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Sample records for a3 receptor affinity

  1. Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine.

    PubMed

    Bozarov, Andrey; Wang, Yu-Zhong; Yu, Jun Ge; Wunderlich, Jacqueline; Hassanain, Hamdy H; Alhaj, Mazin; Cooke, Helen J; Grants, Iveta; Ren, Tianhua; Christofi, Fievos L

    2009-12-01

    We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl(-) secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (I(sc), Cl(-) secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N(3)-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC(50) for IB-MECA was 0.8 microM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex I(sc) responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that

  2. Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine

    PubMed Central

    Bozarov, Andrey; Wang, Yu-Zhong; Yu, Jun Ge; Wunderlich, Jacqueline; Hassanain, Hamdy H.; Alhaj, Mazin; Cooke, Helen J.; Grants, Iveta; Ren, Tianhua

    2009-01-01

    We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl− secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (Isc, Cl− secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N3-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC50 for IB-MECA was 0.8 μM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex Isc responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that eADO acts at

  3. Synthesis and binding affinity of neuropeptide Y at opiate receptors.

    PubMed

    Kiddle, James J; McCreery, Heather J; Soles, Sonia

    2003-03-24

    Neuropeptide Y and several metabolic fragments were synthesized and evaluated for binding affinity at non-selective opiate receptors. Neuropeptide Y and several C-terminal fragments were shown to bind to non-selective opiate receptors with an affinity similar to that of Leu-enkephalin.

  4. Relationships between chemical structure and affinity for acetylcholine receptors

    PubMed Central

    Abramson, F. B.; Barlow, R. B.; Mustafa, M. G.; Stephenson, R. P.

    1969-01-01

    1. Series of analogues of acetylcholine have been prepared in which the acetyl group was replaced by phenylacetyl, cyclohexylacetyl, diphenylacetyl, dicyclohexylacetyl, (±)-phenylcyclohexylacetyl, benziloyl and (±)-phenylcyclohexylhydroxyacetyl groups and the trimethylammonium group was replaced by Me2EtN+, MeEt2N+, Et3N+, [Formula: see text] Further series were prepared in which the acetoxyethyl group was replaced by ethoxyethyl, phenylethoxyethyl, cyclohexylethoxyethyl, diphenylethoxyethyl, and dicyclohexylethoxyethyl groups, and by n-pentyl, 5-phenylpentyl, 5-cyclohexylpentyl and 5:5-diphenylpentyl groups. 2. The ethoxyethyl and n-pentyl series contain some compounds which are agonists or partial agonists when tested on the isolated guinea-pig ileum, but all the other compounds are antagonists. 3. The affinity of the compounds for the postganglionic (“muscarinesensitive”) acetylcholine receptors has been measured in conditions in which the antagonists have been shown to be acting competitively. There were considerable differences between their affinities, the most active (log K, 9·8) having one million times the affinity of the least active (log K, 3·7). 4. The changes in affinity as the onium group was modified were not entirely independent of changes in the rest of the molecule. Increasing the size of the onium group, as judged from conductivity measurements on simpler onium salts, increased affinity in the series containing one large group (phenyl or cyclohexyl) but, in the series with two large groups, affinity declined when the size was increased beyond -+NMeEt2. 5. In general, the effects of changes in the rest of the molecule on affinity were bigger than the effects of changes in the onium group and there were bigger interactions. Affinity was increased to a greater extent by introducing one phenyl and one cyclohexyl group together than by introducing either two phenyl or two cyclohexyl groups; the increment was greater than the separate

  5. Methods for quantifying T cell receptor binding affinities and thermodynamics

    PubMed Central

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  6. RELATIVE BINDING AFFINITY OF ALKYLPHENOLS TO RAINBOW TROUT ESTROGEN RECEPTOR

    EPA Science Inventory

    RELATIVE BINDING AFFINITY OF ALKYLPHENOLS TO RAINBOW TROUT ESTROGEN RECEPTOR. T R Henry1, J S Denny2 and P K Schmieder2. USEPA, ORD, NHEERL, 1Experimental Toxicology Division and 2Mid-Continent Ecology Division, Duluth, MN, USA.
    The USEPA has been mandated to screen industria...

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

    PubMed

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

    2002-02-11

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

  8. High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

    PubMed Central

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

    2008-01-01

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

  9. The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

    SciTech Connect

    Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

    1990-02-20

    High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

  10. Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

    PubMed Central

    Brom, J; Knöller, J; Köller, M; König, W

    1988-01-01

    Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. PMID:2851543

  11. Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors.

    PubMed Central

    Paolini, R; Kinet, J P

    1993-01-01

    Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected. Images PMID:8382611

  12. Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

    PubMed

    Murase, Akio; Nakao, Kazunari; Takada, Junji

    2008-01-01

    In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

  13. Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

    PubMed

    Leedman, P J; Newman, J D; Harrison, L C

    1989-07-01

    We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

  14. PREDICTING RETINOID RECEPTOR BINDING AFFINITY: COREPA-M APPLICATION

    EPA Science Inventory

    Retinoic acid and associated vitamin A derivatives comprise a class of endogenous hormones that activate different retinoic acid receptors RARs). Transcriptional events subsequent to this activation are key to controlling several aspects of vertebrate development. As such, identi...

  15. Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

    PubMed

    White, Jim F; Grisshammer, Reinhard

    2010-09-07

    Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

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

    PubMed

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

    1997-10-15

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

  17. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

  18. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    PubMed

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

  19. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity

    PubMed Central

    Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.

    2013-01-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

  20. High affinity kainate receptor subunits are necessary for ionotropic but not metabotropic signaling

    PubMed Central

    Fernandes, Herman B.; Catches, Justin S.; Petralia, Ronald S.; Copits, Bryan A.; Xu, Jian; Russell, Theron A.; Swanson, Geoffrey T.; Contractor, Anis

    2009-01-01

    Summary Kainate receptors are atypical members of the glutamate receptor family which are able to signal through both ionotropic and metabotropic pathways. Of the five individual kainate receptor subunits the high-affinity subunits, GluK4 (KA1) and GluK5 (KA2), are unique in that they do not form functional homomeric receptors in recombinant expression systems, but combine with the primary subunits GluK1-3 (GluR5-7) to form heteromeric assemblies. Here we generated a GluK4 mutant mouse by disrupting the Grik4 gene locus. We found that loss of the GluK4 subunit leads to a significant reduction in synaptic kainate receptor currents. Moreover, ablation of both high-affinity subunits in GluK4/GluK5 double knockout mice leads to a complete loss of pre- and postsynaptic ionotropic function of synaptic kainate receptors. The principal subunits remain at the synaptic plasma membrane, but are distributed away from postsynaptic densities and presynaptic active zones. There is also an alteration in the properties of the remaining kainate receptors, as kainic acid application fails to elicit responses in GluK4/GluK5 knockout neurons. Despite the lack of detectable ionotropic synaptic receptors, the kainate receptor-mediated inhibition of the slow afterhyperpolarization current (IsAHP), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown critical role for the high-affinity kainate receptor subunits as obligatory components of ionotropic kainate receptor function, and further, demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels. PMID:19778510

  1. Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

    PubMed Central

    Camps, M; Gumà, A; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1992-01-01

    In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full

  2. Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

    PubMed

    Camps, M; Gumà, A; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1992-08-01

    In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full

  3. High-affinity kainate receptor subunits are necessary for ionotropic but not metabotropic signaling.

    PubMed

    Fernandes, Herman B; Catches, Justin S; Petralia, Ronald S; Copits, Bryan A; Xu, Jian; Russell, Theron A; Swanson, Geoffrey T; Contractor, Anis

    2009-09-24

    Kainate receptors signal through both ionotropic and metabotropic pathways. The high-affinity subunits, GluK4 and GluK5, are unique among the five receptor subunits, as they do not form homomeric receptors but modify the properties of heteromeric assemblies. Disruption of the Grik4 gene locus resulted in a significant reduction in synaptic kainate receptor currents. Moreover, ablation of GluK4 and GluK5 caused complete loss of synaptic ionotropic kainate receptor function. The principal subunits were distributed away from postsynaptic densities and presynaptic active zones. There was also a profound alteration in the activation properties of the remaining kainate receptors. Despite this, kainate receptor-mediated inhibition of the slow afterhyperpolarization current (I(sAHP)), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown obligatory role for the high-affinity subunits for ionotropic kainate receptor function and further demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels.

  4. Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

    PubMed

    Adamczewski, M; Paolini, R; Kinet, J P

    1992-09-05

    The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

  5. IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

    SciTech Connect

    Taketazu, F.; Chiba, S.; Shibuya, K.

    1991-02-01

    The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

  6. Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

    PubMed

    Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

    2017-06-23

    The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Cognitive impairments of alcoholic cirrhotic patients: correlation with endogenous benzodiazepine receptor ligands and increased affinity of platelet receptors.

    PubMed Central

    Kapczinski, F; Curran, H V; Przemioslo, R; Williams, R; Fluck, E; Fernandes, C; File, S E

    1996-01-01

    OBJECTIVES--To determine whether differences in cognitive function between alcoholic and non-alcoholic cirrhotic patients relate to differences in endogenous ligands for the benzodiazepine receptor and/or benzodiazepine binding. METHODS--Seventeen grade-I hepatic encephalopathic patients (nine alcoholic, eight non-alcoholic) were compared with 10 matched controls on plasma concentrations of endogenous ligands for the neuronal benzodiazepine receptor, benzodiazepine binding in platelets, and performance on tests of cognitive function. RESULTS--Both groups of patients were impaired on verbal recall and on reaction time tasks compared with controls; alcoholic patients were also impaired on Reitan's trails test and digit cancellation. Four of the 17 patients had detectable concentrations of endogenous benzodiazepine ligands and they were more impaired than other patients on trails and cancellation tests. The groups did not differ in the density of benzodiazepine platelet receptors, but receptor affinity was higher in alcoholic patients than in controls; furthermore, receptor affinity correlated with the time to complete the cancellation task and with reaction time. CONCLUSION--Alcoholic cirrhotic patients may have enhanced concentrations of ligands for neuronal and peripheral benzodiazepine receptors and these may contribute to cognitive impairments in these patients. PMID:8648337

  8. Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G.

    PubMed

    Takizawa, F; Kinet, J P; Adamczewski, M

    1993-06-18

    Conjugates of R-phycoerythrin are widely used for immunohistochemistry, especially for two-color flow cytometry. Their use is however limited by their apparent tendency to bind non-specifically. Using cells transfected with cDNAs for the murine low affinity receptors for immunoglobulin G (Fc gamma RII and -III) and cells naturally expressing these receptors, we demonstrate that R-phycoerythrin and its conjugates bind specifically and inhibitably to Fc gamma RII and -III. Immunofluorescence stainings of cells bearing these receptors, such as macrophages, monocytes, neutrophils, mast cells, subsets of T cells, and natural killer cells, may therefore not reflect the binding of antibody to antigen, but rather the binding of R-phycoerythrin to the receptors.

  9. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    PubMed

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  10. Development of a Sigma-2 Receptor affinity filter through a Monte Carlo based QSAR analysis.

    PubMed

    Rescifina, Antonio; Floresta, Giuseppe; Marrazzo, Agostino; Parenti, Carmela; Prezzavento, Orazio; Nastasi, Giovanni; Dichiara, Maria; Amata, Emanuele

    2017-08-30

    For the first time in sigma-2 (σ 2 ) receptor field, a quantitative structure-activity relationship (QSAR) model has been built using pK i values of the whole set of known selective σ 2 receptor ligands (548 compounds), taken from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB) (http://www.researchdsf.unict.it/S2RSLDB/), through the Monte Carlo technique and employing the software CORAL. The model has been developed by using a large and structurally diverse set of compounds, allowing for a prediction of different populations of chemical compounds endpoint (σ 2 receptor pK i ). The statistical quality reached, suggested that model for pK i determination is robust and possesses a satisfactory predictive potential. The statistical quality is high for both visible and invisible sets. The screening of the FDA approved drugs, external to our dataset, suggested that sixteen compounds might be repositioned as σ 2 receptor ligands (predicted pK i ≥8). A literature check showed that six of these compounds have already been tested for affinity at σ 2 receptor and, of these, two (Flunarizine and Terbinafine) have shown an experimental σ 2 receptor pK i >7. This suggests that this QSAR model may be used as focusing screening filter in order to prospectively find or repurpose new drugs with high affinity for the σ 2 receptor, and overall allowing for an enhanced hit rate respect to a random screening. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

    NASA Astrophysics Data System (ADS)

    Gober, Isaiah Nathaniel

    This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the

  12. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    SciTech Connect

    Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.

