class of therapeutics with significant advantages over orthosteric ligands, including an improved therapeutic and safety profile. This study investigates our newly developed allosteric modulator, PAOPA, which is a specific modulator of the dopamine D2 receptor. Previous studies have shown PAOPA to attenuate schizophrenia-like behavioral abnormalities in preclinical models. To advance this newly developed allosteric drug from the preclinical to clinical stage, this study examines the pharmacokinetic behavior and toxicological profile of PAOPA. Results from this study prove the effectiveness of PAOPA in reaching the implicated regions of the brain for therapeutic action, particularly the striatum. Pharmacokinetic parameters of PAOPA were found to be comparable to current market antipsychotic drugs. Necropsy and histopathological analyses showed no abnormalities in all examined organs. Acute and chronic treatment of PAOPA indicated no movement abnormalities commonly found with the use of current typical antipsychotic drugs. Moreover, acute and chronic PAOPA treatment revealed no hematological or metabolic abnormalities classically found with the use of atypical antipsychotic drugs. Findings from this study demonstrate a better safety profile of PAOPA, and necessitates the progression of this newly developed therapeutic for the treatment of schizophrenia.

  19. 4-Phenylpyridin-2-one Derivatives: A Novel Class of Positive Allosteric Modulator of the M1 Muscarinic Acetylcholine Receptor.

    PubMed

    Mistry, Shailesh N; Jörg, Manuela; Lim, Herman; Vinh, Natalie B; Sexton, Patrick M; Capuano, Ben; Christopoulos, Arthur; Lane, J Robert; Scammells, Peter J

    2016-01-14

    Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer's and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a diverse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization. PMID:26624844

  20. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  1. Allosteric Modulators for the Treatment of Schizophrenia: Targeting Glutamatergic Networks

    PubMed Central

    Menniti, Frank S.; Lindsley, Craig W.; Conn, P. Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A.

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved. PMID:23409764

  2. Entropic mechanism of large fluctuation in allosteric transition.

    PubMed

    Itoh, Kazuhito; Sasai, Masaki

    2010-04-27

    A statistical mechanical model of allosteric transitions in proteins is developed by extending the structure-based model of protein folding to cases of multiple native conformations. The partition function is calculated exactly within the model and the free-energy surface reflecting allostery is derived. This approach is applied to an example protein, the receiver domain of the bacterial enhancer-binding protein NtrC. The model predicts the large entropy associated with a combinatorial number of preexisting transition routes. This large entropy lowers the free-energy barrier of the allosteric transition, which explains the large structural fluctuation observed in the NMR data of NtrC. The global allosteric transformation of NtrC is explained by the shift of preexisting distribution of conformations upon phosphorylation, but the local structural adjustment around the phosphorylation site is explained by the complementary induced-fit mechanism. Structural disordering accompanied by fluctuating interactions specific to two allosteric conformations underlies a large number of routes of allosteric transition. PMID:20385843

  3. Allosteric modulators for the treatment of schizophrenia: targeting glutamatergic networks.

    PubMed

    Menniti, Frank S; Lindsley, Craig W; Conn, P Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved.

  4. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their Ligands: Beyond CB1 and CB2

    PubMed Central

    Howlett, A. C.; Abood, M. E.; Alexander, S. P. H.; Di Marzo, V.; Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos, G.; Mackie, K.; Mechoulam, R.; Ross, R. A.

    2010-01-01

    There are at least two types of cannabinoid receptors (CB1 and CB2). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ9-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB1, non-CB2 established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB1 and/or CB2 receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel “CB3” cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB1, non-CB2 pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB3 receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB1 receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB1/CB2 receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB1, non-CB2 cannabinoid receptors; and 4) current cannabinoid receptor nomenclature. PMID:21079038

  5. Measurements and Modeling To Determine the Reduction Potential of Uncomplexed Bi(III) in Nitrate Solutions for Application in Bi(III)-Ligand Equilibria Studies by Voltammetry.

    PubMed

    Billing, Caren; Cukrowski, Ignacy

    2016-05-12

    The free metal ion potential, E(M), is a critical parameter in the calculation of formation constants when using voltammetry. When studying complex formation of Bi(III), however, E(Bi) cannot be directly measured. In this work a nitrate background electrolyte was employed to obtain reversible reduction waves. To determine E(Bi), measurements have to be made below pH ∼ 2 before the bismuth-oxy-nitrate species precipitates and thus corrections for the diffusion junction potential (monitored using Tl(I) as an internal reference ion) must be made. Additionally shifts in potential due to both Bi(III) hydrolysis and Bi(III) nitrate formation must also be compensated for before E(Bi) can be evaluated. The value of E(Bi) was determined relative to E(Tl) so that in an experiments where ligand is added to determine formation constants, E(Bi) can be determined as accurately as possible (since E(Tl) can generally still be measured). The value of E(Bi) - E(Tl) was found to be 495.6 ± 1.4 mV for the conditions employed. PMID:27088843

  6. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    PubMed

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-01

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  7. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

    PubMed

    Davis, Sterenn; Davis, Benjamin M; Richens, Joanna L; Vere, Kelly-Ann; Petrov, Peter G; Winlove, C Peter; O'Shea, Paul

    2015-08-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir's affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers.

  8. Positive Allosteric Modulators of Metabotropic Glutamate 2 Receptors in Schizophrenia Treatment

    PubMed Central

    Ellaithy, Amr; Younkin, Jason; Gonzalez-Maeso, Javier; Logothetis, Diomedes E.

    2015-01-01

    The last two decades have witnessed a rise in the “NMDA receptor hypofunction” hypothesis for schizophrenia, a devastating disorder that affects around 1% of the population worldwide. A variety of presynaptic, postsynaptic and regulatory proteins involved in glutamatergic signaling have thus been proposed as potential therapeutic targets. This Review focuses on positive allosteric modulation of metabotropic glutamate 2 receptors (mGlu2Rs) and discusses how recent preclinical epigenetic data may provide a molecular explanation for the discrepant results of clinical studies, further stimulating the field to exploit the promise of mGlu2R as a target for schizophrenia treatment. PMID:26148747

  9. Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

    PubMed

    Court, John J; Poisson, Carl; Ardzinski, Andrzej; Bilimoria, Darius; Chan, Laval; Chandupatla, Kishan; Chauret, Nathalie; Collier, Philip N; Das, Sanjoy Kumar; Denis, Francois; Dorsch, Warren; Iyer, Ganesh; Lauffer, David; L'Heureux, Lucille; Li, Pan; Luisi, Brian S; Mani, Nagraj; Nanthakumar, Suganthi; Nicolas, Olivier; Rao, B Govinda; Ronkin, Steven; Selliah, Subajini; Shawgo, Rebecca S; Tang, Qing; Waal, Nathan D; Yannopoulos, Constantin G; Green, Jeremy

    2016-07-14

    The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

  10. A Novel Class of Succinimide-Derived Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 1 Provides Insight into a Disconnect in Activity between the Rat and Human Receptors

    PubMed Central

    2014-01-01

    Recent progress in the discovery of mGlu1 allosteric modulators has suggested the modulation of mGlu1 could offer possible treatment for a number of central nervous system disorders; however, the available chemotypes are inadequate to fully investigate the therapeutic potential of mGlu1 modulation. To address this issue, we used a fluorescence-based high-throughput screening assay to screen an allosteric modulator-biased library of compounds to generate structurally diverse mGlu1 negative allosteric modulator hits for chemical optimization. Herein, we describe the discovery and characterization of a novel mGlu1 chemotype. This series of succinimide negative allosteric modulators, exemplified by VU0410425, exhibited potent inhibitory activity at rat mGlu1 but was, surprisingly, inactive at human mGlu1. VU0410425 and a set of chemically diverse mGlu1 negative allosteric modulators previously reported in the literature were utilized to examine this species disconnect between rat and human mGlu1 activity. Mutation of the key transmembrane domain residue 757 and functional screening of VU0410425 and the literature compounds suggests that amino acid 757 plays a role in the activity of these compounds, but the contribution of the residue is scaffold specific, ranging from critical to minor. The operational model of allosterism was used to estimate the binding affinities of each compound to compare to functional data. This novel series of mGlu1 negative allosteric modulators provides valuable insight into the pharmacology underlying the disconnect between rat and human mGlu1 activity, an issue that must be understood to progress the therapeutic potential of allosteric modulators of mGlu1. PMID:24798819

  11. The Tertiary Origin of the Allosteric Activation of E. coli Glucosamine-6-Phosphate Deaminase Studied by Sol-Gel Nanoencapsulation of Its T Conformer

    PubMed Central

    Zonszein, Sergio; Álvarez-Añorve, Laura I.; Vázquez-Núñez, Roberto J.; Calcagno, Mario L.

    2014-01-01

    The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known

  12. SOD1 exhibits allosteric frustration to facilitate metal binding affinity.

    PubMed

    Das, Atanu; Plotkin, Steven S

    2013-03-01

    Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free-radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative disease. Over 150 mutations in SOD1 have been identified with a familial form of the disease, but it is presently not clear what unifying features, if any, these mutants share to make them pathogenic. Here, we develop several unique computational assays for probing the thermo-mechanical properties of both ALS-associated and rationally designed SOD1 variants. Allosteric interaction-free energies between residues and metals are calculated, and a series of atomic force microscopy experiments are simulated with variable tether positions to quantify mechanical rigidity "fingerprints" for SOD1 variants. Mechanical fingerprinting studies of a series of C-terminally truncated mutants, along with an analysis of equilibrium dynamic fluctuations while varying native constraints, potential energy change upon mutation, frustratometer analysis, and analysis of the coupling between local frustration and metal binding interactions for a glycine scan of 90 residues together, reveal that the apo protein is internally frustrated, that these internal stresses are partially relieved by mutation but at the expense of metal-binding affinity, and that the frustration of a residue is directly related to its role in binding metals. This evidence points to apo SOD1 as a strained intermediate with "self-allostery" for high metal-binding affinity. Thus, the prerequisites for the function of SOD1 as an antioxidant compete with apo state thermo-mechanical stability, increasing the susceptibility of the protein to misfold in the apo state.

  13. Evaluating the potential of a new isotope-labelled glyco-ligand for estimating the remnant liver function of schistosoma-infected mice.