    2011-02-01

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17{beta} to the four rainbow trout ER isoformsmore » with that of three known environmental estrogens 17{alpha}-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ER{alpha} subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17{beta}, bisphenol A binds less strongly to all four receptors, 17{alpha}-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the {alpha} subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.« less

  13. Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

    PubMed

    Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

    2010-01-01

    This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

  14. Synthesis and binding affinity of new 1,4-disubstituted triazoles as potential dopamine D(3) receptor ligands.

    PubMed

    Insua, Ignacio; Alvarado, Mario; Masaguer, Christian F; Iglesias, Alba; Brea, José; Loza, María I; Carro, Laura

    2013-10-15

    A series of new 1,4-disubstituted triazoles was prepared from appropriate arylacetylenes and aminoalkylazides using click chemistry methodology. These compounds were evaluated as potential ligands on several subtypes of dopamine receptors in in vitro competition assays, showing high affinity for dopamine D3 receptors, lower affinity for D2 and D4, and no affinity for the D1 receptors. Compound 18 displayed the highest affinity at the D3 receptor with a Ki value of 2.7 nM, selectivity over D2 (70-fold) and D4 (200-fold), and behaviour as a competitive antagonist in the low nanomolar range. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-04-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

  16. Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

    PubMed Central

    Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

    2012-01-01

    Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

  17. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    NASA Astrophysics Data System (ADS)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  18. Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

    PubMed Central

    Taft, W C; DeLorenzo, R J

    1984-01-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  20. Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

    PubMed Central

    Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

    1998-01-01

    The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding

  1. DOTA-Derivatives of Octreotide Dicarba-Analogs with High Affinity for Somatostatin sst2,5 Receptors.

    PubMed

    Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

    2017-01-01

    In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumors and their metastases. In fact, peptide ligands of somatostatin receptors (sst's) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogs, which show interesting binding profiles at sst's. In this context, it was mandatory to explore the possibility that our analogs could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogs of octreotide. Interestingly, two conjugated analogs exhibited nanomolar affinities on sst 2 and sst 5 somatostatin receptor subtypes.

  2. Neuroprotection Profile of the High Affinity NMDA Receptor Antagonist Conantokin-G

    DTIC Science & Technology

    2002-01-01

    antagonist dextromethorphan reduced infarction a maxi- TABLE 4 Physiological parameters for (0.5 nmol) Con-G and vehicle-treated rats with and without MCAo...experience, only AHN649, an analog of dextromethorphan , has produced comparable reductions in cerebral infarction (Tortella et al. 1999). Although...ischemia) and neurotoxicity (electroencephalographic) studies in rats with AHN649, a 3-amino analog of dextromethorphan and low-affinity N-methyl-D

  3. Affinity States of Striatal Dopamine D2 Receptors in Antipsychotic-Free Patients with Schizophrenia

    PubMed Central

    Kubota, Manabu; Nagashima, Tomohisa; Takano, Harumasa; Kodaka, Fumitoshi; Fujiwara, Hironobu; Takahata, Keisuke; Moriguchi, Sho; Higuchi, Makoto; Okubo, Yoshiro; Takahashi, Hidehiko; Ito, Hiroshi

    2017-01-01

    Abstract Background Dopamine D2 receptors are reported to have high-affinity (D2High) and low-affinity (D2Low) states. Although an increased proportion of D2High has been demonstrated in animal models of schizophrenia, few clinical studies have investigated this alteration of D2High in schizophrenia in vivo. Methods Eleven patients with schizophrenia, including 10 antipsychotic-naive and 1 antipsychotic-free individuals, and 17 healthy controls were investigated. Psychopathology was assessed by Positive and Negative Syndrome Scale, and a 5-factor model was used. Two radioligands, [11C]raclopride and [11C]MNPA, were employed to quantify total dopamine D2 receptor and D2High, respectively, in the striatum by measuring their binding potentials. Binding potential values of [11C]raclopride and [11C]MNPA and the binding potential ratio of [11C]MNPA to [11C]raclopride in the striatal subregions were statistically compared between the 2 diagnostic groups using multivariate analysis of covariance controlling for age, gender, and smoking. Correlations between binding potential and Positive and Negative Syndrome Scale scores were also examined. Results Multivariate analysis of covariance demonstrated a significant effect of diagnosis (schizophrenia and control) on the binding potential ratio (P=.018), although the effects of diagnosis on binding potential values obtained with either [11C]raclopride or [11C]MNPA were nonsignificant. Posthoc test showed that the binding potential ratio was significantly higher in the putamen of patients (P=.017). The Positive and Negative Syndrome Scale “depressed” factor in patients was positively correlated with binding potential values of both ligands in the caudate. Conclusions The present study indicates the possibilities of: (1) a higher proportion of D2High in the putamen despite unaltered amounts of total dopamine D2 receptors; and (2) associations between depressive symptoms and amounts of caudate dopamine D2 receptors in patients

  4. Comparison of receptor affinity of natSc-DOTA-TATE versus natGa-DOTA-TATE.

    PubMed

    Koumarianou, Eftychia; Pawlak, Dariusz; Korsak, Agnieszka; Mikolajczak, Renata

    2011-01-01

    44Sc as a positron emitter can be an interesting alternative to 68Ga (T½=67.71 min) due to its longer half-life (T½=3.97 h). Moreover, the b-emitter 47Sc can be used for therapy when attached to the same biomolecule vectors. DOTA as a chelating agent has been proven suitable for the radiolabelling of peptides recognising tumour cell receptors in vivo with M3+ radiometals. DOTA-derivatized peptides have been successfully labelled with 90Y and 177Lu for therapy, and with 68Ga for PET imaging. However, published data on 44Sc-labelled DOTA-biomolecules as potential PET radiotracers are still very limited. The aim of this study was to compare the affinity of natGa- and natSc-labelled DOTA-TATE to somatostatin receptors subtype 2 expressed in rat pancreatic cancer cell line AR42J. The cold complexes of DOTA-TATE with natGa and natSc were synthesized and identified by HPLC and MS analysis and evaluated in vitro for competitive binding to cancer cell line AR42J expressing somatostatin receptors subtype 2 (sstr2). The IC50 values calculated from the displacement curve of {125I-Tyr11}-SST-14 were: 0.20±0.18, 0.70±0.20, 0.64±0.22 and 0.67±0.12 for natGa-DOTA-TATE, natSc-DOTA-TATE, DOTA-TATE, and {Tyr11}-SST-14 complexes, respectively, with the affinity lowering in the decreasing order: natGa-DOTA-TATE>DOTA-TATE>Tyr11-SST-14>natSc-DOTA-TATE. The binding affinity of natGa-DOTA-TATE appeared higher than that of natSc-DOTA-TATE. Further in vitro and in vivo studies are needed to verify the influence of the chelated metal on the affinity and uptake of the respective radiolabelled compounds. This information might be crucial when the in vivo applications of peptides labelled with 68Ga and 44Sc for PET, as well as the use of 47Sc for radiotherapy are considered.

  5. Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

    PubMed

    Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

    2016-12-01

    The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

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

    PubMed

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

    2002-07-01

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

  7. Affinity and Efficacy Studies of Tetrahydrocannabinolic Acid A at Cannabinoid Receptor Types One and Two.

    PubMed

    McPartland, John M; MacDonald, Christa; Young, Michelle; Grant, Phillip S; Furkert, Daniel P; Glass, Michelle

    2017-01-01

    Introduction: Cannabis biosynthesizes Δ 9 -tetrahydrocannabinolic acid (THCA-A), which decarboxylates into Δ 9 -tetrahydrocannabinol (THC). There is growing interest in the therapeutic use of THCA-A, but its clinical application may be hampered by instability. THCA-A lacks cannabimimetic effects; we hypothesize that it has little binding affinity at cannabinoid receptor 1 (CB 1 ). Materials and Methods: Purity of certified reference standards were tested with high performance liquid chromatography (HPLC). Binding affinity of THCA-A and THC at human (h) CB 1 and hCB 2 was measured in competition binding assays, using transfected HEK cells and [ 3 H]CP55,940. Efficacy at hCB 1 and hCB 2 was measured in a cyclic adenosine monophosphase (cAMP) assay, using a Bioluminescence Resonance Energy Transfer (BRET) biosensor. Results: The THCA-A reagent contained 2% THC. THCA-A displayed small but measurable binding at both hCB 1 and hCB 2 , equating to approximate K i values of 3.1μM and 12.5μM, respectively. THC showed 62-fold greater affinity at hCB 1 and 125-fold greater affinity at hCB 2 . In efficacy tests, THCA-A (10μM) slightly inhibited forskolin-stimulated cAMP at hCB 1 , suggestive of weak agonist activity, and no measurable efficacy at hCB 2 . Discussion: The presence of THC in our THCA-A certified standard agrees with decarboxylation kinetics (literature reviewed herein), which indicate contamination with THC is nearly unavoidable. THCA-A binding at 10μM approximated THC binding at 200nM. We therefore suspect some of our THCA-A binding curve was artifact-from its inevitable decarboxylation into THC-and the binding affinity of THCA-A is even weaker than our estimated values. We conclude that THCA-A has little affinity or efficacy at CB 1 or CB 2 .

  8. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou; Song, Yan-Ling

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the idealmore » antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.« less

  9. Melatonin administration alters nicotine preference consumption via signaling through high-affinity melatonin receptors.

    PubMed

    Horton, William J; Gissel, Hannah J; Saboy, Jennifer E; Wright, Kenneth P; Stitzel, Jerry A

    2015-07-01

    While it is known that tobacco use varies across the 24-h day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however, the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. The objective of this study is examine the role of melatonin and melatonin receptors in nicotine free-choice consumption A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin-deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin-proficient mice lacking both or one of the high-affinity melatonin receptors (MT1 and MT2; double-null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild-type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin-proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However, single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and potential mechanistic

  10. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    PubMed

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  11. Relationships between chemical structure and affinity for postganglionic acetylcholine receptors of the guinea-pig ileum

    PubMed Central

    Abramson, F.B.; Barlow, R.B.; Franks, Fiona M.; Pearson, J.D.M.

    1974-01-01

    1 Some phenylacetyl, diphenylacetyl, benziloyl and (±)-cyclohexylphenylglycolloyl esters have been made with 2- and 3-hydroxymethylpyrrolidines, 3-hydroxymethyl-N-methylpiperidine, piperidin-3-ols, piperidin-4-ols, 2,2,6,6-tetramethyl-N-methylpiperidin-4-ol, tropine, pseudotropine and quinuclidin-3-ol, and the affinity of these compounds and of their metho- and etho- derivatives has been measured for postganglionic acetylcholine receptors of the guinea-pig isolated ileum. 2 Some of the compounds were very active indeed; the benziloyl esters of N-methylpiperidin-4-ol methiodide, tropine methiodide, and quinculidin-3-ol, and the (±)-cyclohexylphenylglycolloyl esters of N-methylpiperidin-4-ol and its methiodide had affinity constants greater than 1010. 3 The effects of inserting an additional methylene group onto the nitrogen were extremely variable, ranging from a decrease in log K of 1.64 units to an increase of 0.97 units. The effects of replacing hydrogen by phenyl in the acid portion ranged from an increase of 1.04 units to an increase of 3.06 units and of replacing hydrogen by hydroxyl from a decrease of 0.09 units to an increase of 1.94 units. 4 The extent of the variation in the effects of a particular change in structure on affinity does not appear to be any different in these relatively rigid compounds from that observed with the same changes in open-chain aminoalcohols. 5 Reasons for the variable effects of groups on affinity are discussed. If differences in effects on preferred conformations of these particular compounds in solution are of secondary importance, the effect of a group on affinity will be the net result of what it could contribute to binding, offset by the disturbance it causes to existing binding. The maximum effect observed in a large number of comparisons may indicate the contribution in the absence of disturbance and for groups containing only carbon and hydrogen it appears to be related to size, assessed from the increments in apparent

  12. Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

    PubMed

    Rosenfeld, M R; Dvorkin, B; Klein, P N; Makman, M H

    1982-03-04

    Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

  13. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    PubMed

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

  14. HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

    PubMed Central

    Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

    2010-01-01

    Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

  15. Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis

    PubMed Central

    Jenkinson, Karl M; Southwell, Bridget R; Furness, John B

    1999-01-01

    In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r.We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum.SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis.The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leuψ[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-βAla) were both more potent in inhibiting endocytosis (50× and 8× greater respectively) against septide than against SP.The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor. PMID:10051129

  16. Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis.

    PubMed

    Jenkinson, K M; Southwell, B R; Furness, J B

    1999-01-01

    1. In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. 2. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. 3. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. 4. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leu,[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-betaAla) were both more potent in inhibiting endocytosis (50 x and 8 x greater respectively) against septide than against SP. 5. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor.

  17. Estrogen Receptor Binding Affinity of Food Contact Material Components Estimated by QSAR.

    PubMed

    Sosnovcová, Jitka; Rucki, Marián; Bendová, Hana

    2016-09-01

    The presented work characterized components of food contact materials (FCM) with potential to bind to estrogen receptor (ER) and cause adverse effects in the human organism. The QSAR Toolbox, software application designed to identify and fill toxicological data gaps for chemical hazard assessment, was used. Estrogen receptors are much less of a lock-and-key interaction than highly specific ones. The ER is nonspecific enough to permit binding with a diverse array of chemical structures. There are three primary ER binding subpockets, each with different requirements for hydrogen bonding. More than 900 compounds approved as of FCM components were evaluated for their potential to bind on ER. All evaluated chemicals were subcategorized to five groups with respect to the binding potential to ER: very strong, strong, moderate, weak binder, and no binder to ER. In total 46 compounds were characterized as potential disturbers of estrogen receptor. Among the group of selected chemicals, compounds with high and even very high affinity to the ER binding subpockets were found. These compounds may act as gene activators and cause adverse effects in the organism, particularly during pregnancy and breast-feeding. It should be considered to carry out further in vitro or in vivo tests to confirm their potential to disturb the regulation of physiological processes in humans by abnormal ER signaling and subsequently remove these chemicals from the list of approved food contact materials. Copyright© by the National Institute of Public Health, Prague 2016

  18. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    PubMed

    Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

    2016-08-01

    Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    PubMed

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Enhancement of GABAergic transmission by zolpidem, an imidazopyridine with preferential affinity for type I benzodiazepine receptors.