    PubMed

    Cheng, P-C; Chiang, P-F; Lee, K-M; Yeh, C-H; Hsu, K-L; Liu, S-W; Shen, L-H; Peng, C-L; Fan, C-K; Luo, T-Y

    2013-01-01

    A new glyco-derivative compound (OCTAM) was developed and labelled with isotope to form (188) Re-OCTAM as a candidate nuclear medicine imaging agent for testing the liver function. We evaluated the potential of isotope-labelled OCTAM for estimating the remnant liver function in vitro and in vivo schistosoma-infected mice. The affinity of OCTAM to liver asialoglycoprotein receptors (ASGPR) was assessed by competitive inhibition assay in vitro. In vivo assessments were performed to score the remnant liver function in mice at different schistosomal infection stages. OCTAM binds specifically to ASGPR and showed competitive inhibition of anti-ASGPR antibody binding to hepatocytes, and was higher than that of other galactosyl ligands. Micro-SPECT/CT images of uninfected mice revealed strong liver uptake. Quantified serial images of mice infected for 9, 12 and 18 weeks showed delayed liver uptake, and the retention of uptake was inversely correlated with stage and grade of schistosoma infection. Pathological and biochemical analysis demonstrated that gradually accumulating liver injury caused by infection significantly influenced uptake of (188) Re-OCTAM. Hepatic ASGPR expression diminished only in the chronic infection stage. This study demonstrated that the isotope-labelled OCTAM could accumulate in the liver, might have potential as an imaging agent for in vivo hepatic function evaluation of schistosomiasis.

  14. Targeting TNF-related apoptosis-inducing ligand (TRAIL) receptor by natural products as a potential therapeutic approach for cancer therapy

    PubMed Central

    Dai, Xiaoyun; Zhang, Jingwen; Arfuso, Frank; Chinnathambi, Arunachalam; Zayed, ME; Alharbi, Sulaiman Ali; Kumar, Alan Prem

    2015-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to selectively induce apoptotic cell death in various tumor cells by engaging its death-inducing receptors (TRAIL-R1 and TRAIL-R2). This property has led to the development of a number of TRAIL–receptor agonists such as the soluble recombinant TRAIL and agonistic antibodies, which have shown promising anticancer activity in preclinical studies. However, besides activating caspase-dependent apoptosis in several cancer cells, TRAIL may also activate nonapoptotic signal transduction pathways such as nuclear factor-kappa B, mitogen-activated protein kinases, AKT, and signal transducers and activators of transcription 3, which may contribute to TRAIL resistance that is being now frequently encountered in various cancers. TRAIL resistance can be overcome by the application of efficient TRAIL-sensitizing pharmacological agents. Natural compounds have shown a great potential in sensitizing cells to TRAIL treatment through suppression of distinct survival pathways. In this review, we have summarized both apoptotic and nonapoptotic pathways activated by TRAIL, as well as recent advances in developing TRAIL–receptor agonists for cancer therapy. We also briefly discuss combination therapies that have shown great potential in overcoming TRAIL resistance in various tumors. PMID:25854879

  15. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    PubMed

    Wang, Wei; Hong, Jeong S; Rab, Andras; Sorscher, Eric J; Kirk, Kevin L

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

  16. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias.

    PubMed

    Bolognini, Daniele; Moss, Catherine E; Nilsson, Karolina; Petersson, Annika U; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B; Milligan, Graeme

    2016-09-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11 Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

  17. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

    PubMed Central

    Wang, Wei; Hong, Jeong S.; Rab, Andras; Sorscher, Eric J.; Kirk, Kevin L.

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

  18. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias*

    PubMed Central

    Bolognini, Daniele; Moss, Catherine E.; Nilsson, Karolina; Petersson, Annika U.; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M.; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B.

    2016-01-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11. Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

  19. Ligand Binding and Structural Changes Associated with Allostery in Yeast NAD+-specific Isocitrate Dehydrogenase

    PubMed Central

    Lee, McAlister-Henn

    2011-01-01

    Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four each of regulatory IDH1 and catalytic IDH2 subunits that share 42% sequence identity. IDH2 contains catalytic isocitrate/Mg2+ and NAD+ binding sites whereas IDH1 contains homologous binding sites, respectively, for cooperative binding of isocitrate and for allosteric binding of AMP. Ligand binding is highly ordered in vitro, and IDH exhibits the unusual property of half-site binding for all ligands. The structures of IDH solved in the absence or presence of ligands have shown: (a) a heterodimer to be the basic structural/functional unit of the enzyme, (b) the organization of heterodimers to form tetramer and octamer structures, (c) structural differences that may underlie cooperative and allosteric regulatory mechanisms, and (d) the possibility for formation of a disulfide bond that could reduce catalytic activity. In vivo analyses of mutant enzymes have elucidated the physiological importance of catalytic activity and allosteric regulation of this tricarboxylic acid cycle enzyme. Other studies have established the importance of a disulfide bond in regulation of IDH activity in vivo, as well as contributions of this bond to the property of half-site ligand binding exhibited by the wild-type enzyme. PMID:22008468

  20. Behavioral effects of the cannabinoid CB1 receptor allosteric modulator ORG27569 in rats

    PubMed Central

    Ding, Yuanyuan; Qiu, Yanyan; Jing, Li; Thorn, David A; Zhang, Yanan; Li, Jun-Xu

    2014-01-01

    The cannabinoid CB1 receptor system is involved in feeding behaviors and the CB1 receptor antagonist SR141716A is an effective antiobesity drug. However, SR141716A also has serious side effects, which prompted the exploration of alternative strategies to modulate this important drug target. Recently a CB1 receptor allosteric modulating site has been discovered and the allosteric modulating activity of several modulators including ORG27569 has been characterized in vitro. Yet, little is known of the in vivo pharmacological effects of ORG27569. This study examined the behavioral pharmacology of ORG27569 in rats. ORG27569 (3.2–10 mg/kg, i.p.) selectively attenuated the hypothermic effects of CB1 receptor agonists CP55940 (0.1–1 mg/kg) and anandamide (3.2–32 mg/kg). In contrast, SR141716A only attenuated the hypothermic effects of CP55940 but not anandamide. SR141716A but not ORG27569 blocked CP55940-induced catalepsy and antinociception. In addition, ORG27569 did not modify SR141716A-elicited grooming and scratching behaviors. In feeding studies, ORG27569 decreased palatable and plain food intake which was partially blocked by CP55940. The hypophagic effect of ORG27569 developed tolerance after 4 days of daily 5.6 mg/kg treatment; however, the effect on body weight gain outlasted the drug treatment for 10 days. These data suggest that ORG27569 may not function as a CB1 receptor allosteric modulator in vivo, although its hypophagic activity still has potential therapeutic utility. PMID:25431655

  1. The therapeutic promise of positive allosteric modulation of nicotinic receptors.

    PubMed

    Uteshev, Victor V

    2014-03-15

    In the central nervous system, deficits in cholinergic neurotransmission correlate with decreased attention and cognitive impairment, while stimulation of neuronal nicotinic acetylcholine receptors improves attention, cognitive performance and neuronal resistance to injury as well as produces robust analgesic and anti-inflammatory effects. The rational basis for the therapeutic use of orthosteric agonists and positive allosteric modulators (PAMs) of nicotinic receptors arises from the finding that functional nicotinic receptors are ubiquitously expressed in neuronal and non-neuronal tissues including brain regions highly vulnerable to traumatic and ischemic types of injury (e.g., cortex and hippocampus). Moreover, functional nicotinic receptors do not vanish in age-, disease- and trauma-related neuropathologies, but their expression and/or activation levels decline in a subunit- and brain region-specific manner. Therefore, augmenting the endogenous cholinergic tone by nicotinic agents is possible and may offset neurological impairments associated with cholinergic hypofunction. Importantly, because neuronal damage elevates extracellular levels of choline (a selective agonist of α7 nicotinic acetylcholine receptors) near the site of injury, α7-PAM-based treatments may augment pathology-activated α7-dependent auto-therapies where and when they are most needed (i.e., in the penumbra, post-injury). Thus, nicotinic-PAM-based treatments are expected to augment the endogenous cholinergic tone in a spatially and temporally restricted manner creating the potential for differential efficacy and improved safety as compared to exogenous orthosteric nicotinic agonists that activate nicotinic receptors indiscriminately. In this review, I will summarize the existing trends in therapeutic applications of nicotinic PAMs.

  2. Metal ion coupled protein folding and allosteric motions

    NASA Astrophysics Data System (ADS)

    Wang, Wei

    2014-03-01

    Many proteins need the help of cofactors for their successful folding and functioning. Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors. Binding of metal ions can reshape the energy landscapes of proteins, thereby modifying the folding and allosteric motions. For example, such binding may make the intrinsically disordered proteins have funneled energy landscapes, consequently, ensures their spontaneous folding. In addition, the binding may activate certain biological processes by inducing related conformational changes of regulation proteins. However, how the local interactions involving the metal ion binding can induce the global conformational motions of proteins remains elusive. Investigating such question requires multiple models with different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we have been developing such multiscale methods which can reasonably model the metal ion binding induced charge transfer, protonation/deprotonation, and large conformational motions of proteins. With such multiscale model, we elucidated the zinc-binding induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric motions of calmodulin domains. In this talk, I will introduce the above progresses on the metal ion coupled protein folding and allosteric motions. We thank the finacial support from NSFC and the 973 project.

  3. The structure and allosteric regulation of glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2011-09-01

    Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.

  4. An allosteric inhibitor of protein arginine methyltransferase 3.

    PubMed

    Siarheyeva, Alena; Senisterra, Guillermo; Allali-Hassani, Abdellah; Dong, Aiping; Dobrovetsky, Elena; Wasney, Gregory A; Chau, Irene; Marcellus, Richard; Hajian, Taraneh; Liu, Feng; Korboukh, Ilia; Smil, David; Bolshan, Yuri; Min, Jinrong; Wu, Hong; Zeng, Hong; Loppnau, Peter; Poda, Gennadiy; Griffin, Carly; Aman, Ahmed; Brown, Peter J; Jin, Jian; Al-Awar, Rima; Arrowsmith, Cheryl H; Schapira, Matthieu; Vedadi, Masoud

    2012-08-01

    PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

  5. Deeper Insights into the Allosteric Modulation of Ionotropic Glutamate Receptors.

    PubMed

    Regan, Michael C; Furukawa, Hiro

    2016-09-21

    Two articles in this issue of Neuron (Yelshanskaya et al., 2016; Yi et al., 2016) explore the structural basis of allosteric inhibition in ionotropic glutamate receptors, providing key insights into how iGluRs function in the brain as well as how they might be pharmacologically modulated in neurological disorders and disease. PMID:27657445

  6. Oxazolidinone-based allosteric modulators of mGluR5: Defining molecular switches to create a pharmacological tool box.