    PubMed

    Biggio, G; Concas, A; Corda, M G; Serra, M

    1989-02-28

    The effect of zolpidem, an imidazopyridine derivative with high affinity at the type I benzodiazepine recognition site, on the function of the GABAA/ionophore receptor complex was studied in vitro. Zolpidem, mimicking the action of diazepam, increased [3H]GABA binding, enhanced muscimol-stimulated 36Cl- uptake and reduced [35S]TBPS binding in rat cortical membrane preparations. Zolpidem was less effective than diazepam on the above parameters. Zolpidem induced a lower increase of [3H]GABA binding (23 vs. 35%) and muscimol-stimulated 36Cl- uptake (22 vs. 40%) and a smaller decrease of [35S]TBPS binding (47 vs. 77%) than diazepam. The finding that zolpidem enhanced the function of GABAergic synapses with an efficacy qualitatively and quantitatively different from that of diazepam suggests that this compound is a partial agonist at the benzodiazepine recognition site. Thus, our results are consistent with the view that the biochemical and pharmacological profile of a benzodiazepine recognition site ligand reflects its efficacy to enhance GABAergic transmission. Whether the preferential affinity of zolpidem at the type I site is involved in its atypical biochemical and pharmacological profile remains to be clarified.

  1. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    SciTech Connect

    Lach, E.; Trifilieff, A.; Landry, Y.

    1991-01-01

    The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin inmore » an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.« less

  2. Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

    PubMed Central

    Boursalian, Tamar E.; Fink, Pamela J.

    2003-01-01

    Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

  3. Introduction of D-phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides.

    PubMed

    Lu, Jie; Hathaway, Helen J; Royce, Melanie E; Prossnitz, Eric R; Miao, Yubin

    2014-02-01

    The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys(6)-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys(6)-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys(6) to generate new DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) or DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys(6)-GnRH1) (36.1 nM), the introduction of D-Phe improved the GnRH receptor binding affinities of DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) (16.3 nM) and DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1), (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Contributions of pocket depth and electrostatic interactions to affinity and selectivity of receptors for methylated lysine in water.

    PubMed

    Beaver, Joshua E; Peacor, Brendan C; Bain, Julianne V; James, Lindsey I; Waters, Marcey L

    2015-03-21

    Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0-3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1-1.5 kcal mol(-1), with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.

  5. Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities.

    PubMed

    Harland, Aubrie A; Yeomans, Larisa; Griggs, Nicholas W; Anand, Jessica P; Pogozheva, Irina D; Jutkiewicz, Emily M; Traynor, John R; Mosberg, Henry I

    2015-11-25

    In a previously described peptidomimetic series, we reported the development of bifunctional μ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the κ-opioid receptor (KOR), and (3) improving in vivo efficacy. Here, we establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h.

  6. New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

    PubMed

    Barocelli, E; Ballabeni, V; Bertoni, S; Dallanoce, C; De Amici, M; De Micheli, C; Impicciatore, M

    2000-06-30

    Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

  7. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    PubMed

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  8. Use of receptor chimeras to identify small molecules with high affinity for the dynorphin A binding domain of the kappa opioid receptor.

    PubMed

    Kumar, Virendra; Guo, Deqi; Marella, Michael; Cassel, Joel A; Dehaven, Robert N; Daubert, Jeffrey D; Mansson, Erik

    2008-06-15

    A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.

  9. Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

    PubMed

    Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

  10. Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

    PubMed

    Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

    2014-01-01

    Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected] Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

    PubMed

    Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

    2015-11-01

    Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. DOTA-derivatives of octreotide dicarba-analogues with high affinity for somatostatin sst2,5 receptors

    NASA Astrophysics Data System (ADS)

    Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M.; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

    2017-02-01

    In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumours and their metastases. In fact, peptide ligands of somatostatin receptors (sst’s) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogues, which show interesting binding profiles at sst’s. In this context, it was mandatory to explore the possibility that our analogues could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogues of octreotide. Interestingly, two conjugated analogues exhibited nanomolar affinities on sst2 and sst5 somatostatin receptor subtypes.

  13. N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

    PubMed

    Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

    2017-07-05

    The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

  14. Direct Measurement of T Cell Receptor Affinity and Sequence from Naïve Anti-Viral T Cells

    PubMed Central

    Zhang, Shuqi; Parker, Patricia; Ma, Keyue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad; Wendel, Ben S.; Meriwether, Amanda; Salazar, Mary A.; Jiang, Ning

    2016-01-01

    T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCR). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. Here, we introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8+ T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications. PMID:27252176

  15. Seasonal changes in cortisol sensitivity and glucocorticoid receptor affinity and number in leukocytes of coho salmon

    USGS Publications Warehouse

    Maule, Alec G.; Schreck, Carl B.; Sharpe, Cameron

    1993-01-01

    To determine if there were organ-specific changes in immune responses or immune-endocrine interaction, we monitored in vitro immune response, cortisol sensitivity and number and affinity of glucocorticoid receptors (GR) in leukocytes from freshwater-adapted juvenile coho salmon (Oncorhynchus kisutch) during the physiological changes that prepare them to enter the marine environment. During this period, absolute immune response declined, but splenic leukocytes generated more antibody-producing cells than did cells from anterior kidney. Splenic leukocytes were initially more sensitive to the suppressive effects of cortisol and had fewer GR than leukocytes from the anterior kidney. Leukocytes from the anterior kidney were initially insensitive to cortisol but developed sensitivity at about the same time as the dissociation constant and number of GR increased. In vitro incubation of anterior kidney leukocytes in cortisol altered GR variables when experiments were conducted during March through September but not during November through February. In some years, changes in GR or immune responses were correlated with plasma cortisol titers, but in other years there was no correlation. Thus, the exact relation between cortisol, GR and immune response in anadromous salmonids is unclear and other factors are involved.

  16. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

    PubMed Central

    Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

    2010-01-01

    The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

  17. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one classmore » of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.« less

  18. Improved binding affinity and interesting selectivities of aminopyrimidine-bearing carbohydrate receptors in comparison with their aminopyridine analogues.

    PubMed

    Lippe, Jan; Seichter, Wilhelm; Mazik, Monika

    2015-12-28

    Due to the problems with the exact prediction of the binding properties of an artificial carbohydrate receptor, the identification of characteristic structural features, having the ability to influence the binding properties in a predictable way, is of high importance. The purpose of our investigation was to examine whether the previously observed higher affinity of 2-aminopyrimidine-bearing carbohydrate receptors in comparison with aminopyridine substituted analogues represents a general tendency of aminopyrimidine-bearing compounds. Systematic binding studies on new compounds consisting of 2-aminopyrimidine groups confirmed such a tendency and allowed the identification of interesting structure-activity relationships. Receptors having different symmetries showed systematic preferences for specific glycosides, which are remarkable for such simple receptor systems. Particularly suitable receptor architectures for the recognition of selected glycosides were identified and represent a valuable base for further developments in this field.

  19. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    SciTech Connect

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing

    2013-09-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

  20. Structure-activity relationships for hallucinogenic N,N-dialkyltryptamines: photoelectron spectra and serotonin receptor affinities of methylthio and methylenedioxy derivatives

    SciTech Connect

    Kline, T.B.; Benington, F.; Morin, R.D.

    1982-11-01

    Serotonin receptor affinity and photelectron spectral data were obtained on a number of substituted N,N-dimethyltryptamines. Evidence is presented that electron-donating substituents in the 5-position lead to enhanced behavioral disruption activity and serotonin receptor affinity as compared to unsubstituted N,N-dimethyltryptamine and analogues substituted in the 4- or 6-position. Some correlation was found between ionization potentials and behavioral activity, which may have implications concerning the mechanism of receptor binding.

  1. [125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

    PubMed Central

    Dumont, Yvan; Quirion, Rémi

    2000-01-01

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

  2. Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands.

    PubMed

    Bender, Aaron M; Clark, Mary J; Agius, Michael P; Traynor, John R; Mosberg, Henry I

    2014-01-15

    In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Meta-analysis of cannabinoid ligand binding affinity and receptor distribution: interspecies differences

    PubMed Central

    McPartland, J M; Glass, M; Pertwee, R G

    2007-01-01

    A meta-analysis, unlike a literature review, synthesizes previous studies into new results. Pooled data from 211 studies measured ligand binding affinities at human (Hs) or rat (Rn) cannabinoid receptors CB1 and CB2. Cochrane methods were modified for this non-clinical analysis. Meta-regression detected data heterogeneity arising from methodological factors: use of sectioned tissues, lack of PMSF and choice of radioligand. Native brain tissues exhibited greater affinity (lower nM) than transfected cells, but the trend fell short of significance, as did the trend between centrifugation and filtration methods. Correcting for heterogeneity, mean Ki values for Δ9-tetrahydrocannabinol differed significantly between HsCB1 and RnCB1 (25.1 and 42.6 nM, respectively) but not between HsCB1 and HsCB2 (25.1 and 35.2). Mean Kd values for HsCB1, RnCB1 and HsCB2 of CP55,940 (2.5, 0.98, 0.92) and WIN55,212-2 (16.7, 2.4, 3.7) differed between HsCB1 and RnCB1 and between HsCB1 and HsCB2. SR141716A differed between HsCB1 and RnCB1 (2.9 and 1.0 nM). Anandamide at HsCB1, RnCB1 and HsCB2 (239.2, 87.7, 439.5) fell short of statistical differences due to heterogeneity. We consider these Kd and Ki values to be the most valid estimates in the literature. Sensitivity analyses did not support the numerical validity of cannabidiol, cannabinol, 2-arachidonoyl glycerol and all ligands at RnCB2. Aggregate rank order analysis of CB1 distribution in the brain (pooled from 119 autoradiographic, immunohistochemical and in situ hybridization studies) showed denser HsCB1 expression in cognitive regions (cerebral cortex) compared to RnCB1, which was relatively richer in movement-associated areas (cerebellum, caudate-putamen). Implications of interspecies differences are discussed. PMID:17641667

  4. The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

    PubMed

    Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

    1999-09-17

    The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

  5. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    PubMed Central

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  6. Development of QSAR models for predicting the binding affinity of endocrine disrupting chemicals to eight fish estrogen receptor.

    PubMed

    He, Junyi; Peng, Tao; Yang, Xianhai; Liu, Huihui

    2018-02-01

    Endocrine disrupting effect has become a central point of concern, and various biological mechanisms involve in the disruption of endocrine system. Recently, we have explored the mechanism of disrupting hormonal transport protein, through the binding affinity of sex hormone-binding globulin in different fish species. This study, serving as a companion article, focused on the mechanism of activating/inhibiting hormone receptor, by investigating the binding interaction of chemicals with the estrogen receptor (ER) of different fish species. We collected the relative binding affinity (RBA) of chemicals with 17β-estradiol binding to the ER of eight fish species. With this parameter as the endpoints, quantitative structure-activity relationship (QSAR) models were established using DRAGON descriptors. Statistical results indicated that the developed models had satisfactory goodness of fit, robustness and predictive ability. The Euclidean distance and Williams plot verified that these models had wide application domains, which covered a large number of structurally diverse chemicals. Based on the screened descriptors, we proposed an appropriate mechanism interpretation for the binding potency. Additionally, even though the same chemical had different affinities for ER from different fish species, the affinity of ER exhibited a high correlation for fish species within the same Order (i.e., Salmoniformes, Cypriniformes, Perciformes), which consistent with that in our previous study. Hence, when performing the endocrine disrupting effect assessment, the species diversity should be taken into account, but maybe the fish species in the same Order can be grouped together. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

    PubMed

    Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

    2014-11-01

    The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. The size of the hydroxyl group and its contribution to the affinity of atropine for muscarine-sensitive acetylcholine receptors.

    PubMed Central

    Barlow, R. B.; Ramtoola, S.

    1980-01-01

    1 From measurements of the affinity constants of hydratropyltropine and its methiodide for muscarine-sensitive acetylcholine receptors in the guinea-pig ileum, the increment in log K for the hydroxyl group in atropine is 2.06 and in the methiodide it is 2.16. These effects are slightly bigger than any so far recorded with these receptors. 2 The estimate of the increment in apparent molal volume for the hydroxyl group is 1.1 cm3/mol in atropine and 1.0 cm3/mol in the methobromide. 3 The large effect of the group on affinity may be linked to its small apparent size in water as suggested in the previous paper. PMID:7470742

  9. The Affinity of D2-Like Dopamine Receptor Antagonists Determines the Time to Maximal Effect on Cocaine Self-Administration

    PubMed Central

    Tabet, Michael R.; Norman, Mantana K.; Fey, Brittney K.; Tsibulsky, Vladimir L.; Millard, Ronald W.