    PubMed

    Huang, Hong; Degnan, Andrew P; Balakrishnan, Anand; Easton, Amy; Gulianello, Michael; Huang, Yanling; Matchett, Michele; Mattson, Gail; Miller, Regina; Santone, Kenneth S; Senapati, Arun; Shields, Eric E; Sivarao, Digavalli V; Snyder, Lawrence B; Westphal, Ryan; Whiterock, Valerie J; Yang, Fukang; Bronson, Joanne J; Macor, John E

    2016-09-01

    Herein we describe the structure activity relationships uncovered in the pursuit of an mGluR5 positive allosteric modulator (PAM) for the treatment of schizophrenia. It was discovered that certain modifications of an oxazolidinone-based chemotype afforded predictable changes in the pharmacological profile to give analogs with a wide range of functional activities. The discovery of potent silent allosteric modulators (SAMs) allowed interrogation of the mechanism-based liabilities associated with mGluR5 activation and drove our medicinal chemistry effort toward the discovery of low efficacy (fold shift) PAMs devoid of agonist activity. This work resulted in the identification of dipyridyl 22 (BMS-952048), a compound with a favorable free fraction, efficacy in a rodent-based cognition model, and low potential for convulsions in mouse. PMID:27496211

  7. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

    PubMed Central

    Buller, Andrew R.; Brinkmann-Chen, Sabine; Romney, David K.; Herger, Michael; Murciano-Calles, Javier; Arnold, Frances H.

    2015-01-01

    Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope. PMID:26553994

  8. Ultraviolet light converts propranolol, a nonselective β-blocker and potential lupus-inducing drug, into a proinflammatory AhR ligand.

    PubMed

    Dorgham, Karim; Amoura, Zahir; Parizot, Christophe; Arnaud, Laurent; Frances, Camille; Pionneau, Cédric; Devilliers, Hervé; Pinto, Sandra; Zoorob, Rima; Miyara, Makoto; Larsen, Martin; Yssel, Hans; Gorochov, Guy; Mathian, Alexis

    2015-11-01

    UV light and some medications are known to trigger lupus erythematosus (LE). A common mechanism underlying the immunopathologic effect, resulting from exposure to these two seemingly unrelated factors, remains unknown. The aryl hydrocarbon receptor (AhR) plays a key role in the regulation of IL-22 production in humans and can be activated by both xenobiotics and naturally occurring photoproducts. A significant expansion of Th17 and Th22 cells was observed in the peripheral blood of active systemic LE (SLE) patients, compared to inactive patients and controls. We also show that propranolol, a potential lupus-inducing drug, induced stronger AhR activation in PBMCs of SLE patients than in those of controls. AhR agonist activity of propranolol was enhanced by UV light exposure. MS analysis of irradiated propranolol revealed the generation of a proinflammatory photoproduct. This compound behaves like the prototypic AhR ligand 6-formylindolo[3,2-b]carbazole, a cutaneous UV light-induced tryptophan metabolite, both promoting IL-22, IL-8, and CCL2 secretion by T-cells and macrophages. Finally, LE patients exhibit signs of cutaneous AhR activation that correlate with lesional expression of the same proinflammatory cytokines, suggesting a role for photometabolites in the induction of skin inflammation. The AhR might therefore represent a target for therapeutic intervention in LE. PMID:26354876

  9. Cell-specific targeting in the mouse inner ear using nanoparticles conjugated with a neurotrophin-derived peptide ligand: potential tool for drug delivery.

    PubMed

    Roy, Soumen; Johnston, Alex H; Newman, Tracey A; Glueckert, Rudolf; Dudas, Jozsef; Bitsche, Mario; Corbacella, Elisa; Rieger, Gunde; Martini, Alessandro; Schrott-Fischer, Anneliese

    2010-05-10

    Cell specific targeting is an emerging field in nanomedicine. Homing of the multifunctional nanoparticles (MFNPs) is achieved by the conjugation of targeting moieties on the nanoparticle surface. The inner ear is an attractive target for new drug delivery strategies as it is hard to access and hearing loss is a significant worldwide problem. In this work we investigated the utility of a Nerve Growth Factor-derived peptide (hNgf_EE) functionalized nanoparticles (NPs) to target cells of the inner ear. These functionalized NPs were introduced to organotypic explant cultures of the mouse inner ear and to PC-12 rat pheochromocytoma cells. The NPs did not show any signs of toxicity. Specific targeting and higher binding affinity to spiral ganglion neurons, Schwann cells and nerve fibers of the explant cultures were achieved through ligand mediated multivalent binding to tyrosine kinase receptors and to p75 neurotrophin receptors. Unspecific uptake of NPs was investigated using NPs conjugated with scrambled hNgf_EE peptide. Our results indicate a selective cochlear cell targeting by MFNPs, which may be a potential tool for cell specific drug and gene delivery to the inner ear.

  10. Two-ligand priming mechanism for potentiated phosphoinositide synthesis is an evolutionarily conserved feature of Sec14-like phosphatidylinositol and phosphatidylcholine exchange proteins

    PubMed Central

    Huang, Jin; Ghosh, Ratna; Tripathi, Ashutosh; Lönnfors, Max; Somerharju, Pentti; Bankaitis, Vytas A.

    2016-01-01

    Lipid signaling, particularly phosphoinositide signaling, plays a key role in regulating the extreme polarized membrane growth that drives root hair development in plants. The Arabidopsis AtSFH1 gene encodes a two-domain protein with an amino-terminal Sec14-like phosphatidylinositol transfer protein (PITP) domain linked to a carboxy-terminal nodulin domain. AtSfh1 is critical for promoting the spatially highly organized phosphatidylinositol-4,5-bisphosphate signaling program required for establishment and maintenance of polarized root hair growth. Here we demonstrate that, like the yeast Sec14, the AtSfh1 PITP domain requires both its phosphatidylinositol (PtdIns)- and phosphatidylcholine (PtdCho)-binding properties to stimulate PtdIns-4-phosphate [PtdIns(4)P] synthesis. Moreover, we show that both phospholipid-binding activities are essential for AtSfh1 activity in supporting polarized root hair growth. Finally, we report genetic and biochemical evidence that the two-ligand mechanism for potentiation of PtdIns 4-OH kinase activity is a broadly conserved feature of plant Sec14-nodulin proteins, and that this strategy appeared only late in plant evolution. Taken together, the data indicate that the PtdIns/PtdCho-exchange mechanism for stimulated PtdIns(4)P synthesis either arose independently during evolution in yeast and in higher plants, or a suitable genetic module was introduced to higher plants from a fungal source and subsequently exploited by them. PMID:27193303

  11. Water-soluble and photo-stable silver(I) dicarboxylate complexes containing 1,10-phenanthroline ligands: Antimicrobial and anticancer chemotherapeutic potential, DNA interactions and antioxidant activity.

    PubMed

    Thornton, Laura; Dixit, Vidya; Assad, Letícia O N; Ribeiro, Thales P; Queiroz, Daniela D; Kellett, Andrew; Casey, Alan; Colleran, John; Pereira, Marcos D; Rochford, Garret; McCann, Malachy; O'Shea, Denis; Dempsey, Rita; McClean, Siobhán; Kia, Agnieszka Foltyn-Arfa; Walsh, Maureen; Creaven, Bernadette; Howe, Orla; Devereux, Michael

    2016-06-01

    The complexes [Ag2(OOC-(CH2)n-COO)] (n=1-10) (1-10) were synthesised and reacted with 1,10-phenanthroline (phen) to yield derivatives formulating as [Ag2(phen)x(OOC-(CH2)y-COO)]·zH2O (x=2 or 3; y=1-10; z=1-4) (11-20) which are highly water-soluble and photo-stable in aqueous solution. The phen derivatives 11-20 exhibit chemotherapeutic potential against Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and against cisplatin-sensitive breast (MCF-7) and resistant ovarian (SKOV-3) cancer cell lines. Cyclic voltammetric analysis and DNA binding and intercalation studies indicate that the mechanism of action of 11-20 is significantly different to that of their silver(I) dicarboxylate precursors and they do not induce DNA damage or ROS generation in mammalian cells. The representative complexes 9 and 19 (containing the undecanedioate ligand) were both found to significantly reduce superoxide and hydrogen peroxide induced oxidative stress in the yeast S. cerevisiae. PMID:26986979

  12. Ultraviolet light converts propranolol, a nonselective β-blocker and potential lupus-inducing drug, into a proinflammatory AhR ligand.

    PubMed

    Dorgham, Karim; Amoura, Zahir; Parizot, Christophe; Arnaud, Laurent; Frances, Camille; Pionneau, Cédric; Devilliers, Hervé; Pinto, Sandra; Zoorob, Rima; Miyara, Makoto; Larsen, Martin; Yssel, Hans; Gorochov, Guy; Mathian, Alexis

    2015-11-01

    UV light and some medications are known to trigger lupus erythematosus (LE). A common mechanism underlying the immunopathologic effect, resulting from exposure to these two seemingly unrelated factors, remains unknown. The aryl hydrocarbon receptor (AhR) plays a key role in the regulation of IL-22 production in humans and can be activated by both xenobiotics and naturally occurring photoproducts. A significant expansion of Th17 and Th22 cells was observed in the peripheral blood of active systemic LE (SLE) patients, compared to inactive patients and controls. We also show that propranolol, a potential lupus-inducing drug, induced stronger AhR activation in PBMCs of SLE patients than in those of controls. AhR agonist activity of propranolol was enhanced by UV light exposure. MS analysis of irradiated propranolol revealed the generation of a proinflammatory photoproduct. This compound behaves like the prototypic AhR ligand 6-formylindolo[3,2-b]carbazole, a cutaneous UV light-induced tryptophan metabolite, both promoting IL-22, IL-8, and CCL2 secretion by T-cells and macrophages. Finally, LE patients exhibit signs of cutaneous AhR activation that correlate with lesional expression of the same proinflammatory cytokines, suggesting a role for photometabolites in the induction of skin inflammation. The AhR might therefore represent a target for therapeutic intervention in LE.

  13. Positive homotropic allosteric receptors for neutral guests: annulated tetrathiafulvalene-calix[4]pyrroles as colorimetric chemosensors for nitroaromatic explosives.

    PubMed

    Park, Jung Su; Le Derf, Franck; Bejger, Christopher M; Lynch, Vincent M; Sessler, Jonathan L; Nielsen, Kent A; Johnsen, Carsten; Jeppesen, Jan O

    2010-01-18

    The study of positive homotropic allosterism in supramolecular receptors is important for elucidating design strategies that can lead to increased sensitivity in various molecular recognition applications. In this work, the cooperative relationship between tetrathiafulvalene (TTF)-calix[4]pyrroles and several nitroaromatic guests is examined. The design and synthesis of new annulated TTF-calix[4]pyrrole receptors with the goal of rigidifying the system to accommodate better nitroaromatic guests is outlined. These new derivatives, which display significant improvement in terms of binding constants, also display a positive homotropic allosteric relationship, as borne out from the sigmoidal nature of the binding isotherms and analysis by using the Hill equation, Adair equation, and Scatchard plots. The host-guest complexes themselves have been characterized by single-crystal X-ray diffraction analyses and studied by means of UV-spectroscopic titrations. Investigations into the electronic nature of the receptors were made by using cyclic voltammetry; this revealed that the binding efficiency was not strictly related to the redox potential of the receptor. On the other hand, this work serves to illustrate how cooperative effects may be used to enhance the recognition ability of TTF-calix[4]pyrrole receptors. It has led to new allosteric systems that function as rudimentary colorimetric chemosensors for common nitroaromatic-based explosives, and which are effective even in the presence of potentially interfering anions.

  14. Phenyl-tetrazolyl acetophenones: discovery of positive allosteric potentiatiors for the metabotropic glutamate 2 receptor.

    PubMed

    Pinkerton, Anthony B; Vernier, Jean-Michel; Schaffhauser, Hervé; Rowe, Blake A; Campbell, Una C; Rodriguez, Dana E; Lorrain, Daniel S; Baccei, Christopher S; Daggett, Lorrie P; Bristow, Linda J

    2004-08-26

    Herein we disclose the discovery of a new class of positive allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2), phenyl-tetrazolyl acetophenones, e.g. 1-(2-hydroxy-3-propyl-4-[4-[4-(2H-tetrazol-5-yl)phenoxy]butoxy]phenyl) ethanone (4). These potentiators were shown to have no effect in the absence of glutamate as well as no effect at mGlu3 or the other mGlu receptors. The compounds were also evaluated in rodent models with potential relevance for schizophrenia, and 4 was shown to have activity in the inhibition of ketamine-induced norepinephrine release and ketamine-induced hyperactivity. This represents the first example of the efficacy of mGlu2 receptor potentiators in these models. PMID:15317469

  15. Crystal structure of a GroEL-ADP complex in the relaxed allosteric state at 2.7 Å resolution

    PubMed Central

    Fei, Xue; Yang, Dong; LaRonde-LeBlanc, Nicole; Lorimer, George H.

    2013-01-01

    The chaperonin proteins GroEL and GroES are cellular nanomachines driven by the hydrolysis of ATP that facilitate the folding of structurally diverse substrate proteins. In response to ligand binding, the subunits of a ring cycle in a concerted manner through a series of allosteric states (T, R, and R″), enabling work to be performed on the substrate protein. Removing two salt bridges that ordinarily break during the allosteric transitions of the WT permitted the structure of GroEL-ADP in the R state to be solved to 2.7 Å resolution. Whereas the equatorial domain displays almost perfect sevenfold symmetry, the apical domains, to which substrate proteins bind, and to a lesser extent, the intermediate domains display a remarkable asymmetry. Freed of intersubunit contacts, the apical domain of each subunit adopts a different conformation, suggesting a flexibility that permits interaction with diverse substrate proteins. This result contrasts with a previous cryo-EM study of a related allosteric ATP-bound state at lower resolution. After artificially imposing sevenfold symmetry it was concluded that a GroEL ring in the R-ATP state existed in six homogeneous but slightly different states. By imposing sevenfold symmetry on each of the subunits of the crystal structure of GroEL-ADP, we showed that the synthetic rings of (X-ray) GroEL-ADP and (cryo-EM) GroEL-ATP are structurally closely related. A deterministic model, the click stop mechanism, that implied temporal transitions between these states was proposed. Here, however, these conformational states are shown to exist as a structurally heterogeneous ensemble within a single ring. PMID:23861496

  16. Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis

    PubMed Central

    Ferreira, Tatiana Paula Teixeira; Mariano, Lívia Lacerda; Ghilosso-Bortolini, Roberta; de Arantes, Ana Carolina Santos; Fernandes, Andrey Junior; Berni, Michelle; Cecchinato, Valentina; Uguccioni, Mariagrazia; Maj, Roberto; Barberis, Alcide; Silva, Patricia Machado Rodrigues e; Martins, Marco Aurélio

    2016-01-01

    Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice

  17. Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis.

    PubMed

    Ferreira, Tatiana Paula Teixeira; Mariano, Lívia Lacerda; Ghilosso-Bortolini, Roberta; de Arantes, Ana Carolina Santos; Fernandes, Andrey Junior; Berni, Michelle; Cecchinato, Valentina; Uguccioni, Mariagrazia; Maj, Roberto; Barberis, Alcide; Silva, Patricia Machado Rodrigues E; Martins, Marco Aurélio

    2016-01-01

    Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice

  18. Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs.

    PubMed

    Dwivedi, Varun; Manickam, Cordelia; Dhakal, Santosh; Binjawadagi, Basavaraj; Ouyang, Kang; Hiremath, Jagadish; Khatri, Mahesh; Hague, Jacquelyn Gervay; Lee, Chang Won; Renukaradhya, Gourapura J

    2016-04-15

    Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract.

  19. Quantifying the allosteric properties of Escherichia coli carbamyl phosphate synthetase: determination of thermodynamic linked-function parameters in an ordered kinetic mechanism.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1992-03-01

    The effects of the allosteric ligands UMP, IMP, and ornithine on the partial reactions catalyzed by Escherichia coli carbamyl phosphate synthetase have been examined. Both of these reactions, a HCO3(-)-dependent ATP synthesis reaction and a carbamyl phosphate-dependent ATP synthesis reaction, follow bimolecular ordered sequential kinetic mechanisms. In the ATPase reaction, MgATP binds before HCO3- as established previously for the overall reaction catalyzed by carbamyl phosphate synthetase [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587-5591]. The initial velocity kinetics for the ATP synthesis reaction indicate that MgADP binds before carbamyl phosphate in an equilibrium ordered mechanism except in the presence of ornithine. Determination of true thermodynamic linked-function parameters describing the impact of allosteric ligands on the binding interactions of the first substrate to bind in an ordered mechanism requires experiments to be performed in which both substrates are varied even if only one is apparently affected by the allosteric ligands. In so doing, we have found that IMP has little effect on the overall reaction of either of these two partial reactions. UMP and ornithine, which have a pronounced effect on the apparent Km for MgATP in the overall reaction, both substantially change the thermodynamic dissociation constant for MgADP from the binary E-MgADP complex, Kia, in the ATP synthesis reaction, with UMP increasing Kia 15-fold and ornithine decreasing Kia by 18-fold. By contrast, only UMP substantially affects the Kia for MgATP in the ATPase reaction, increasing it by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1531767

  20. Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo

    PubMed Central

    Deshayes, Caroline; Bielecka, Magdalena K; Cain, Robert J; Scortti, Mariela; de las Heras, Aitor; Pietras, Zbigniew; Luisi, Ben F; Núñez Miguel, Ricardo; Vázquez-Boland, José A

    2012-01-01

    The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the β-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfAallo substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues. PMID:22646689

  1. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

    PubMed

    DeVree, Brian T; Mahoney, Jacob P; Vélez-Ruiz, Gisselle A; Rasmussen, Soren G F; Kuszak, Adam J; Edwald, Elin; Fung, Juan-Jose; Manglik, Aashish; Masureel, Matthieu; Du, Yang; Matt, Rachel A; Pardon, Els; Steyaert, Jan; Kobilka, Brian K; Sunahara, Roger K

    2016-07-01

    G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. PMID:27362234

  2. 3-Benzyl-1,3-oxazolidin-2-ones as mGluR2 positive allosteric modulators: Hit-to lead and lead optimization.

    PubMed

    Duplantier, Allen J; Efremov, Ivan; Candler, John; Doran, Angela C; Ganong, Alan H; Haas, Jessica A; Hanks, Ashley N; Kraus, Kenneth G; Lazzaro, John T; Lu, Jiemin; Maklad, Noha; McCarthy, Sheryl A; O'Sullivan, Theresa J; Rogers, Bruce N; Siuciak, Judith A; Spracklin, Douglas K; Zhang, Lei

    2009-05-01

    The discovery, synthesis and SAR of a novel series of 3-benzyl-1,3-oxazolidin-2-ones as positive allosteric modulators (PAMs) of mGluR2 is described. Expedient hit-to-lead work on a single HTS hit led to the identification of a ligand-efficient and structurally attractive series of mGluR2 PAMs. Human microsomal clearance and suboptimal physicochemical properties of the initial lead were improved to give potent, metabolically stable and orally available mGluR2 PAMs.

  3. NbIT--a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT.

    PubMed

    LeVine, Michael V; Weinstein, Harel

    2014-05-01

    Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems. PMID:24785005

  4. NbIT--a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT.

    PubMed

    LeVine, Michael V; Weinstein, Harel

    2014-05-01

    Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems.

  5. Novel Scaffold Identification of mGlu1 Receptor Negative Allosteric Modulators Using a Hierarchical Virtual Screening Approach.

    PubMed

    Jang, Jae Wan; Cho, Nam-Chul; Min, Sun-Joon; Cho, Yong Seo; Park, Ki Duk; Seo, Seon Hee; No, Kyoung Tai; Pae, Ae Nim

    2016-02-01

    Metabotropic glutamate receptor 1 (mGluR1) is considered as an attractive drug target for neuropathic pain treatments. The hierarchical virtual screening approach for identifying novel scaffolds of mGluR1 allosteric modulators was performed using a homology model built with the dopamine D3 crystal structure as template. The mGluR1 mutagenesis data, conserved amino acid sequences across class A and class C GPCRs, and previously reported multiple sequence alignments of class C GPCRs to the rhodopsin template, were employed for the sequence alignment to overcome difficulties of model generation with low sequence identity of mGluR1 and dopamine D3. The structures refined by molecular dynamics simulations were employed for docking of Asinex commercial libraries after hierarchical virtual screening with pharmacophore and naïve Bayesian models. Five of 35 compounds experimentally evaluated using a calcium mobilization assay exhibited micromolar activities (IC50) with chemotype novelty that demonstrated the validity of our methods. A hierarchical structure and ligand-based virtual screening approach with homology model of class C GPCR based on dopamine D3 class A GPCR structure was successfully performed and applied to discover novel negative mGluR1 allosteric modulators.