    2011-01-01

    Differences in the time to maximal effect (Tmax) of a series of dopamine receptor antagonists on the self-administration of cocaine are not consistent with their lipophilicity (octanol-water partition coefficients at pH 7.4) and expected rapid entry into the brain after intravenous injection. It was hypothesized that the Tmax reflects the time required for maximal occupancy of receptors, which would occur as equilibrium was approached. If so, the Tmax should be related to the affinity for the relevant receptor population. This hypothesis was tested using a series of nine antagonists having a 2500-fold range of Ki or Kd values for D2-like dopamine receptors. Rats self-administered cocaine at regular intervals and then were injected intravenously with a dose of antagonist, and the self-administration of cocaine was continued for 6 to 10 h. The level of cocaine at the time of every self-administration (satiety threshold) was calculated throughout the session. The satiety threshold was stable before the injection of antagonist and then increased approximately 3-fold over the baseline value at doses of antagonists selected to produce this approximately equivalent maximal magnitude of effect (maximum increase in the equiactive cocaine concentration, satiety threshold; Cmax). Despite the similar Cmax, the mean Tmax varied between 5 and 157 min across this series of antagonists. Furthermore, there was a strong and significant correlation between the in vivo Tmax values for each antagonist and the affinity for D2-like dopamine receptors measured in vitro. It is concluded that the cocaine self-administration paradigm offers a reliable and predictive bioassay for measuring the affinity of a competitive antagonist for D2-like dopamine receptors. PMID:21606176

  10. Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

    PubMed

    Ciossek, T; Ullrich, A

    1997-01-09

    Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

  11. Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

    PubMed

    Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

    1993-03-10

    Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

  12. New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

    PubMed

    Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

    2017-12-01

    Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

  13. 14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

    PubMed

    Zádor, Ferenc; Balogh, Mihály; Váradi, András; Zádori, Zoltán S; Király, Kornél; Szűcs, Edina; Varga, Bence; Lázár, Bernadette; Hosztafi, Sándor; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

    2017-11-05

    14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Comparison of kappa opioids in rhesus monkeys: behavioral effects and receptor binding affinities.

    PubMed

    France, C P; Medzihradsky, F; Woods, J H

    1994-01-01

    Bremazocine, [5R-(5,7,8 beta)]-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro [4,5]dec-8-yl]-4-benzofuranacetamide (Cl-977), (+-)-trans-3,4-dichloro-N- methyl-(2-(pyrrolidin-1-yl)-5-methoxy-1,2,3,4-tetrahydronapth++ +-1-yl benzeneacetamide methanesulfonate (DUP 747), ethylketocyclazocine (EKC), nalorphine, (+/-)-trans-N-methyl-N-[2-(1- pyrrolidnyl)-cyclohexyl]benzo[b]thiophene-4-acetamide (PD117302), trans-(+/-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide (U-50,488), (5,7,8 beta)-N-methyl-N[2-(1- pyrrolidinyl), 1-oxaspiro[4,5]dec-8-yl benzeneacetamide (U-69,593) and spiradoline were compared in rhesus monkeys for their discriminative stimulus, analgesic and respiratory effects. Selected compounds also were studied for their binding affinities at mu [[3H](D-Ala2-Me-Phe4,Glyol5)enkephalin], kappa ([3H]U-69,593) and delta [[3H](D-Pen2-D-Pen5) enkephalin], opioid receptors in monkey brain membranes. All compounds substituted completely (> or = 90%) for EKC in monkeys discriminating between EKC and saline, with the exception that DUP 747 produced a maximum of 74% EKC responding. None of the compounds reversed naltrexone responding in morphine-abstinent monkeys; all of the compounds substituted for naltrexone in morphine-treated monkeys discriminating between naltrexone and saline, with the exception that spiradoline produced a maximum of 68% naltrexone responding. Eight compounds produced maximum analgesic effects in a tail withdrawal procedure and quadazocine antagonized these effects; nalorphine did not have analgesic effects, but it antagonized analgesic effects of several other compounds. U-50,488 did not decrease respiratory function, whereas U-69,593 decreased frequency of respiration and volume of respiration to less than 40% of control values; Cl-977, DUP 747, PD117302 and spiradoline had limited effects on respiratory function. Larger doses of each compound increased both respiration and motor activity.

  15. In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor.

    PubMed Central

    Alvarez-Maya, I.; Navarro-Quiroga, I.; Meraz-Ríos, M. A.; Aceves, J.; Martinez-Fong, D.

    2001-01-01

    BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases. PMID:11471555

  16. Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

    PubMed

    Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

    2016-03-14

    The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Novel antipsychotics activate recombinant human and native rat serotonin 5-HT1A receptors: affinity, efficacy and potential implications for treatment of schizophrenia.

    PubMed

    Newman-Tancredi, Adrian; Assié, Marie-Bernadette; Leduc, Nathalie; Ormière, Anne-Marie; Danty, Nathalie; Cosi, Cristina

    2005-09-01

    Serotonin 5-HT1A receptors are promising targets in the management of schizophrenia but little information exists about affinity and efficacy of novel antipsychotics at these sites. We addressed this issue by comparing binding affinity at 5-HT1A receptors with dopamine rD2 receptors, which are important targets for antipsychotic drug action. Agonist efficacy at 5-HT1A receptors was determined for G-protein activation and adenylyl cyclase activity. Whereas haloperidol, thioridazine, risperidone and olanzapine did not interact with 5-HT1A receptors, other antipsychotic agents exhibited agonist properties at these sites. E(max) values (% effect induced by 10 microM of 5-HT) for G-protein activation at rat brain 5-HT1A receptors: sarizotan (66.5), bifeprunox (35.9), SSR181507 (25.8), nemonapride (25.7), ziprasidone (20.6), SLV313 (19), aripiprazole (15), tiospirone (8.9). These data were highly correlated with results obtained at recombinant human 5-HT1A receptors in determinations of G-protein activation and inhibition of forskolin-stimulated adenylyl cyclase. In binding-affinity determinations, the antipsychotics exhibited diverse properties at r5-HT1A receptors: sarizotan (pK(i)=8.65), SLV313 (8.64), SSR181507 (8.53), nemonapride (8.35), ziprasidone (8.30), tiospirone (8.22), aripiprazole (7.42), bifeprunox (7.19) and clozapine (6.31). The affinity ratios of the ligands at 5-HT1A vs. D2 receptors also varied widely: ziprasidone, SSR181507 and SLV313 had similar affinities whereas aripiprazole, nemonapride and bifeprunox were more potent at D2 than 5-HT1A receptors. Taken together, these data indicate that aripiprazole has low efficacy and modest affinity at 5-HT1A receptors, whereas bifeprunox has low affinity but high efficacy. In contrast, SSR181507 has intermediate efficacy but high affinity, and is likely to have more prominent 5-HT1A receptor agonist properties. Thus, the contribution of 5-HT1A receptor activation to the pharmacological profile of action of the

  18. Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations

    PubMed Central

    Jones, RL; Woodward, DF; Wang, JW; Clark, RL

    2011-01-01

    BACKGROUND AND PURPOSE The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP3 receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated. EXPERIMENTAL APPROACH Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted. KEY RESULTS L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP3 antagonists. For ligands of low to moderate lipophilicity, potency increments for EP3 and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC50 of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H1 antagonism above a pA2 limit of 8.0. In contrast, L-798106 (EP3), L-826266 (EP3, TP) and the lipophilic H1 antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP3) had a faster onset. Agonism and antagonism on the vas deferens EP3 system were overall much faster, although trends were similar. CONCLUSIONS AND IMPLICATIONS High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP3 antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP3 antagonists is discussed. PMID:20973775

  19. Classifier ensemble based on feature selection and diversity measures for predicting the affinity of A(2B) adenosine receptor antagonists.

    PubMed

    Bonet, Isis; Franco-Montero, Pedro; Rivero, Virginia; Teijeira, Marta; Borges, Fernanda; Uriarte, Eugenio; Morales Helguera, Aliuska

    2013-12-23

    A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

  20. Antidepressant-like activity of VN2222, a serotonin reuptake inhibitor with high affinity at 5-HT1A receptors.

    PubMed

    Tordera, Rosa M; Monge, Antonio; Del Río, Joaquín; Lasheras, Berta

    2002-05-03

    It has been suggested that drugs combining serotonin (5-hydroxytryptamine, 5-HT) transporter blockade and 5-HT1A autoreceptor antagonism could be a novel strategy for a shorter onset of action and higher therapeutic efficacy of antidepressants. The present study was aimed at characterizing the pharmacology of 1-(3-benzo[b]tiophenyl)-3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propanol (VN2222) a new synthetic compound with high affinity at both the 5-HT transporter and 5-HT1A receptors and devoid of high affinity at other receptors studied, with the only exception of alpha1-adrenoceptors. In keeping with the binding affinity at the 5-HT transporter, VN2222 inhibited 5-HT uptake in vitro both in rat cortical synaptosomes and in mesencephalic cultures and also in vivo when administered locally into the rat ventral hippocampus. After systemic administration, VN2222 exhibited an inverted U-shape effect so the inhibition of [3H]5-HT uptake ex vivo and the increase in 5-HT extracellular levels in microdialysis experiments was observed at low doses of 0.01-0.1 mg/kg whereas higher doses were ineffective. In studies related to 5-HT1A receptor function, 0.01-0.1 microM VN2222 produced a partial inhibition of forskolin-stimulated cAMP formation behaving as a weak agonist of 5-HT1A receptors. In body temperature studies, 5 mg/kg VN2222 produced a mild hypothermic effect in mice, suggesting a weak agonist activity at presynaptic 5-HT1A receptors; much lower doses (0.01-0.5 mg/kg) partially antagonized the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) possibly through 5-HT transporter blockade. In the learned helplessness test in rats, an animal model for antidepressants, 1-5 mg/kg VN2222 reduced significantly the number of escape failures. Consequently, VN2222 is a new compound with a dual effect on the serotonergic system, as 5-HT uptake blocker and 5-HT1A receptor partial agonist, and with a remarkable activity in an animal model of depression with

  1. Cyclic peptide unguisin A is an anion receptor with high affinity for phosphate and pyrophosphate.

    PubMed

    Daryl Ariawan, A; Webb, James E A; Howe, Ethan N W; Gale, Philip A; Thordarson, Pall; Hunter, Luke

    2017-04-05

    Unguisin A (1) is a marine-derived, GABA-containing cyclic heptapeptide. The biological function of this flexible macrocycle is obscure. Here we show that compound 1 lacks any detectable activity in antimicrobial growth inhibition assays, a result that runs contrary to a previous report. However, we find that 1 functions as a promiscuous host molecule in a variety of anion-binding interactions, with high affinity particularly for phosphate and pyrophosphate. We also show that a series of rigidified, backbone-fluorinated analogues of 1 displays altered affinity for chloride ions.

  2. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice

    PubMed Central

    Liu, Xiaojun; Jiang, Shuguang; Fang, Chongyun; Yang, Shiyu; Olalere, Devvora; Pequignot, Edward C.; Cogdill, Alexandria P.; Li, Na; Ramones, Melissa; Granda, Brian; Zhou, Li; Loew, Andreas; Young, Regina M.; June, Carl H.; Zhao, Yangbing

    2015-01-01

    Target-mediated toxicity is a major limitation in the development of chimeric antigen T cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues which express it at physiologic levels. A CAR-expressing T cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach. PMID:26330166

  3. RELATIVE BINDING AFFINITY OF ENDOCRINE DISRUPTING CHEMICALS TO ESTROGEN RECEPTOR IN TWO SPECIES OF FRESHWATER FISH

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  4. Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

    PubMed

    Gillis, Caitlin M; Jönsson, Friederike; Mancardi, David A; Tu, Naxin; Beutier, Héloïse; Van Rooijen, Nico; Macdonald, Lynn E; Murphy, Andrew J; Bruhns, Pierre

    2017-04-01

    Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

  5. ( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

    SciTech Connect

    Branchek, T.; Adham, N.; Macchi, M.

    1990-11-01

    The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

  6. Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

    PubMed Central

    Erchegyi, Judit; Grace, Christy Rani R.; Samant, Manoj; Cescato, Renzo; Piccand, Veronique; Riek, Roland; Reubi, Jean Claude; Rivier, Jean E.

    2009-01-01

    The synthesis, biological testing and NMR studies of several analogues of H-c[Cys3-Phe6-Phe7-dTrp8-Lys9-Thr10-Phe11-Cys14]-OH (ODT-8, a pan-somatostatin analogue) (1), have been performed to assess the effect of changing the stereochemistry and the number of the atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (SRIF numbering) were/was substituted with d-cysteine, Nor-cysteine, d-Nor-cysteine, Homo-cysteine and/or d-Homo-cysteine. The 3D structures of selected partially selective, bioactive analogues (3, 18, 19 and 21) were carried out in DMSO. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst4 in all cases). PMID:18410084

  7. What reaches the antenna? How to calibrate odor flux and ligand-receptor affinities.

    PubMed

    Andersson, Martin N; Schlyter, Fredrik; Hill, Sharon Rose; Dekker, Teun

    2012-06-01

    Physiological studies on olfaction frequently ignore the airborne quantities of stimuli reaching the sensory organ. We used a gas chromatography-calibrated photoionization detector to estimate quantities released from standard Pasteur pipette stimulus cartridges during repeated puffing of 27 compounds and verified how lack of quantification could obscure olfactory sensory neuron (OSN) affinities. Chemical structure of the stimulus, solvent, dose, storage condition, puff interval, and puff number all influenced airborne quantities. A model including boiling point and lipophilicity, but excluding vapor pressure, predicted airborne quantities from stimuli in paraffin oil on filter paper. We recorded OSN responses of Drosophila melanogaster, Ips typographus, and Culex quinquefasciatus, to known quantities of airborne stimuli. These demonstrate that inferred OSN tuning width, ligand affinity, and classification can be confounded and require stimulus quantification. Additionally, proper dose-response analysis shows that Drosophila AB3A OSNs are not promiscuous, but highly specific for ethyl hexanoate, with other earlier proposed ligands 10- to 10 000-fold less potent. Finally, we reanalyzed published Drosophila OSN data (DoOR) and demonstrate substantial shifts in affinities after compensation for quantity and puff number. We conclude that consistent experimental protocols are necessary for correct OSN classification and present some simple rules that make calibration, even retroactively, readily possible.

  8. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

    PubMed

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G; Marini, Pietro; Pertwee, Roger G; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-07-14

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

  9. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency

    PubMed Central

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M.; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G.; Marini, Pietro; Pertwee, Roger G.; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-01-01

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ9-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3–4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [3H]CP55,940 displacement and its effect on [35S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [35S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes. PMID:26124120

  10. Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain.

    PubMed

    Boudin, H; Lazaroff, B; Bachelet, C M; Pélaprat, D; Rostène, W; Beaudet, A

    2000-09-11

    Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry. Copyright 2000 Wiley-Liss, Inc.

  11. Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: the binding isotherm of STD initial growth rates.

    PubMed

    Angulo, Jesús; Enríquez-Navas, Pedro M; Nieto, Pedro M

    2010-07-12

    The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the

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

    PubMed

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

    2006-12-14

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

  13. Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

    PubMed Central

    O'Herrin, Sean M.; Lebowitz, Michael S.; Bieler, Joan G.; al-Ramadi, Basel K.; Utz, Ursula; Bothwell, Alfred L.M.; Schneck, Jonathan P.

    1997-01-01

    Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis

  14. A three-parameter two-state model of receptor function that incorporates affinity, efficacy, and signal amplification.

    PubMed

    Buchwald, Peter

    2017-06-01

    A generalized model of receptor function is proposed that relies on the essential assumptions of the minimal two-state receptor theory (i.e., ligand binding followed by receptor activation), but uses a different parametrization and allows nonlinear response (transduction) for possible signal amplification. For the most general case, three parameters are used: K d , the classic equilibrium dissociation constant to characterize binding affinity; ε , an intrinsic efficacy to characterize the ability of the bound ligand to activate the receptor (ranging from 0 for an antagonist to 1 for a full agonist); and γ , a gain (amplification) parameter to characterize the nonlinearity of postactivation signal transduction (ranging from 1 for no amplification to infinity). The obtained equation, E/Emax=εγLεγ+1-εL+Kd, resembles that of the operational (Black and Leff) or minimal two-state (del Castillo-Katz) models, E/Emax=τLτ+1L+Kd, with εγ playing a role somewhat similar to that of the τ efficacy parameter of those models, but has several advantages. Its parameters are more intuitive as they are conceptually clearly related to the different steps of binding, activation, and signal transduction (amplification), and they are also better suited for optimization by nonlinear regression. It allows fitting of complex data where receptor binding and response are measured separately and the fractional occupancy and response are mismatched. Unlike the previous models, it is a true generalized model as simplified forms can be reproduced with special cases of its parameters. Such simplified forms can be used on their own to characterize partial agonism, competing partial and full agonists, or signal amplification.

  15. In Silico Molecular Interaction of Bisphenol Analogues with Human Nuclear Receptors Reveals their Stronger Affinity vs. Classical Bisphenol A.

    PubMed

    Sharma, Shikha; Ahmad, Shahzad; Faraz Khan, Mohemmed; Parvez, Suhel; Raisuddin, Sheikh

    2018-06-21

    Bisphenol A (BPA) is known for endocrine disrupting activity. In order to replace BPA a number of bisphenol analogues have been designed. However, their activity profile is poorly described and little information exists about their endocrine disrupting potential and interactions with nuclear receptors. An understanding of such interaction may unravel mechanism of their molecular action and provide valuable inputs for risk assessment. BPA binds and activates peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) which act as transcription factors and regulate genes involved in glucose, lipid, and cholesterol metabolism and adipogenesis. We studied binding efficiency of 18 bisphenol analogues and BPA with human PPARs and RXRs. Using Maestro Schrodinger 9.4, docking scores of bisphenols were compared with the known endogenous and exogenous ligands of hPPARs and hRXRs. BPA showed good binding efficiency. Several analogues also showed higher binding efficiency than BPA. BPPH which has high tendency to be absorbed in tissues showed the strongest binding with hPPARα, hPPARβ, hPPARγ and hRXRα whereas two of the most toxic bisphenols, BPM and BPAF showed strongest binding with hRXRβ and hRXRγ. Some of the bisphenol analogues showed a stronger binding affinity with PPAR and RXR compared to BPA implying that BPA substitutes may not be fully safe and chemico-biological interactions indicate their toxic potential. These results may also serve to plan further studies for determining safety profile of bisphenol analogues and be helpful in risk characterization.

  16. Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

    PubMed

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E; Kozyraki, Renata

    2013-06-07

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

  17. Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

    PubMed Central

    Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

    2003-01-01

    Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539

  18. Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

    PubMed

    Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

    2014-01-01

    The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

  19. SLP-76 is required for high-affinity IgE receptor- and IL-3 receptor-mediated activation of basophils.

    PubMed

    Hidano, Shinya; Kitamura, Daisuke; Kumar, Lalit; Geha, Raif S; Goitsuka, Ryo

    2012-11-01

    Basophils have been reported to play a critical role in allergic inflammation by secreting IL-4 in response to IL-3 or high-affinity IgE receptor (FcεRI)-cross-linking. However, the signaling pathways downstream of FcεRI and the IL-3 receptor in basophils have yet to be determined. In the present study, we used mice deficient in SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76kDa) to demonstrate critical functions of this adaptor molecule in transducing FcεRI- and IL-3 receptor-mediated signals that induce basophil activation. Although SLP-76 was dispensable for in vivo differentiation, as well as IL-3-induced in vitro proliferation of basophils, IL-4 production induced by both stimuli was completely ablated by SLP-76 deficiency. Biochemical analyses revealed that IL-3-induced phosphorylation of phospholipase C (PLC) γ2 and Akt, but not STAT5, was severely reduced in SLP-76-deficient basophils, whereas FcεRI cross-linking phosphorylation of PLCγ2, but not Akt, was abrogated by SLP-76 deficiency, suggesting important differences in the requirement of SLP-76 for Akt activation between FcεRI- and IL-3 receptor-mediated signaling pathways in basophils. Because IL-3-induced IL-4 production was sensitive to calcineurin inhibitors and an intracellular calcium chelator, in addition to PI3K inhibitors, SLP-76 appears to regulate FcεRI- and IL-3 receptor-induced IL-4 production via mediating PLCγ2 activation in basophils. Taken together, these findings indicate that SLP-76 is an essential signaling component for basophil activation downstream of both FcεRI and the IL-3 receptor.

  20. Structure-Based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists

    EPA Science Inventory

    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

  1. Structure-based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists.

    EPA Science Inventory

    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

  2. Relative binding affinity prediction of farnesoid X receptor in the D3R Grand Challenge 2 using FEP+

    NASA Astrophysics Data System (ADS)

    Schindler, Christina; Rippmann, Friedrich; Kuhn, Daniel

    2018-01-01

    Physics-based free energy simulations have increasingly become an important tool for predicting binding affinity and the recent introduction of automated protocols has also paved the way towards a more widespread use in the pharmaceutical industry. The D3R 2016 Grand Challenge 2 provided an opportunity to blindly test the commercial free energy calculation protocol FEP+ and assess its performance relative to other affinity prediction methods. The present D3R free energy prediction challenge was built around two experimental data sets involving inhibitors of farnesoid X receptor (FXR) which is a promising anticancer drug target. The FXR binding site is predominantly hydrophobic with few conserved interaction motifs and strong induced fit effects making it a challenging target for molecular modeling and drug design. For both data sets, we achieved reasonable prediction accuracy (RMSD ≈ 1.4 kcal/mol, rank 3-4 according to RMSD out of 20 submissions) comparable to that of state-of-the-art methods in the field. Our D3R results boosted our confidence in the method and strengthen our desire to expand its applications in future in-house drug design projects.

  3. Relative binding affinity prediction of farnesoid X receptor in the D3R Grand Challenge 2 using FEP.

    PubMed

    Schindler, Christina; Rippmann, Friedrich; Kuhn, Daniel

    2018-01-01

    Physics-based free energy simulations have increasingly become an important tool for predicting binding affinity and the recent introduction of automated protocols has also paved the way towards a more widespread use in the pharmaceutical industry. The D3R 2016 Grand Challenge 2 provided an opportunity to blindly test the commercial free energy calculation protocol FEP+ and assess its performance relative to other affinity prediction methods. The present D3R free energy prediction challenge was built around two experimental data sets involving inhibitors of farnesoid X receptor (FXR) which is a promising anticancer drug target. The FXR binding site is predominantly hydrophobic with few conserved interaction motifs and strong induced fit effects making it a challenging target for molecular modeling and drug design. For both data sets, we achieved reasonable prediction accuracy (RMSD ≈ 1.4 kcal/mol, rank 3-4 according to RMSD out of 20 submissions) comparable to that of state-of-the-art methods in the field. Our D3R results boosted our confidence in the method and strengthen our desire to expand its applications in future in-house drug design projects.

  4. Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

    SciTech Connect

    Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

    1986-05-01

    The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions andmore » subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.« less

  5. Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

    PubMed

    Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep

    2014-07-01

    To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.

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

    PubMed Central

    Politi, Regina; Rusyn, Ivan; Tropsha, Alexander

    2016-01-01

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

  7. Affinity purification of angiotensin type 2 receptors from N1E-115 cells: evidence for agonist-induced formation of multimeric complexes.

    PubMed

    Siemens, I R; Yee, D K; Reagan, L P; Fluharty, S J

    1994-01-01

    The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (AngII) during affinity purification of AT2 receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

    PubMed

    Piirainen, Henni; Hellman, Maarit; Tossavainen, Helena; Permi, Perttu; Kursula, Petri; Jaakola, Veli-Pekka

    2015-02-17

    Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    SciTech Connect

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

    2014-10-01

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

  10. Production of a Purified Marine Neurotoxin and Demonstration of its Binding Affinity to Ion Channel Receptors

    DTIC Science & Technology

    1989-06-10

    the ciguatera Implicated toxins, maitotoxin, does not displace brevetoxin from its unique receptor and therefore must produce its toxic 49octs with a...James R. Balthrop, John A. Babinchak, Penny B. Travis, Teresa L. Herring and Pam Y. Brown-Eyo Ciguatera is a tropical fish-borne disease in which both a...synaptosome bound toxin from free toxin following in vitro bindina. we have demonstruted that one of the ciguatera implicated toxins, maitotoxin

  11. Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases.

    PubMed

    Checler, F; Vincent, J P; Kitabgi, P

    1986-07-31

    Neuromedin N (NN) is a novel neurotensin (NT)-like hexapeptide recently isolated from porcine spinal cord. NN competitively inhibited the binding of monoiodinated [Trp11]NT to rat brain synaptic membranes with a 19-fold lower potency than NT. In the presence of 1 mM 1,10-phenanthroline or 10 microM bestatin, the potency of NN relative to NT was increased about 5-fold. NN was readily degraded by rat brain synaptic membranes, and NN-(2-6) was the major degradation product. NN-(2-6) did not bind to NT receptors at concentrations up to 1 microM whether or not peptidase inhibitors were present in the binding assay. The rate of degradation by synaptic membranes was nearly 2.5 times higher for NN than for NT. NN degradation by membranes was totally prevented by 1,10-phenanthroline and markedly inhibited by bestatin. The presence of NN in the central nervous system, its high potency to interact with brain NT receptors and its rapid inactivation by brain synaptic peptidases make it a potential neurotransmitter candidate acting at the NT receptor.

  12. 3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

    PubMed

    Werle, E; Lenz, T; Strobel, G; Weicker, H

    1988-07-01

    The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

  13. Humoral immunity provides resident intestinal eosinophils access to luminal antigen via eosinophil-expressed low affinity Fc gamma receptors

    PubMed Central

    Smith, Kalmia M.; Rahman, Raiann S.; Spencer, Lisa A.

    2016-01-01

    Eosinophils are native to the healthy gastrointestinal tract, and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g. food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct antigen engagement elicits eosinophil effector functions including degranulation and antigen presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food antigens by a columnar epithelium might similarly engage food antigens. Using an intestinal ligated loop model in mice, here we determined that resident intestinal eosinophils acquire antigen from the lumen of antigen-sensitized but not naïve mice in vivo. Antigen acquisition was immunoglobulin-dependent; intestinal eosinophils were unable to acquire antigen in sensitized immunoglobulin-deficient mice, and passive immunization with immune serum or antigen-specific IgG was sufficient to enable intestinal eosinophils in otherwise naïve mice to acquire antigen in vivo. Intestinal eosinophils expressed low affinity IgG receptors, and the activating receptor FcγRIII was necessary for immunoglobulin-mediated acquisition of antigens by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food antigens in sensitized mice via FcγRIII antigen focusing, and may therefore participate in antigen-driven secondary immune responses to oral antigens. PMID:27683752

  14. Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.

    PubMed

    Adamczewski, M; Numerof, R P; Koretzky, G A; Kinet, J P

    1995-04-01

    Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.