  6. Novel Scaffold Identification of mGlu1 Receptor Negative Allosteric Modulators Using a Hierarchical Virtual Screening Approach.

    PubMed

    Jang, Jae Wan; Cho, Nam-Chul; Min, Sun-Joon; Cho, Yong Seo; Park, Ki Duk; Seo, Seon Hee; No, Kyoung Tai; Pae, Ae Nim

    2016-02-01

    Metabotropic glutamate receptor 1 (mGluR1) is considered as an attractive drug target for neuropathic pain treatments. The hierarchical virtual screening approach for identifying novel scaffolds of mGluR1 allosteric modulators was performed using a homology model built with the dopamine D3 crystal structure as template. The mGluR1 mutagenesis data, conserved amino acid sequences across class A and class C GPCRs, and previously reported multiple sequence alignments of class C GPCRs to the rhodopsin template, were employed for the sequence alignment to overcome difficulties of model generation with low sequence identity of mGluR1 and dopamine D3. The structures refined by molecular dynamics simulations were employed for docking of Asinex commercial libraries after hierarchical virtual screening with pharmacophore and naïve Bayesian models. Five of 35 compounds experimentally evaluated using a calcium mobilization assay exhibited micromolar activities (IC50) with chemotype novelty that demonstrated the validity of our methods. A hierarchical structure and ligand-based virtual screening approach with homology model of class C GPCR based on dopamine D3 class A GPCR structure was successfully performed and applied to discover novel negative mGluR1 allosteric modulators. PMID:26343933

  7. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    DOE PAGES

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F.

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Twomore » hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.« less

  8. Low-frequency normal modes that describe allosteric transitions in biological nanomachines are robust to sequence variations.

    PubMed

    Zheng, Wenjun; Brooks, Bernard R; Thirumalai, D

    2006-05-16

    By representing the high-resolution crystal structures of a number of enzymes using the elastic network model, it has been shown that only a few low-frequency normal modes are needed to describe the large-scale domain movements that are triggered by ligand binding. Here we explore a link between the nearly invariant nature of the modes that describe functional dynamics at the mesoscopic level and the large evolutionary sequence variations at the residue level. By using a structural perturbation method (SPM), which probes the residue-specific response to perturbations (or mutations), we identify a sparse network of strongly conserved residues that transmit allosteric signals in three structurally unrelated biological nanomachines, namely, DNA polymerase, myosin motor, and the Escherichia coli chaperonin. Based on the response of every mode to perturbations, which are generated by interchanging specific sequence pairs in a multiple sequence alignment, we show that the functionally relevant low-frequency modes are most robust to sequence variations. Our work shows that robustness of dynamical modes at the mesoscopic level is encoded in the structure through a sparse network of residues that transmit allosteric signals.

  9. Designing Allosteric Regulators of Thrombin. Monosulfated Benzofuran Dimers Selectively Interact With Arg173 of Exosite II to Induce Inhibition

    PubMed Central

    Abdel Aziz, May H.; Sidhu, Preetpal Singh; Liang, Aiye; Kim, Ji Yeong; Mosier, Philip D.; Zhou, Qibing; Farrell, David H.; Desai, Umesh R.

    2012-01-01

    Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. (2011) J Med Chem 54: 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC50 of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ’ fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175 and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC50. Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. Absence of Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially non-toxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants. PMID:22788964

  10. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    SciTech Connect

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F.

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.

  11. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation.

    PubMed

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-09-18

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  12. Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

    PubMed Central

    Doshi, Urmi; Holliday, Michael J.; Eisenmesser, Elan Z.; Hamelberg, Donald

    2016-01-01

    Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general. PMID:27071107

  13. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation

    PubMed Central

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-01-01

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  14. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  15. Complexes of trivalent metal ions with potentially heptadentate N{sub 4}O{sub 3} Schiff base and amine phenol ligands of varying rigidity

    SciTech Connect

    Yang, L.W.; Liu, S.; Wong, E.; Rettig, S.J.; Orvig, C.

    1995-04-12

    The synthesis and characterization of several potentially heptadentate N{sub 4}O{sub 3} Schiff bases and amine phenols, as well as a series of their mononuclear and dinuclear complexes with indium and the lanthanides are reported. Schiff bases containing imidazolidine rings were the products of the known condensation reaction of triethylenetetramine with 3 equiv of 5-substituted salicylaldehydes to form H{sub 3}api (5-H-substituent), H{sub 3}Clapi (5-Cl-substituent), or H{sub 3}Brapi (5-Br-substituent); KBH{sub 4} reduction of these Schiff bases gave the appropriate isomeric N{sub 4}O{sub 3} amine phenols H{sub 3}(1,2,4-btt) and H{sub 3}(1,1,4-btt), as well as an acetone adduct, H{sub 3}(1,2,4-ahi). The Schiff bases reacted with 1 equiv of a lanthanide (Ln{sup 3+}) nitrate to produce mononuclear nine-coordinated [Ln(H{sub 3}Xapi)-(NO{sub 3}){sub 3}] complex wherein the ligand adopts a tridentate capping coordination mode, whereas the amine phenols formed mononuclear seven-coordinate complexes with the lanthanides and indium; homodinuclear complexes [LnL]{sub 2} were also obtained with the Schiff bases. The X-ray structures of the Schiff bases H{sub 3}api and H{sub 3}Clapi, the mononuclear amine phenol complexes Yb(1,2,4-btt){center_dot}0.5CH{sub 3}OH and In(1,1,4-btt), and the homodinuclear Schiff base complex [La(Brapi)]{sub 2}{center_dot}2CHCl{sub 3} have been determined.

  16. Solution Structure of the PAS Domain of a Thermophilic YybT Protein Homolog Reveals a Potential Ligand-binding Site*

    PubMed Central

    Tan, Edward; Rao, Feng; Pasunooti, Swathi; Pham, Thi Huong; Soehano, Ishin; Turner, Mark S.; Liew, Chong Wai; Lescar, Julien; Pervushin, Konstantin; Liang, Zhao-Xun

    2013-01-01

    The Bacillus subtilis protein YybT (or GdpP) and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP. YybT homologs contain a small Per-ARNT-Sim (PAS) domain (∼80 amino acids) that can bind b-type heme with 1:1 stoichiometry despite the small size of the domain and the lack of a conserved heme iron-coordinating residue. We determined the solution structure of the PAS domain of GtYybT from Geobacillus thermodenitrificans by NMR spectroscopy to further probe its function. The solution structure confirms that PASGtYybT adopts the characteristic PAS fold composed of a five-stranded antiparallel β sheet and a few short α-helices. One α-helix and three central β-strands of PASGtYybT are noticeably shorter than those of the typical PAS domains. Despite the small size of the protein domain, a hydrophobic pocket is formed by the side chains of nonpolar residues stemming from the β-strands and α-helices. A set of residues in the vicinity of the pocket and in the C-terminal region at the dimeric interface exhibits perturbed NMR parameters in the presence of heme or zinc protoporphyrin. Together, the results unveil a compact PAS domain with a potential ligand-binding pocket and reinforce the view that the PASYybT domains function as regulatory domains in the modulation of cellular cyclic di-AMP concentration. PMID:23504327

  17. Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.

    PubMed

    Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; M Allewell, Norma; Tuchman, Mendel; Shi, Dashuang

    2013-08-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

  18. Pharmacological Modulation of NMDA Receptor Activity and the Advent of Negative and Positive Allosteric Modulators

    PubMed Central

    Monaghan, Daniel T.; Irvine, Mark W.; Costa, Blaise Mathias; Fang, Guangyu; Jane, David E.

    2012-01-01

    The NMDA receptor (NMDAR) family of L-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the L-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identifed new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C & GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the L-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics. PMID:22269804

  19. Light-activated DNA binding in a designed allosteric protein

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  20. Looking for the Origin of Allosteric Cooperativity in Metallopolymers.

    PubMed

    Babel, Lucille; Hoang, Thi Nhu Y; Guénée, Laure; Besnard, Céline; Wesolowski, Tomasz A; Humbert-Droz, Marie; Piguet, Claude

    2016-06-01

    The basic concept of allosteric cooperativity used in biology, chemistry and physics states that any change in the intermolecular host-guest interactions operating in multisite receptors can be assigned to intersite interactions. Using lanthanide metals as guests and linear multi-tridentate linear oligomers of variable lengths and geometries as hosts, this work shows that the quantitative modeling of metal loadings requires the consideration of a novel phenomenon originating from solvation processes. It stepwise modulates the intrinsic affinity of each isolated site in multisite receptors, and this without resorting to allosteric cooperativity. An easy-to-handle additive model predicts a negative power law dependence of the intrinsic affinity on the length of the linear metallopolymer. Applied to lanthanidopolymers, the latter common analysis overestimates cooperativity factors by more than two orders of magnitude. PMID:27142083

  1. A general framework improving teaching ligand binding to a macromolecule.

    PubMed

    Haiech, Jacques; Gendrault, Yves; Kilhoffer, Marie-Claude; Ranjeva, Raoul; Madec, Morgan; Lallement, Christophe

    2014-10-01

    The interaction of a ligand with a macromolecule has been modeled following different theories. The tenants of the induced fit model consider that upon ligand binding, the protein-ligand complex undergoes a conformational change. In contrast, the allosteric model assumes that only one among different coexisting conformers of a given protein is suitable to bind the ligand optimally. In the present paper, we propose a general framework to model the binding of ligands to a macromolecule. Such framework built on the binding polynomial allows opening new ways to teach in a unified manner ligand binding, enzymology and receptor binding in pharmacology. Moreover, we have developed simple software that allows building the binding polynomial from the schematic description of the biological system under study. Taking calmodulin as a canonical example, we show here that the proposed tool allows the easy retrieval of previously experimental and computational reports. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

  2. Discovery of GPCR ligands for probing signal transduction pathways

    PubMed Central

    Brogi, Simone; Tafi, Andrea; Désaubry, Laurent; Nebigil, Canan G.

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven integral transmembrane proteins that are the primary targets of almost 30% of approved drugs and continue to represent a major focus of pharmaceutical research. All of GPCR targeted medicines were discovered by classical medicinal chemistry approaches. After the first GPCR crystal structures were determined, the docking screens using these structures lead to discovery of more novel and potent ligands. There are over 360 pharmaceutically relevant GPCRs in the human genome and to date about only 30 of structures have been determined. For these reasons, computational techniques such as homology modeling and molecular dynamics simulations have proven their usefulness to explore the structure and function of GPCRs. Furthermore, structure-based drug design and in silico screening (High Throughput Docking) are still the most common computational procedures in GPCRs drug discovery. Moreover, ligand-based methods such as three-dimensional quantitative structure–selectivity relationships, are the ideal molecular modeling approaches to rationalize the activity of tested GPCR ligands and identify novel GPCR ligands. In this review, we discuss the most recent advances for the computational approaches to effectively guide selectivity and affinity of ligands. We also describe novel approaches in medicinal chemistry, such as the development of biased agonists, allosteric modulators, and bivalent ligands for class A GPCRs. Furthermore, we highlight some knockout mice models in discovering biased signaling selectivity. PMID:25506327

  3. The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.

    PubMed

    Zheng, Fudan; Robertson, Alan P; Abongwa, Melanie; Yu, Edward W; Martin, Richard J

    2016-04-01

    Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization. PMID:27054065

  4. The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening

    PubMed Central

    Zheng, Fudan; Robertson, Alan P.; Abongwa, Melanie; Yu, Edward W.; Martin, Richard J.

    2016-01-01

    Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization. PMID:27054065

  5. A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by its dodecameric structure

    PubMed Central

    Filippova, Ekaterina V.; Kuhn, Misty L.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Ballicora, Miguel A.

    2015-01-01

    Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key towards understanding the regulation of polyamine levels in bacteria during pathogenesis. PMID:25623305

  6. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  7. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  8. Enzyme Inhibition by Allosteric Capture of an Inactive Conformation

    PubMed Central

    Lee, Gregory M.; Shahian, Tina; Baharuddin, Aida; Gable, Jonathan E.; Craik, Charles S.

    2011-01-01

    All members of the human herpesvirus protease family are active as weakly associating dimers, but inactive as monomers. A small molecule allosteric inhibitor of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low μM affinity. A 2.0 Å resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease (HCMV Pr) via a similar mechanism. As all HHV proteases are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics which allosterically regulate enzymatic activity by disrupting protein-protein interactions. PMID:21723875

  9. Computational approaches to detect allosteric pathways in transmembrane molecular machines.

    PubMed

    Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V; Weinstein, Harel; Shi, Lei

    2016-07-01

    Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

  10. Signal peptides are allosteric activators of the protein translocase

    PubMed Central

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G.; Economou, Anastassios

    2010-01-01

    Extra-cytoplasmic polypeptides are usually synthesized as “preproteins” carrying aminoterminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, “triggering” that drives the translocase to a lower activation energy state; then “trapping” that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, “secretion” during which trapped mature domains undergo multiple turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  11. Synthesis and Characterization of New Binuclear Co(0) Complexes with Diphosphinoamine Ligands. A Potential Approach for Asymmetric Pauson-Khand Reactions.

    PubMed

    Gimbert, Yves; Robert, Frédéric; Durif, André; Averbuch, Marie-Thérèse; Kann, Nina; Greene, Andrew E.

    1999-05-14

    The synthesis of P-N-P bidentate ligands and the evaluation, based on IR and X-ray data, of their pi-acceptor properties in the complexes derived from phenylacetylene-dicobalt hexacarbonyl have been carried out. In addition, the reactivity of these complexes in the Pauson-Khand reaction has been examined.

  12. Chemokine (C-C motif) Ligand 2 is a potential biomarker of inflammation & physical fitness in obese children: a cross-sectional study

    PubMed Central

    2013-01-01

    Background Obesity is a global epidemic that is impacting children around the world. Obesity is a chronic inflammatory state with enhanced production of multiple cytokines and chemokines. Chemokine (C-C motif) Ligand 2 (CCL2) is produced by immune and metabolic cells and attracts immune cells into liver, muscle and adipose tissue, resulting in initiation and propagation of the inflammatory response in obesity. How obesity and fitness affect the production of this chemokine in children is unknown. This study tested the hypotheses that CCL2 levels are higher in obese children when compared to lean controls, and that fitness modulates CCL2 levels allowing its use as a biomarker of fitness. Methods This was a cross sectional case–control study conducted in a Pediatric Tertiary care center in Hamilton, Ontario, Canada. Controls were recruited from the community. This study recruited overweight/obese children (BMI ≥ 85th percentile, n = 18, 9 female, mean age 14.0 ± 2.6 years) and lean controls (BMI < 85th percentile, n = 18, 8 female, mean age 14.0 ± 2.6 years) matched for age, sex and biological maturation. Aerobic fitness test was done using a cycle ergometer performing the McMaster All-Out Progressive Continuous Cycling test to exhaustion to determine peak oxygen uptake. Fasting CCL2 samples were taken prior to test. Categorical variables including subject categorization into different aerobic fitness levels in overweight/obese and lean children was reported based on the median split in each group. Results Obese participants had significantly higher CCL2 levels when compared to lean group (150.4 ± 61.85 pg/ml versus 112.7 ± 38 pg/ml, p-value 0.034). To establish if CCL2 is a biomarker of fitness, we divided the groups based on their fitness levels. There was a main effect for group (F (3,32) = 3.2, p = 0.036). Obese high fitness group were similar to lean unfit and fit participants. Post-hoc analysis revealed that

  13. Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition in phosphofructokinase from Escherichia coli.

    PubMed

    Fenton, Aron W; Reinhart, Gregory D

    2009-12-29

    This study quantifies the contribution of each of the four unique inhibiting heterotropic interactions between the allosteric inhibitor, phosphoenolpyruvate (PEP), and the substrate, fructose 6-phosphate (Fru-6-P), in phosphofructokinase from Escherichia coli (EcPFK). The unique heterotropic interactions, previously labeled by the distances between ligand binding sites, were isolated independently by constructing hybrid tetramers. Of the four unique heterotropic PEP-Fru-6-P interactions, the 45 A interaction contributed 25%, the 30 A interaction contributed 31%, and the 23 A interaction contributed 42% of the total PEP inhibition. The 33 A interaction actually causes a small activation of Fru-6-P binding by PEP and therefore contributed -8% of the total observed PEP inhibition. The pattern of relative contribution to PEP inhibition from each interaction in EcPFK does not follow the same pattern seen in MgADP activation of EcPFK. This observation supports the conclusion that although PEP and MgADP bind to the same site, they do not use the same communication pathways to influence the active site. The pattern of relative contribution describing PEP inhibition observed in this study also does not follow the pattern determined for PEP inhibition in phosphofructokinase from Bacillus stearothermophilus, suggesting that these two highly homologous isoforms are not inhibited in the same manner by PEP.

  14. InaD PDZs 4-5 Act as an Allosterically-Regulated Dynamic Scaffold

    NASA Astrophysics Data System (ADS)

    Helms, Stephen; Mishra, Prashant; Socolich, Michael; Ranganathan, Rama

    2011-03-01

    The Drosophila scaffolding protein InaD is required for proper visual signaling. We previously identified that the fifth PDZ domain of InaD undergoes light-dependent PKC-mediated formation of a disulfide bond which disrupts the binding site. We investigated the interaction of this switch with the adjacent PDZ4 of InaD. We showed that PDZ4 destabilizes the disulfide bond and promotes binding of PDZ5 to its ligand, indicating a previously unidentified allosteric interaction between the two domains. We solved the structure of PDZ45 to 2.4Å, which revealed that PDZ4 forms an extensive interface with PDZ5 but does not alter its conformation. NMR HSQC spectra, however, indicated that nearly all of PDZ5 is in a different chemical environment in PDZ45. Finally, we identified that PDZ45 is phosphorylated by PKC in vitro at a site located near the domain interface. Intriguingly, the disulfide bond in PDZ5 is an evolutionary adaptation of just fast-flying flies, revealing the remarkable ability of evolution to rapidly build novel regulatory features into scaffolding proteins.

  15. Profiling two indole-2-carboxamides for allosteric modulation of the CB1 receptor.

    PubMed

    Ahn, Kwang H; Mahmoud, Mariam M; Samala, Sushma; Lu, Dai; Kendall, Debra A

    2013-03-01

    Allosteric modulation of G-protein coupled receptors (GPCRs) represents a novel approach for fine-tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole-2-carboxamides:5-chloro-3-ethyl-1-methyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-a) and 5-chloro-3-pentyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-b). Although both ICAM-a and ICAM-b enhanced CP55, 940 binding, ICAM-b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G-protein coupling to CB1, ICAM-b induced β-arrestin-mediated downstream activation of extracellular signal-regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.

  16. Allosteric Model of Maraviroc Binding to CC Chemokine Receptor 5 (CCR5)*

    PubMed Central

    Garcia-Perez, Javier; Rueda, Patricia; Alcami, Jose; Rognan, Didier; Arenzana-Seisdedos, Fernando; Lagane, Bernard; Kellenberger, Esther

    2011-01-01

    Maraviroc is a nonpeptidic small molecule human immunodeficiency virus type 1 (HIV-1) entry inhibitor that has just entered the therapeutic arsenal for the treatment of patients. We recently demonstrated that maraviroc binding to the HIV-1 coreceptor, CC chemokine receptor 5 (CCR5), prevents it from binding the chemokine CCL3 and the viral envelope glycoprotein gp120 by an allosteric mechanism. However, incomplete knowledge of ligand-binding sites and the lack of CCR5 crystal structures have hampered an in-depth molecular understanding of how the inhibitor works. Here, we addressed these issues by combining site-directed mutagenesis (SDM) with homology modeling and docking. Six crystal structures of G-protein-coupled receptors were compared for their suitability for CCR5 modeling. All CCR5 models had equally good geometry, but that built from the recently reported dimeric structure of the other HIV-1 coreceptor CXCR4 bound to the peptide CVX15 (Protein Data Bank code 3OE0) best agreed with the SDM data and discriminated CCR5 from non-CCR5 binders in a virtual screening approach. SDM and automated docking predicted that maraviroc inserts deeply in CCR5 transmembrane cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct yet overlapped regions of the CCR5 extracellular loop 2. Data suggesting that the transmembrane cavity remains accessible for maraviroc in CCL3-bound and gp120-bound CCR5 help explain our previous observation that the inhibitor enhances dissociation of preformed ligand-CCR5 complexes. Finally, we identified residues in the predicted CCR5 dimer interface that are mandatory for gp120 binding, suggesting that receptor dimerization might represent a target for new CCR5 entry inhibitors. PMID:21775441

  17. Discovery of Novel Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Reveals Chemical and Functional Diversity and In Vivo Activity in Rat Behavioral Models of Anxiolytic and Antipsychotic ActivityS⃞

    PubMed Central

    Rodriguez, Alice L.; Grier, Mark D.; Jones, Carrie K.; Herman, Elizabeth J.; Kane, Alexander S.; Smith, Randy L.; Williams, Richard; Zhou, Ya; Marlo, Joy E.; Days, Emily L.; Blatt, Tasha N.; Jadhav, Satyawan; Menon, Usha N.; Vinson, Paige N.; Rook, Jerri M.; Stauffer, Shaun R.; Niswender, Colleen M.; Lindsley, Craig W.; Weaver, C. David

    2010-01-01

    Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenylethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phencyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl)phenyl)methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl)ethynyl)nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have

  18. Ligand exclusion on acetylcholinesterase.

    PubMed

    Berman, H A; Leonard, K

    1990-11-27

    This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values. PMID:2271673

  19. Recent progress in the synthesis and characterization of group II metabotropic glutamate receptor allosteric modulators.