  15. A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

    PubMed

    Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

    2015-06-05

    Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  16. Studies on molecular properties prediction and histamine H3 receptor affinities of novel ligands with uracil-based motifs.

    PubMed

    Lipani, Luca; Odadzic, Dalibor; Weizel, Lilia; Schwed, Johannes-Stephan; Sadek, Bassem; Stark, Holger

    2014-10-30

    The histamine H3 receptor (H3R) plays a role in cognitive and memory processes and is involved in different neurological disorders, including Alzheimer's disease, schizophrenia, and narcolepsy. Therefore, several hH3R antagonists/inverse agonists entered clinical phases for a broad spectrum of mainly centrally occurring diseases. However, many other promising candidates failed due to their pharmacokinetic profile, mostly because of their strong lipophilicity accompanied with low solubility. Analysis of previous potential H3R selective antagonists/inverse agonists, e.g. pitolisant, revealed promising results concerning physicochemical properties and drug-likeness. Herein, a series of new hH3R ligands 8-20 consisting of piperidin-1-yl or piperidin-1-yl-propoxyphenyl coupled to different uracil, thymine, and 5,6-dimethyluracil related moieties, were synthesized, evaluated on their binding properties at the hH3R and the estimation of different physicochemical and drug-likeness properties. Due to the coupling to various positions at pyrimidine-2,4-(1H,3H)-dione, affinity at hH3Rs and drug-likeness parameters have been improved. For instance, compound 9 showed in addition to high affinity at the hH3R (pKi (hH3R) = 8.14) clog S, clog P, LE, LipE, and drug-likeness score values of -4.36, 3.47, 0.34, 4.63, and 1.54, respectively. Also, the methyl substituted analog 17 (pKi (hH3R) = 8.15) revealed LE, LipE and drug-likeness score values of -3.29, 2.47, 0.49, 5.52, and 1.76, respectively. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    PubMed

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction

    PubMed Central

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J.

    2015-01-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine’s enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2–4 mins prior to each extinction session. Our results showed that the that mice lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. PMID:26688111

  19. A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

    PubMed

    Arruda, Maria Augusta; Stoddart, Leigh A; Gherbi, Karolina; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J

    2017-01-01

    Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected

  20. Nicotine Ameliorates NMDA Receptor Antagonist-Induced Deficits in Contextual Fear Conditioning through High Affinity Nicotinic Acetylcholine Receptors in the Hippocampus

    PubMed Central

    André, Jessica M.; Leach, Prescott T.; Gould, Thomas J.

    2011-01-01

    NMDA glutamate receptors (NMDARs) and nicotinic acetylcholine receptors (nAChRs) are both involved in learning and synaptic plasticity. Increasing evidence suggests processes mediated by these receptors may interact to modulate learning; however, little is known about the neural substrates involved in these interactive processes. The present studies investigated the effects of nicotine on MK-801 hydrogen maleate (MK-801) and DL-2-Amino-5-phosphonovaleric acid (APV) induced disruption of contextual fear conditioning in male C57BL/6J mice, using direct drug infusion and selective nAChR antagonists to define the brain regions and the nAChR subtypes involved. Mice treated with MK-801 showed a deficit in contextual fear conditioning that was ameliorated by nicotine. Direct drug infusion demonstrated that the NMDAR antagonists disrupted hippocampal function and that nicotine acted in the dorsal hippocampus to ameliorate the deficit in learning. The high-affinity nAChR antagonist Dihydro-β-erythroidine hydrobromide (DhβE) blocked the effects of nicotine on MK-801-induced deficits while the α7 nAChR antagonist methyllycaconitine citrate salt hydrate (MLA) did not. These results suggest that NMDARs and nAChRs may mediate similar hippocampal processes involved in contextual fear conditioning. Furthermore, these results may have implications for developing effective therapeutics for the cognitive deficits associated with schizophrenia because a large subset of patients with schizophrenia exhibit cognitive deficits that may be related to NMDAR dysfunction and smoke at much higher rates than the healthy population, which may be an attempt to ameliorate cognitive deficits. PMID:21167848

  1. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    SciTech Connect

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsicmore » activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights:

  2. Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

    PubMed

    Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

    2007-05-15

    Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

  3. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

    PubMed

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

  4. Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content.

    PubMed

    Benítez, Sonia; Villegas, Virtudes; Bancells, Cristina; Jorba, Oscar; González-Sastre, Francesc; Ordóñez-Llanos, Jordi; Sánchez-Quesada, José Luis

    2004-12-21

    The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

  5. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

    PubMed

    Hastrup, H; Schwartz, T W

    1996-12-16

    The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

  6. Basophils, high-affinity IgE receptors, and CCL2 in human anaphylaxis.

    PubMed

    Korosec, Peter; Turner, Paul J; Silar, Mira; Kopac, Peter; Kosnik, Mitja; Gibbs, Bernhard F; Shamji, Mohamed H; Custovic, Adnan; Rijavec, Matija

    2017-09-01

    The role of basophils in anaphylaxis is unclear. We sought to investigate whether basophils have an important role in human anaphylaxis. In an emergency department study we recruited 31 patients with acute anaphylaxis, predominantly to Hymenoptera venom. We measured expression of basophil activation markers (CD63 and CD203c); the absolute number of circulating basophils; whole-blood FCER1A, carboxypeptidase A3 (CPA3), and L-histidine decarboxylase (HDC) gene expression; and serum markers (CCL2, CCL5, CCL11, IL-3, and thymic stromal lymphopoietin) at 3 time points (ie, during the anaphylactic episode and in convalescent samples 7 and 30 days later). We recruited 134 patients with Hymenoptera allergy and 76 healthy control subjects for comparison. We then investigated whether the changes observed during venom-related anaphylaxis also occur during allergic reactions to food in 22 patients with peanut allergy undergoing double-blind, placebo-controlled food challenge to peanut. The number of circulating basophils was significantly lower during anaphylaxis (median, 3.5 cells/μL) than 7 and 30 days later (17.5 and 24.7 cells/μL, P < .0001) and compared with those in patients with venom allergy and healthy control subjects (21 and 23.4 cells/μL, P < .0001). FCER1A expression during anaphylaxis was also significantly lower than in convalescent samples (P ≤ .002) and control subjects with venom allergy (P < .0001). CCL2 levels (but not those of other serum markers) were significantly higher during anaphylaxis (median, 658 pg/mL) than in convalescent samples (314 and 311 pg/mL at 7 and 30 days, P < .001). Peanut-induced allergic reactions resulted in a significant decrease in circulating basophil counts compared with those in prechallenge samples (P = .016), a decrease in FCER1A expression (P = .007), and an increase in CCL2 levels (P = .003). Our findings imply an important and specific role for basophils in the pathophysiology of human

  7. [125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

    PubMed

    Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

    2002-07-01

    The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway

  8. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    PubMed

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

  9. Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif.

    PubMed

    Seet, Bruce T; Berry, Donna M; Maltzman, Jonathan S; Shabason, Jacob; Raina, Monica; Koretzky, Gary A; McGlade, C Jane; Pawson, Tony

    2007-02-07

    The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.

  10. Adenosine A3 receptors regulate heart rate, motor activity and body temperature

    PubMed Central

    Yang, Jiangning; Wang, Yingqing; Garcia-Roves, Pablo; Björnholm, Marie; Fredholm, Bertil B.

    2010-01-01

    Aim We wanted to examine the phenotype of mice that lack the adenosine A3 receptor (A3R). Methods We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition the effect of the adenosine analogue R - N6- phenylisopropyl-adenosine (R-PIA) was examined. In addition, we examined heat production and food intake. Results We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A3R knockout mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R-PIA in wild type mice was virtually eliminated in the A3R knock-out mice. The marked reduction in activity was associated with a decreased heat production, as expected. However, the A3R knock-out mice, surprisingly, had a higher food intake but no difference in body weight compared to wild type mice. Conclusions The mice lacking adenosine A3 receptors exhibit a surprisingly clear phenotype with changes in e.g. diurnal rhythm and temperature regulation. Whether these effects are due to a physiological role of A3 receptors in these processes or if they represent a role in development remains to be elucidated. PMID:20121716

  11. The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening

    PubMed Central

    Fu, Junjie; Lee, Timothy; Qi, Xin

    2014-01-01

    G protein-coupled receptors (GPCRs), which are involved in virtually every biological process, constitute the largest family of transmembrane receptors. Many top-selling and newly approved drugs target GPCRs. In this review, we aim to recapitulate efforts and progress in combinatorial library-assisted GPCR ligand discovery, particularly focusing on one-bead-one-compound library synthesis and quantum dot-labeled cell-based assays, which both effectively enhance the rapid identification of GPCR ligands with higher affinity and specificity. PMID:24941874

  12. A DFT and semiempirical model-based study of opioid receptor affinity and selectivity in a group of molecules with a morphine structural core.

    PubMed

    Bruna-Larenas, Tamara; Gómez-Jeria, Juan S

    2012-01-01

    We report the results of a search for model-based relationships between mu, delta, and kappa opioid receptor binding affinity and molecular structure for a group of molecules having in common a morphine structural core. The wave functions and local reactivity indices were obtained at the ZINDO/1 and B3LYP/6-31G(∗∗) levels of theory for comparison. New developments in the expression for the drug-receptor interaction energy expression allowed several local atomic reactivity indices to be included, such as local electronic chemical potential, local hardness, and local electrophilicity. These indices, together with a new proposal for the ordering of the independent variables, were incorporated in the statistical study. We found and discussed several statistically significant relationships for mu, delta, and kappa opioid receptor binding affinity at both levels of theory. Some of the new local reactivity indices incorporated in the theory appear in several equations for the first time in the history of model-based equations. Interaction pharmacophores were generated for mu, delta, and kappa receptors. We discuss possible differences regulating binding and selectivity in opioid receptor subtypes. This study, contrarily to the statistically backed ones, is able to provide a microscopic insight of the mechanisms involved in the binding process.

  13. Expression of σ receptors of human urinary bladder tumor cells (RT-4 cells) and development of a competitive receptor binding assay for the determination of ligand affinity to human σ(2) receptors.

    PubMed

    Schepmann, Dirk; Lehmkuhl, Kirstin; Brune, Stefanie; Wünsch, Bernhard

    2011-07-15

    A selective competitive binding assay for the determination of the affinity of compounds to the human σ(2) receptor using 96-well multiplates and a solid state scintillator was developed. In the assay system, [(3)H]ditolylguanidine (DTG) was used as radioligand and membrane homogenates from human RT-4 cells physiologically expressing σ(2) receptors served as receptor material. In order to block the interaction of the unselective radioligand [(3)H]DTG with σ(1) receptors, all experiments were performed in the presence of the σ(1) selective ligand (+)-pentazocine. The density of σ(2) receptors of the cells was analyzed by a saturation experiment with [(3)H]DTG. The radioligand [(3)H]DTG was bound to a single, saturable site on human σ(2) receptors, resulting in a B(max) value of 2108±162fmol/mg protein and K(d)-value of 8.3±2.0nM. The expression of competing σ(1) receptors was evaluated by performing a saturation experiment using the σ(1) selective radioligand [(3)H](+)-pentazocine, which resulted in a B(max) value of 279±40fmol/mg protein and K(d) value of 13.4±1.6nM. For validation of the σ(2) binding assay, the K(i)-values of four σ(2) ligands (ditolylguanidine, haloperidol, rimczole and BMY-14802) were determined with RT-4 cell membrane preparations. The K(i) values obtained from these experiments are in good accordance with the K(i)-values obtained with rat liver membrane preparations as receptor material and with K(i) values given in the literature. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  15. Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice.

    PubMed

    Dziubina, Anna; Szmyd, Karina; Zygmunt, Małgorzata; Sapa, Jacek; Dudek, Magdalena; Filipek, Barbara; Drabczyńska, Anna; Załuski, Michał; Pytka, Karolina; Kieć-Kononowicz, Katarzyna

    2016-12-01

    It has recently been suggested that the adenosine A 2A receptor plays a role in several animal models of depression. Additionally, A 2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with affinity to A 2A receptors but poor A 1 affinity were evaluated for their antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. Available data support the proposition that the examined compounds with adenosine A 2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  16. Clobazam and its active metabolite N-desmethylclobazam display significantly greater affinities for α₂- versus α₁-GABA(A)-receptor complexes.

    PubMed

    Jensen, Henrik Sindal; Nichol, Kathryn; Lee, Deborah; Ebert, Bjarke

    2014-01-01

    Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α₁-subunit-selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α₂ subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α₁, α₂, α₃, or α₅), β₂, and γ₂ subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α₂- vs. α₁-receptor complexes, a difference not observed for CLN, for which no distinction between α₂ and α₁ receptors was observed. Our experiments with ZOL confirmed the high preference for α₁ receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB.

  17. Clobazam and Its Active Metabolite N-desmethylclobazam Display Significantly Greater Affinities for α2- versus α1-GABAA–Receptor Complexes

    PubMed Central

    Jensen, Henrik Sindal; Nichol, Kathryn; Lee, Deborah; Ebert, Bjarke

    2014-01-01

    Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α1-subunit–selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α2 subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α1, α2, α3, or α5), β2, and γ2 subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α2- vs. α1-receptor complexes, a difference not observed for CLN, for which no distinction between α2 and α1 receptors was observed. Our experiments with ZOL confirmed the high preference for α1 receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB. PMID:24533090

  18. Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor.

    PubMed

    Berger, Bryan W; García, Roxana Y; Lenhoff, Abraham M; Kaler, Eric W; Robinson, Clifford R

    2005-07-01

    The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.

  19. Imprinting the Fate of Antigen-Reactive B Cells through the Affinity of the B Cell Receptor

    PubMed Central

    O'Connor, Brian P.; Vogel, Laura A.; Zhang, Weijun; Loo, William; Shnider, Danielle; Lind, Evan F.; Ratliff, Michelle; Noelle, Randolph J.; Erickson, Loren D.

    2010-01-01

    Long-lived plasma cells (PCs) and memory B cells (Bmem) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short-or long-lived PCs and Bmem. Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither Bmem nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but Bmem remain extinct. In contrast, lower affinity interactions show tempered GCs, producing Bmem and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity. PMID:17114443

  20. A critical role for the EphA3 receptor tyrosine kinase in heart development.

    PubMed

    Stephen, Lesley J; Fawkes, Amy L; Verhoeve, Adam; Lemke, Greg; Brown, Arthur

    2007-02-01

    Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3(-/-) mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3(-/-) atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3(-/-) endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.

  1. Design and Synthesis of N6-Substituted-4′-thioadenosine-5′-uronamides As Potent and Selective Human A3 Adenosine Receptor Agonists

    PubMed Central

    Choi, Won Jun; Lee, Hyuk Woo; Kim, Hea Ok; Chinn, Moshe; Gao, Zhan-Guo; Patel, Amit; Jacobson, Kenneth A.; Moon, Hyung Ryong; Jung, Young Hoon; Jeong, Lak Shin

    2009-01-01

    On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA, which is a potent and selective A3 adenosine receptor agonist (AR), were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (Ki = 0.25 nM) at the human A3AR and was more potent than IB-MECA (Ki = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2- methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A3AR binding, although small alkyl analogues were more potent. PMID:19879151

  2. Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

    PubMed

    Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

    2018-05-24

    We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

  3. Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

    PubMed Central

    Alder, J Tracy; Hacksell, Uli; Strange, Philip G

    2003-01-01

    Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT1A receptor: Ki values for agonists were determined in competition versus the binding of the agonist [3H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model.Ki values for agonists were also determined in competition versus the binding of the antagonist [3H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (Kh) and lower affinity (Kl) sites were determined. The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [35S]-GTPγS binding. Maximal effects of agonists (Emax) and their potencies (EC50) were determined from concentration/response curves for stimulation of [35S]-GTPγS binding. Kl/Kh determined from ligand binding assays correlated with the relative efficacy (relative Emax) of agonists determined in [35S]-GTPγS binding assays. There was also a correlation between Kl/Kh and Kl/EC50 for agonists determined from ligand binding and [35S]-GTPγS binding assays. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of Kl/Kh for predicting the relative efficacy of agonists. PMID:12684269

  4. Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

    PubMed Central

    Labonté, Eric D.; Howles, Philip N.; Granholm, Norman A.; Rojas, Juan C.; Davies, Joanna P.; Ioannou, Yiannis A.; Hui, David Y.

    2007-01-01

    Recent studies have documented the importance of Niemann Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1−/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1−/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1−/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1. PMID:17442616

  5. Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

    PubMed Central

    Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

    2006-01-01

    The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

  6. Synthesis, modelling, and mu-opioid receptor affinity of N-3(9)-arylpropenyl-N-9(3)-propionyl-3,9-diazabicycl.

    PubMed

    Pinna, G A; Murineddu, G; Curzu, M M; Villa, S; Vianello, P; Borea, P A; Gessi, S; Toma, L; Colombo, D; Cignarella, G

    2000-08-01

    A series of N-3-arylpropenyl-N-9-propionyl-3,9-diazabicyclo[3.3.1]nonanes (1a-g) and of reverted N-3-propionyl-N-9-arylpropenyl isomers (2a-g), as homologues of the previously reported analgesic 3,8-diazabicyclo[3.2.1]octanes (I-II), were synthesized and evaluated for the binding affinity towards opioid receptor subtypes mu, delta and kappa. Compounds 1a-g and 2a-g exhibited a strong selective mu-affinity with Ki values in the nanomolar range, which favourably compared with those of I and II. In addition, contrary to the trend observed for DBO-I, II, the mu-affinity of series 2 is markedly higher than that of the isomeric series 1. This aspect was discussed on the basis of the conformational studies performed on DBN which allowed hypotheses on the mode of interaction of these compounds with the mu receptor.

  7. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    PubMed

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  8. Kynurenic acid analogues with improved affinity and selectivity for the glycine site on the N-methyl-D-aspartate receptor from rat brain.

    PubMed

    Foster, A C; Kemp, J A; Leeson, P D; Grimwood, S; Donald, A E; Marshall, G R; Priestley, T; Smith, J D; Carling, R W

    1992-05-01

    The glycine site on the N-methyl-D-aspartate (NMDA) subtype of receptors for the excitatory neurotransmitter glutamate is a potential target for the development of neuroprotective drugs. We report here two chemical series of glycine site antagonists derived from kynurenic acid (KYNA), with greatly improved potency and selectivity. Disubstitution with chlorine or bromine in the 5- and 7-positions of KYNA increased affinity for [3H]glycine binding sites in rat cortex/hippocampus P2 membranes, with a parallel increase of potency for antagonism of NMDA-evoked responses in the rat cortical wedge preparation. The optimal compound was 5-I,7-Cl-KYNA, with an IC50 for [3H]glycine binding of 29 nM and an apparent Kb in the cortical wedge preparation of 0.41 microM. Reduction of the right-hand ring of 5,7-diCl-KYNA reduced affinity by 10-fold, but this was restored by substitution in the 4-position with the trans-phenylamide and further improved in the trans-benzylamide. The optimal compound was the transphenylurea (L-689,560), with an IC50 of 7.4 nM and an apparent Kb of 0.13 microM. Both series of compounds displayed a high degree of selectivity for the glycine site, having IC50 values of greater than 10 microM versus radioligand binding to the glutamate recognition sites of NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate receptors and the strychnine-sensitive glycine receptor. Selectivity versus AMPA receptor-mediated responses was also apparent in the rat cortical wedge and in patch-clamp recordings of cortical neurons in culture. Experiments using [3H]dizocilpine (MK-801) binding indicated that 5,7-diBr-KYNA, 5,7-diCl-KYNA, 5-I,7-Cl-KYNA, and L-689,560 all behaved as full antagonists and were competitive with glycine. Patch-clamp recordings of cortical neurons in culture also indicated that NMDA-induced currents were antagonized by competition for the glycine site, and gave no evidence for partial agonist activity. pKi values for 5,7-di

  9. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    PubMed Central

    Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

    2017-01-01

    The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously

  10. Molecular basis for subtype-specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino terminal domain

    PubMed Central

    Romero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan

    2016-01-01

    Summary Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete due to lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc binding site and reveals distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability supporting the general model where the bi-lobe motion in ATD regulates the channel activity in NMDA receptors. PMID:27916457

  11. Structure-Activity Relationships of Truncated C2- or C8-Substituted Adenosine Derivatives as Dual Acting A2A and A3 Adenosine Receptor Ligands

    PubMed Central

    Hou, Xiyan; Majik, Mahesh S.; Kim, Kyunglim; Pyee, Yuna; Lee, Yoonji; Alexander, Varughese; Chung, Hwa-Jin; Lee, Hyuk Woo; Chandra, Girish; Lee, Jin Hee; Park, Seul-gi; Choi, Won Jun; Kim, Hea Ok; Phan, Khai; Gao, Zhan-Guo; Jacobson, Kenneth A.; Choi, Sun; Lee, Sang Kook; Jeong, Lak Shin

    2011-01-01

    Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA2AAR, but hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of synthesized compounds displayed medium to high binding affinity at the hA3AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR agonists. C2 substitution probed geometrically through hA2AAR-docking, was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases. PMID:22142423

  12. Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor

    PubMed Central

    Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G.M.

    2017-01-01

    Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. Here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker. The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. PMID:27867058

  13. Inhibition of neurotensin receptor 1 induces intrinsic apoptosis via let-7a-3p/Bcl-w axis in glioblastoma.

    PubMed

    Dong, Zhen; Lei, Qian; Yang, Rui; Zhu, Shunqin; Ke, Xiao-Xue; Yang, Liqun; Cui, Hongjuan; Yi, Liang

    2017-06-06

    Backgroud:Glioblastoma is a kind of highly malignant and aggressive tumours in the central nervous system. Previously, we found that neurotensin (NTS) and its high-affinity receptor 1 (NTSR1) had essential roles in cell proliferation and invasiveness of glioblastoma. Unexpectedly, cell death also appeared by inhibition of NTSR1 except for cell cycle arrest. However, the mechanisms were remained to be further explored. Cells treated with SR48692, a selective antagonist of NTSR1, or NTSR1 shRNA were stained with Annexin V-FITC/PI and the apoptosis was assessed by flow cytometry. Cytochrome c release was detected by using immunofluorescence. Mitochondrial membrane potential (MMP, ΔΨm) loss was stained by JC-1 and detected by immunofluorescence or flow cytometry. Apoptosis antibody array and microRNA microarray were performed to seek the potential regulators of NTSR1 inhibition-induced apoptosis. Interaction between let-7a-3p and Bcl-w 3'UTR was evaluated by using luciferase assay. SR48692 induced massive apoptosis, which was related to mitochondrial cytochrome c release and MMP loss. Knockdown of NTSR1 induced slight apoptosis and significant MMP loss. In addition, NTSR1 inhibition sensitised glioblastoma cells to actinomycin D or doxorubicin-induced apoptosis. Consistently, NTSR1 inhibition-induced mitochondrial apoptosis was accompanied by downregulation of Bcl-w and Bcl-2. Restoration of Bcl-w partly rescued NTSR1 deficiency-induced apoptosis. In addition, NTSR1 deficiency promoted higher let-7a-3p expression and inhibition let-7a-3p partly rescued NTSR1 inhibition-induced apoptosis. In addition, let-7a-3p inhibition promoted 3'UTR activities of Bcl-w and the expression of c-Myc and LIN28, which were the upstream of let-7a-3p, decreased after NTSR1 inhibition. NTSR1 had an important role in protecting glioblastoma from intrinsic apoptosis via c-Myc/LIN28/let-7a-3p/Bcl-w axis.

  14. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

    EPA Science Inventory

    The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

  15. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays, oral

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  16. A 3D QSAR CoMFA study of non-peptide angiotensin II receptor antagonists

    NASA Astrophysics Data System (ADS)

    Belvisi, Laura; Bravi, Gianpaolo; Catalano, Giovanna; Mabilia, Massimo; Salimbeni, Aldo; Scolastico, Carlo

    1996-12-01

    A series of non-peptide angiotensin II receptor antagonists was investigated with the aim of developing a 3D QSAR model using comparative molecular field analysis descriptors and approaches. The main goals of the study were dictated by an interest in methodologies and an understanding of the binding requirements to the AT1 receptor. Consistency with the previously derived activity models was always checked to contemporarily test the validity of the various hypotheses. The specific conformations chosen for the study, the procedures invoked to superimpose all structures, the conditions employed to generate steric and electrostatic field values and the various PCA/PLS runs are discussed in detail. The effect of experimental design techniques to select objects (molecules) and variables (descriptors) with respect to the predictive power of the QSAR models derived was especially analysed.

  17. Synthesis, NMR conformational analysis and pharmacological evaluation of 7,7a,13,14-tetrahydro-6H-cyclobuta[b]pyrimido[1,2-a:3,4-a']diindole analogues as melatonin receptor ligands.

    PubMed

    Attia, Mohamed I; Güclü, Deniz; Hertlein, Barbara; Julius, Justin; Witt-Enderby, Paula A; Zlotos, Darius P

    2007-07-07

    A structure for the self-condensation product of 2-(1H-indol-2-yl)ethyl tosylate 2a, previously proposed as 6,7,14,15-tetrahydro-15aH-azocino[1,2-a:6,5-b]diindole 3a, was revised based on the (13)C-2D-INADEQUATE experiment, and proved to be 7,7a,13,14-tetrahydro-6H-cyclobuta[b]pyrimido[1,2-a:3,4-a']diindole 4a. A mechanism for the unexpected formation of this novel hexacyclic heterocycle was proposed and its NMR solution structure was elucidated. Five derivatives of the title ring skeleton 12-16 designed as melatonin receptor ligands were synthesized and their affinities for the human MT(1) and MT(2) receptors were determined. Both butyramides 13 and 15, as well as the non-methoxy acetamide 12 exhibited micromolar binding affinities for both receptors being slightly MT(2) selective. The methoxy acetamide 14 showed the best pharmacological profile exhibiting a five times higher affinity for MT(1) (K(i) = 49 nM) than for MT(2) (K(i) = 246 nM) receptor.