    PubMed

    Sheffler, Douglas J; Pinkerton, Anthony B; Dahl, Russell; Markou, Athina; Cosford, Nicholas D P

    2011-08-17

    Group II metabotropic glutamate (mGlu) receptors consist of the metabotropic glutamate 2 (mGlu(2)) and metabotropic glutamate 3 (mGlu(3)) receptor subtypes which modulate glutamate transmission by second messenger activation to negatively regulate the activity of adenylyl cyclase. Excessive accumulation of glutamate in the perisynaptic extracellular region triggers mGlu(2) and mGlu(3) receptors to inhibit further release of glutamate. There is growing evidence that the modulation of glutamatergic neurotransmission by small molecule modulators of Group II mGlu receptors has significant potential for the treatment of several neuropsychiatric and neurodegenerative diseases. This review provides an overview of recent progress on the synthesis and pharmacological characterization of positive and negative allosteric modulators of the Group II mGlu receptors. PMID:22860167

  20. Recent Progress in the Synthesis and Characterization of Group II Metabotropic Glutamate Receptor Allosteric Modulators

    PubMed Central

    2011-01-01

    Group II metabotropic glutamate (mGlu) receptors consist of the metabotropic glutamate 2 (mGlu2) and metabotropic glutamate 3 (mGlu3) receptor subtypes which modulate glutamate transmission by second messenger activation to negatively regulate the activity of adenylyl cyclase. Excessive accumulation of glutamate in the perisynaptic extracellular region triggers mGlu2 and mGlu3 receptors to inhibit further release of glutamate. There is growing evidence that the modulation of glutamatergic neurotransmission by small molecule modulators of Group II mGlu receptors has significant potential for the treatment of several neuropsychiatric and neurodegenerative diseases. This review provides an overview of recent progress on the synthesis and pharmacological characterization of positive and negative allosteric modulators of the Group II mGlu receptors. PMID:22860167

  1. Ligand regulation of a constitutively dimeric EGF receptor

    NASA Astrophysics Data System (ADS)

    Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

    2015-06-01

    Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

  2. Behavior of the potential antitumor V(IV)O complexes formed by flavonoid ligands. 3. Antioxidant properties and radical production capability.

    PubMed

    Sanna, Daniele; Ugone, Valeria; Fadda, Angela; Micera, Giovanni; Garribba, Eugenio

    2016-08-01

    The radical production capability and the antioxidant properties of some V(IV)O complexes formed by flavonoid ligands were examined. In particular, the bis-chelated species of quercetin (que), [VO(que)2](2-), and morin (mor), [VO(mor)2], were evaluated for their capability to reduce the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and produce the hydroxyl radical (•)OH by Fenton-like reactions, where the reducing agent is V(IV)O(2+). The results were compared with those displayed by other V(IV)O complexes, such as [VO(H2O)5](2+), [VO(acac)2] (acac=acetylacetonate) and [VO(cat)2](2-) (cat=catecholate). The capability of the V(IV)O flavonoids complexes to reduce DPPH is much larger than that of the V(IV)O species formed by non-antioxidant ligands and it is due mainly to the flavonoid molecule. Through the 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trapping assay of the hydroxyl radical it was possible to demonstrate that in acidic solution V(IV)O(2+) has an effectiveness in producing (•)OH radicals comparable to that of Fe(2+). When V(IV)O complexes of flavonoids were taken into account, the amount of hydroxyl radicals produced in Fenton-like reactions depends on the specific structure of the ligand and on their capability to reduce H2O2 to give (•)OH. Both the formation of reactive oxygen species (ROS) under physiological conditions by V(IV)O complexes of flavonoid ligands and their radical scavenging capability can be put in relationship with their antitumor effectiveness and it could be possible to modulate these actions by changing the features of the flavonoid coordinated to the V(IV)O(2+) ion, such as the entity, nature and position of the substituents and the number of phenolic groups.

  3. Characterization of a potentially axially symmetric europium(III) complex of a tetraacetate,tetraaza, macrocyclic ligand by luminescence excitation, emission and lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Albin, Michael; de, William; Horrocks, W., Jr.; Liotta, Frank J.

    1982-01-01

    The Eu(III) complex of the octadentate macrocyclic ligand, 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' -tetraacetate, DOTA, has been examined by luminescence excitation, emission, and lifetime spectroscopy using pulsed dye laser techniques. The results confirm the expected axially symmetric nature of the major component in solution and reveal that 1.2 ± 0.4 water molecules arc coordinatcd to the Eu(III) ion in the complex.

  4. Behavior of the potential antitumor V(IV)O complexes formed by flavonoid ligands. 1. Coordination modes and geometry in solution and at the physiological pH.

    PubMed

    Sanna, Daniele; Ugone, Valeria; Lubinu, Giuseppe; Micera, Giovanni; Garribba, Eugenio

    2014-11-01

    The coordination modes and geometry assumed in solution by the potent antitumor oxidovanadium(IV) complexes formed by different flavonoids were studied by spectroscopic (Electron Paramagnetic Resonance, EPR) and computational (Density Functional Theory, DFT) methods. A series of bidentate flavonoid ligands (L) with increasing structural complexity was examined, which can involve (CO, O(-)) donors and formation of five- and six-membered chelate rings, or (O(-), O(-)) donors and five-membered chelate rings. The geometry corresponding to these coordination modes can be penta-coordinated, [VOL2], or cis-octahedral, cis-[VOL2(H2O)]. The results show that, at physiological pH, ligands provided with (CO, O(-)) donor set yield cis-octahedral species with "maltol-like" coordination when five-membered chelate rings are formed (as with 3-hydroxyflavone), while penta-coordinated structures with "acetylacetone-like" coordination are preferred when the chelate rings are six-membered (as with chrysin). When both the binding modes are possible, as with morin, the "acetylacetone-like" coordination is observed. For the ligands containing a catecholic donor set, such as 7,8-dihydroxyflavone, baicalein, fisetin, quercetin and rutin, the formation of square pyramidal complexes with (O(-), O(-)) "catechol-like" coordination and five-membered chelate rings is preferred at physiological pH. The determination of the different coordination modes and geometry is important to define the biotransformation in the blood and the interaction of these complexes with the biological membranes.

  5. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

    Kennedy, Dylan P.; McRobb, Fiona M.; Leonhardt, Susan A.; Purdy, Michael; Figler, Heidi; Marshall, Melissa A.; Chordia, Mahendra; Figler, Robert; Linden, Joel

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  6. Oxygen-linked S-nitrosation in fish myoglobins: a cysteine-specific tertiary allosteric effect.

    PubMed

    Helbo, Signe; Gow, Andrew J; Jamil, Amna; Howes, Barry D; Smulevich, Giulietta; Fago, Angela

    2014-01-01

    The discovery that cysteine (Cys) S-nitrosation of trout myoglobin (Mb) increases heme O2 affinity has revealed a novel allosteric effect that may promote hypoxia-induced nitric oxide (NO) delivery in the trout heart and improve myocardial efficiency. To better understand this allosteric effect, we investigated the functional effects and structural origin of S-nitrosation in selected fish Mbs differing by content and position of reactive cysteine (Cys) residues. The Mbs from the Atlantic salmon and the yellowfin tuna, containing two and one reactive Cys, respectively, were S-nitrosated in vitro by reaction with Cys-NO to generate Mb-SNO to a similar yield (∼0.50 SH/heme), suggesting reaction at a specific Cys residue. As found for trout, salmon Mb showed a low O2 affinity (P50 = 2.7 torr) that was increased by S-nitrosation (P50 = 1.7 torr), whereas in tuna Mb, O2 affinity (P50 = 0.9 torr) was independent of S-nitrosation. O2 dissociation rates (koff) of trout and salmon Mbs were not altered when Cys were in the SNO or N-ethylmaleimide (NEM) forms, suggesting that S-nitrosation should affect O2 affinity by raising the O2 association rate (kon). Taken together, these results indicate that O2-linked S-nitrosation may occur specifically at Cys107, present in salmon and trout Mb but not in tuna Mb, and that it may relieve protein constraints that limit O2 entry to the heme pocket of the unmodified Mb by a yet unknown mechanism. UV-Vis and resonance Raman spectra of the NEM-derivative of trout Mb (functionally equivalent to Mb-SNO and not photolabile) were identical to those of the unmodified Mb, indicating that S-nitrosation does not affect the extent or nature of heme-ligand stabilization of the fully ligated protein. The importance of S-nitrosation of Mb in vivo is confirmed by the observation that Mb-SNO is present in trout hearts and that its level can be significantly reduced by anoxic conditions. PMID:24879536

  7. A linkage analysis toolkit for studying allosteric networks in ion channels

    PubMed Central

    2013-01-01

    A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca2+-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy WH[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy WC[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (VM) of normalized capacitance (slope of Q-V curve). Plots of WH[g] and WC[q] versus voltage and Ca2+ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca2+-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%. PMID:23250867

  8. Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: Lead optimization.

    PubMed

    Nógrádi, Katalin; Wágner, Gábor; Domány, György; Bobok, Amrita; Magdó, Ildikó; Kolok, Sándor; Mikó-Bakk, Mónika L; Vastag, Mónika; Sághy, Katalin; Gyertyán, István; Kóti, János; Gál, Krisztina; Farkas, Sándor; Keserű, György M; Greiner, István; Szombathelyi, Zsolt

    2015-04-15

    An HTS campaign of our corporate compound library, and hit-to lead development resulted in thieno[2,3-b]pyridine derivative leads with mGluR5 negative allosteric modulator effects. During the lead optimization process, our objective was to improve affinity and metabolic stability. Modification of the first two targeted regions resulted in compounds with nanomolar affinity, then optimal substitution of the third region improved metabolic stability. One of the most promising compounds showed excellent in vivo efficacy and is a potential development candidate.

  9. X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel.

    PubMed

    Nury, Hugues; Van Renterghem, Catherine; Weng, Yun; Tran, Alphonso; Baaden, Marc; Dufresne, Virginie; Changeux, Jean-Pierre; Sonner, James M; Delarue, Marc; Corringer, Pierre-Jean

    2011-01-20

    General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.

  10. Identification of ligand analogues that control c-di-GMP riboswitches.

    PubMed

    Furukawa, Kazuhiro; Gu, Hongzhou; Sudarsan, Narasimhan; Hayakawa, Yoshihiro; Hyodo, Mamoru; Breaker, Ronald R

    2012-08-17

    Riboswitches for the bacterial second messenger c-di-GMP control the expression of genes involved in numerous cellular processes such as virulence, competence, biofilm formation, and flagella synthesis. Therefore, the two known c-di-GMP riboswitch classes represent promising targets for developing novel modulators of bacterial physiology. Here, we examine the binding characteristics of circular and linear c-di-GMP analogues for representatives of both class I and II c-di-GMP riboswitches derived from the pathogenic bacterium Vibrio choleae (class I) and Clostridium difficile (class II). Some compounds exhibit values for apparent dissociation constant (K(D)) below 1 μM and associate with riboswitch RNAs during transcription with a speed that is sufficient to influence riboswitch function. These findings are consistent with the published structural models for these riboswitches and suggest that large modifications at various positions on the ligand can be made to create novel compounds that target c-di-GMP riboswitches. Moreover, we demonstrate the potential of an engineered allosteric ribozyme for the rapid screening of chemical libraries for compounds that bind c-di-GMP riboswitches. PMID:22646696

  11. Using Mutant Cycle Analysis to Elucidate Long-Range Functional Coupling in Allosteric Receptors

    PubMed Central

    Shanata, Jai A. P.; Frazier, Shawnalea J.; Lester, Henry A.; Dougherty, Dennis A.

    2014-01-01

    The functional coupling of residues that are far apart in space is the quintessential property of allosteric receptors. Data from functional studies of allosteric receptors, such as whole-cell dose-response relations, can be used to determine if mutation to a receptor significantly impacts agonist potency. However, the classification of perturbations as primarily impacting binding or allosteric function is more challenging, often requiring detailed kinetic studies. This protocol describes a simple strategy, derived from mutant cycle analysis, for elucidating long-range functional coupling in allosteric receptors (ELFCAR). Introduction of a gain-of-function reporter mutation, followed by a mutant cycle analysis of the readily-measured macroscopic EC50 values can provide insight into the role of many physically distant targets. This new method should find broad application in determining the functional roles of residues in allosteric receptors. PMID:22052487

  12. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

  13. In vitro screening strategies for nicotinic receptor ligands.

    PubMed

    Dunlop, John; Roncarati, Renza; Jow, Brian; Bothmann, Hendrick; Lock, Tim; Kowal, Dianne; Bowlby, Mark; Terstappen, Georg C

    2007-10-15

    A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat alpha7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC(50) values of 5.5 and 1.6 microM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known alpha7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration-response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC(50) values for acetylcholine-evoked currents in GH4C1/alpha7 cells were 55 and 576 microM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of alpha7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using

  14. The allosteric modulation of lipases and its possible biological relevance

    PubMed Central

    Köhler, Jens; Wünsch, Bernhard

    2007-01-01

    Background During the development of an enantioselective synthesis using the lipase from Mucor miehei an unusual reaction course was observed, which was analyzed precisely. For the first time an allosteric modulation of a lipase changing its selectivity was shown. Theory Considering the biological relevance of the discovered regulation mechanism we developed a theory that describes the regulation of energy homeostasis and fat metabolism. Conclusion This theory represents a new approach to explain the cause of the metabolic syndrome and provides an innovative basis for further research activity. PMID:17825093

  15. Indole-based allosteric inhibitors of HIV-1 integrase.

    PubMed

    Patel, Pratiq A; Kvaratskhelia, Nina; Mansour, Yara; Antwi, Janet; Feng, Lei; Koneru, Pratibha; Kobe, Mathew J; Jena, Nivedita; Shi, Guqin; Mohamed, Mosaad S; Li, Chenglong; Kessl, Jacques J; Fuchs, James R

    2016-10-01

    Employing a scaffold hopping approach, a series of allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) have been synthesized based on an indole scaffold. These compounds incorporate the key elements utilized in quinoline-based ALLINIs for binding to the IN dimer interface at the principal LEDGF/p75 binding pocket. The most potent of these compounds displayed good activity in the LEDGF/p75 dependent integration assay (IC50=4.5μM) and, as predicted based on the geometry of the five- versus six-membered ring, retained activity against the A128T IN mutant that confers resistance to many quinoline-based ALLINIs. PMID:27568085

  16. Bioinformatic scaling of allosteric interactions in biomedical isozymes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-09-01

    Allosteric (long-range) interactions can be surprisingly strong in proteins of biomedical interest. Here we use bioinformatic scaling to connect prior results on nonsteroidal anti-inflammatory drugs to promising new drugs that inhibit cancer cell metabolism. Many parallel features are apparent, which explain how even one amino acid mutation, remote from active sites, can alter medical results. The enzyme twins involved are cyclooxygenase (aspirin) and isocitrate dehydrogenase (IDH). The IDH results are accurate to 1% and are overdetermined by adjusting a single bioinformatic scaling parameter. It appears that the final stage in optimizing protein functionality may involve leveling of the hydrophobic limits of the arms of conformational hydrophilic hinges.

  17. Allosteric modulators of the hERG K{sup +} channel

    SciTech Connect

    Yu, Zhiyi Klaasse, Elisabeth Heitman, Laura H. IJzerman, Adriaan P.

    2014-01-01

    Drugs that block the cardiac K{sup +} channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K{sup +} channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [{sup 3}H]astemizole and [{sup 3}H]dofetilide to the hERG K{sup +} channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC{sub 50} values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC{sub 50} values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K{sup +} channel, which is discussed in the light of findings on other ion channels. - Highlights: • Allosteric modulators on the hERG K{sup +} channel were evaluated in binding assays. • LUF6200 was identified as a potent allosteric inhibitor. • Potassium ions were found to behave as allosteric enhancers. • Positive cooperativity and distinct allosteric sites for them were proposed.

  18. Chemistry and pharmacology of GABAB receptor ligands.

    PubMed

    Froestl, Wolfgang

    2010-01-01

    This chapter presents new clinical applications of the prototypic GABA(B) receptor agonist baclofen for the treatment of addiction by drugs of abuse, such as alcohol, cocaine, nicotine, morphine, and heroin, a novel baclofen prodrug Arbaclofen placarbil, the GABA(B) receptor agonist AZD3355 (Lesogabaran) currently in Phase 2 clinical trials for the treatment of gastroesophageal reflux disease, and four positive allosteric modulators of GABA(B) receptors (CGP7930, GS39783, NVP-BHF177, and BHFF), which have less propensity for the development of tolerance due to receptor desensitization than classical GABA(B) receptor agonists. All four compounds showed anxiolytic affects. In the presence of positive allosteric modulators the "classical" GABA(B) receptor antagonists CGP35348 and 2-hydroxy-saclofen showed properties of partial GABA(B) receptor agonists. Seven micromolar affinity GABA(B) receptor antagonists, phaclofen; 2-hydroxy-saclofen; CGP's 35348, 36742, 46381, 51176; and SCH50911, are discussed. CGP36742 (SGS742) showed statistically significant improvements of working memory and attention in a Phase 2 clinical trial in mild, but not in moderate Alzheimer patients. Eight nanomolar affinity GABA(B) receptor antagonists are presented (CGP's 52432, 54626, 55845, 56433, 56999, 61334, 62349, and 63360) that were used by pharmacologists for numerous in vitro and in vivo investigations. CGP's 36742, 51176, 55845, and 56433 showed antidepressant effects. Several compounds are also available as radioligands, such as [(3)H]CGP27492, [(3)H]CGP54626, [(3)H]CGP5699, and [(3)H]CGP62349. Three novel fluorescent and three GABA(B) receptor antagonists with very high specific radioactivity (>2,000 Ci/mmol) are presented. [(125)I]CGP64213 and the photoaffinity ligand [(125)I]CGP71872 allowed the identification of GABA(B1a) and GABA(B1b) receptors in the expression cloning work. PMID:20655477

  19. 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidines as mGluR2 positive allosteric modulators for the treatment of psychosis.

    PubMed

    Zhang, Lei; Brodney, Michael A; Candler, John; Doran, Angela C; Duplantier, Allen J; Efremov, Ivan V; Evrard, Edel; Kraus, Kenneth; Ganong, Alan H; Haas, Jessica A; Hanks, Ashley N; Jenza, Keith; Lazzaro, John T; Maklad, Noha; McCarthy, Sheryl A; Qian, Weimin; Rogers, Bruce N; Rottas, Melinda D; Schmidt, Christopher J; Siuciak, Judith A; Tingley, F David; Zhang, Andy Q

    2011-03-24

    A novel series of mGluR2 positive allosteric modulators (PAMs), 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidines, is herein disclosed. Structure-activity relationship studies led to potent, selective mGluR2 PAMs with excellent pharmacokinetic profiles. A representative lead compound (+)-17e demonstrated dose-dependent inhibition of methamphetamine-induced hyperactivity and mescaline-induced scratching in mice, providing support for potential efficacy in treating psychosis.

  20. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

  1. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

  2. Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABA(A) receptors.

    PubMed

    Kumar, Manish; Dillon, Glenn H

    2016-03-15

    Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxβzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1β1γ2 and α1β2γ2 receptors, whereas allosteric effects were enhanced in α1β2 compared to α1β2γ2 receptors. In "extrasynaptic" (α1β3δ and α4β3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted. PMID:26872987

  3. Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4

    PubMed Central

    Niswender, Colleen M.; Johnson, Kari A.; Weaver, C. David; Jones, Carrie K.; Xiang, Zixiu; Luo, Qingwei; Rodriguez, Alice L.; Marlo, Joy E.; de Paulis, Tomas; Thompson, Analisa D.; Days, Emily L.; Nalywajko, Tasha; Aust, Cheryl A.; Williams, Michael Baxter; Ayala, Jennifer E.; Williams, Richard; Lindsley, Craig W.; Conn, P. Jeffrey

    2008-01-01

    Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. PHCCC is a positive allosteric modulator (PAM) of mGluR4 which has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency and poor solubility. Via high-throughput screening, we discovered over 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, VU0155041, contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD. PMID:18664603