  18. Influences of Histidine-1 and Azaphenylalanine-4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators.

    PubMed

    Chignen Possi, Kelvine; Mulumba, Mukandila; Omri, Samy; Garcia-Ramos, Yesica; Tahiri, Houda; Chemtob, Sylvain; Ong, Huy; Lubell, William D

    2017-11-22

    Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A 1 , azaF 4 ]- and [azaY 4 ]-GHRP-6 (1a and 2b) were previously shown to bind selectively to CD36 and exhibited respectively significant antiangiogenic and slight angiogenic activities in a microvascular sprouting assay using choroid explants. The influences of the 1- and 4-position residues on the affinity, anti-inflammatory, and antiangiogenic activity of these azapeptides have now been studied in detail by the synthesis and analysis of a set of 25 analogues featuring Ala 1 or His 1 and a variety of aromatic side chains at the aza-amino acid residue in the 4-position. Although their binding affinities differed only by a factor of 17, the analogues exhibited significant differences in ability to modulate production of nitric oxide (NO) in macrophages and choroidal neovascularization.

  19. Improvement of anemia in W/WV mice by recombinant human erythropoietin (rHuEPO) mediated through EPO receptors with lowered affinity.

    PubMed

    Kabaya, K; Akiyama, H; Nishi, N; Misaizu, T; Okada, Y; Kawagishi, M; Amano, K; Kusaka, M; Seki, M; Uzumaki, H

    1995-01-01

    We studied the effects of recombinant human erythropoietin (rHuEPO) on anemic W/WV mice which manifested severe anemia accompanied by mutation of the W gene encoding tyrosine kinase type receptor (c-kit gene) of bone marrow hematopoietic cells. Nine-week-old male W/WV mice or normal littermates (+/+) were used. Since serum EPO concentration in W/WV mice increased in proportion to severity of anemia, EPO production in the kidneys of these animals was found to be regulated normally. Hematocrit in +/+ mice increased and a maximal response was also obtained with 2,000 IU/kg of rHuEPO. On the other hand, hematocrit in W/WV mice increased in a dose-responsive manner by administration with 2,000 and 10,000 IU/kg, showing different responses to rHuEPO in these two types of mice. The responsiveness of W/WV mice to rHuEPO was low in terms of increases in erythroblastic precursor cells (CFU-E), and immature cells in the bone marrow. Scatchard analysis of the specific binding of 125I-rHuEPO against bone marrow cells revealed that the different responsiveness to rHuEPO between W/WV and +/+ mice may be correlated with differences in affinity of EPO receptor of bone marrow cells in these mice. From these results, a high dose of rHuEPO is capable of improving the anemia in W/WV mice that had EPO receptors with lowered affinity, indicating the possible effectiveness of rHuEPO in anemic patients with EPO receptor abnormality.

  20. Stimulation of ANP secretion by 2-Cl-IB-MECA through A(3) receptor and CaMKII.

    PubMed

    Yuan, Kuichang; Bai, Guang Yi; Park, Woo Hyun; Kim, Sung Zoo; Kim, Suhn Hee

    2008-12-01

    Adenosine is a potent mediator of myocardial protection against hypertrophy via A(1) or A(3) receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A(3) receptor on ANP release. We studied the effects of the A(3) receptor on atrial functions and its modification in hypertrophied atria. A selective A(3) receptor agonist, 2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A(3) receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30muM 2-CI-IB-MECA were not affected. The 100muM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA), A(3) receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA>IB-MECA=HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca(2+) channels, sarcoplasmic reticulum Ca(2+)-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A(3) receptor activation and positive inotropy by intracellular Ca(2+) regulation via the ryanodine receptor and CaMKII.

  1. Iodination of (Tyr11)somatostatin yields a super high affinity ligand for somatostatin receptors in GH4C1 pituitary cells

    SciTech Connect

    Presky, D.H.; Schonbrunn, A.

    1988-11-01

    GH4C1 cells are a clonal strain of rat pituitary tumor cells which contain high affinity receptors for the inhibitory neuropeptide somatostatin (SRIF). In contrast to other peptides that bind to specific receptors on these cells, receptor-bound (125I-Tyr1)SRIF does not undergo rapid endocytosis. Rather, partial degradation to 125I-tyrosine occurs concomitantly with the dissociation of (125I-Tyr1)SRIF from cell surface receptors. In this study we characterize the binding, biological activity and receptor-mediated degradation of (125I-Tyr11)SRIF, a SRIF analog that is radiolabeled in the center of the molecule. The binding of trace concentrations of (125I-Tyr11)SRIF (less than 50 pM) required 6 hr to reachmore » equilibrium at 37 degrees compared with the 60 min required for (125I-Tyr1)SRIF. Analysis of the kinetics of (125I- Tyr11)SRIF binding showed that the rate constant for association (kon = 1.7 x 10(8) M-8min-1) was similar to that for (125I-Tyr1)SRIF (0.8 x 10(8) M-1min-1). However, the two radioligands exhibited markedly different dissociation kinetics; the koff for (125I-Tyr11)SRIF was 0.002 min-1 compared with the value of 0.02 min-1 for (125I-Tyr1) SRIF. In agreement with its much slower rate of dissociation, (125I-Tyr11)SRIF bound to the SRIF receptor with higher affinity (Kd = 70 pM) than did (125I-Tyr1)SRIF (Kd = 350 pM). However, the apparent ED50 for (I-Tyr11)SRIF to inhibit cAMP accumulation (1.9 +/- 0.4 nM) was greater than the ED50 for SRIF (0.19 +/- 0.04 nM). The low potency of (I-Tyr11)SRIF probably resulted from the fact that subsaturating concentrations of this peptide did not achieve equilibrium binding during the 30-min incubation used to assay biological activity. As previously reported for (125I-Tyr1)SRIF, receptor-bound (125I-Tyr11)SRIF was not internalized and was released from the cells as a mixture of intact (125I-Tyr11)SRIF (30%) and the degradation product 125I-tyrosine (65%).« less

  2. The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

    NASA Astrophysics Data System (ADS)

    Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

    2017-04-01

    Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

  3. The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region.

    PubMed

    Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

    2017-04-07

    Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

  4. The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

    PubMed Central

    Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

    2017-01-01

    Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine. PMID:28387240

  5. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

    PubMed

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

  6. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3

    PubMed Central

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M.

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted. PMID:27713686

  7. Austrian Moderate Altitude Study (AMAS 2000): erythropoietic activity and Hb-O(2) affinity during a 3-week hiking holiday at moderate altitude in persons with metabolic syndrome.

    PubMed

    Schobersberger, Wolfgang; Greie, Sven; Humpeler, Egon; Mittermayr, Markus; Fries, Dietmar; Schobersberger, Beatrix; Artner-Dworzak, Erika; Hasibeder, Walter; Klingler, Anton; Gunga, Hanns-Christian

    2005-01-01

    Moderate altitude hypoxia (1500 to 2500 m) is known to stimulate erythropoiesis and to improve oxygen transport to tissue by a reduction of Hb-O(2) affinity. Whether this adaptation also occurs in tourists with metabolic syndrome has not yet been investigated sufficiently. Thus, we performed a prospective field study to measure erythropoietic parameters and oxygen transport properties in 24 male volunteers with metabolic syndrome during a 3- week holiday program at 1700 m consisting of four guided, individually adapted hiking tours per week. The following examinations were performed: baseline investigations at 500 m (T1); examinations at moderate altitude on day 1 (T2), day 4 (T3), day 9 (T4), and day 19 (T5); and postaltitude tests (T6) 7 to 10 days after return. On day 1 and day 19, a walk on a standardized hiking test route with oxygen saturation (SpO(2)) measure points was performed. Hemoglobin, packed cell volume, and red cell count showed changes over time, with higher values at T5 as compared to baseline. Reticulocyte count and erythropoietin (EPO) were increased at T2 and increased further until T5. EPO declined toward prealtitude values. P50-value (blood PO(2) at 50% hemoglobin oxygen saturation at actual pH) increased during the altitude sojourn (maximum increase at T5 by +0.40 kPa). At T5 all volunteers had a higher SpO(2) before, during, and at the end of the test route compared to T1. During adaptation to moderate altitude, persons with metabolic syndrome exhibit an increase in EPO and a rightward shift of the oxygen dissociation curve that is similar to healthy subjects.

  8. The effect of omalizumab treatment on the low affinity immunoglobulin E receptor (CD23/fc epsilon RII) in patients with severe allergic asthma.

    PubMed

    Assayag, Miri; Moshel, Shabtai; Kohan, Martin; Berkman, Neville

    2018-01-01

    Omalizumab is an anti-immunoglobulin E (IgE) monoclonal antibody used in the treatment of severe asthma. Its therapeutic efficacy is primarily attributed to reduction of serum-free IgE and in the expression of high-affinity IgE receptor, fc epsilon RI. However, its effect on the low-affinity IgE receptor fc epsilon RII/CD23 in vivo has not been evaluated. To determine whether CD23 plays a role in the inflammatory process in severe uncontrolled asthma and whether anti-IgE therapy modulates fc epsilon RII/CD23 expression in these patients. We evaluated the expression of IgE receptors fc epsilon RI, fc epsilon RII/CD23, and soluble CD23 (sCD23), and the activation state of peripheral blood monocytes (tumor necrosis factor alpha, interleukin (IL) 1-beta, transforming growth factor (TGF) beta expression) in the patients with severe asthma before and after 24 weeks of omalizumab treatment and in the healthy controls. Cytokine expression of monocytes in response to different stimulation (IL-4, IL-4 plus IgE, IL-4 plus IgE plus anti-IgE, and IL-4 plus IgE plus anti-IgE plus anti-CD23 for 72 hours) was determined by enzyme-linked immunosorbent assay. Treatment with omalizumab (for 24 weeks) improved disease control and pulmonary function (forced expiratory volume in the first second of expiration, 64.5 versus 74%; p = 0.021). Mean ± SE expression of fc epsilon RI on monocytes was higher in the patients with asthma versus the controls (45.7 ± 12.2% versus 18.6 ± 5.8%; p = 0.04) and was reduced after omalizumab treatment (45.7 ± 12.2% versus 15.6 ± 4.4%; p = 0.027). Mean ± SE TGF-beta levels in supernatants from monocytes were reduced in the patients treated with omalizumab (211 ± 6 pg/mL versus 184 ± 9 pg/mL; p = 0.036). Modulation of the low affinity IgE receptor CD23 in severe asthma is complex, and sCD23 may inversely reflect disease activity. Treatment with omalizumab was associated with reduced monocyte activation.

  9. Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

    PubMed Central

    Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T. N.; Gregory, Karen J.; Tosh, Dilip K.; Christopoulos, Arthur; Jacobson, Kenneth A.

    2016-01-01

    Biased agonism at G protein–coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias “fingerprints” for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with significant N6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5′-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

  10. Concomitant alteration in number and affinity of P2X and muscarinic receptors are associated with bladder dysfunction in early stage of diabetic rats.

    PubMed

    Yoshizawa, Tsuyoshi; Hayashi, Yukio; Yoshida, Akira; Yoshida, Shohei; Ito, Yoshihiko; Yamaguchi, Kenya; Yamada, Shizuo; Takahashi, Satoru

    2018-03-01

    To investigate time course of bladder dysfunction and concurrent changes in number and affinity of the muscarinic and P 2 X receptor in the early stage of streptozotocin (STZ)-induced diabetic rats. Diabetic rats were prepared by the intraperitoneal injection of 50 mg/kg of STZ to 7-week-old female Wistar rats. We performed recording of 24-h voiding behavior and cystometry at 1, 4, 8, and 12 weeks after the induction of diabetes. A muscle strip experiments with electrical field stimulation (EFS), carbachol, and α,β-methylene adenosine 5'-triphosphate (α,β-MeATP) were also performed at the same time-points. Additionally, concurrent changes in number and affinity of bladder muscarinic and P 2 X receptor were measured by a radioreceptor assay using [N-methyl- 3 H] scopolamine methyl chloride ([ 3 H]NMS) and α,β-methylene-ATP (2,8- 3 H) tetrasodium salt ([ 3 H]α,β-MeATP). In STZ-induced diabetic rats, polydipsic polyuric pollakiuria were noted on recording of 24-h voiding behavior from early stage. Also, the residual urine volume markedly increased in diabetic rats on cystometry. In the muscle strip experiment, the detrusor contractions induced by EFS, carbachol, and α,β-MeATP were enhanced in STZ-induced diabetic rats. Based on the radioreceptor assay, the maximum number of sites (Bmax) for the specific binding of [ 3 H]NMS and [ 3 H]α,β-MeATP was concurrently increased in the bladder from diabetic rats. Increased bladder contractility is found in early stage of diabetic rats. Then, bladder dysfunction is associated with increased number of muscarinic and P 2 X receptors in STZ-induced diabetic rats.

  11. Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII.

    PubMed

    Takizawa, F; Adamczewski, M; Kinet, J P

    1992-08-01

    In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation.

  12. Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII

    PubMed Central

    1992-01-01

    In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation. PMID:1386873

  13. Evidence that the adenosine A3 receptor may mediate the protection afforded by preconditioning in the isolated rabbit heart.

    PubMed

    Liu, G S; Richards, S C; Olsson, R A; Mullane, K; Walsh, R S; Downey, J M

    1994-07-01

    Agonists selective for the A1 adenosine receptor mimic the protective effect of ischaemic preconditioning against infarction in the rabbit heart. Unselective adenosine antagonists block this protection but, paradoxically, the A1 adenosine receptor selective antagonist 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX) does not. The aim of this study was to test the hypothesis that the newly described A3 adenosine receptor, which has an agonist profile similar to the A1 receptor but is insensitive to DPCPX, might mediate preconditioning. Isolated rabbit hearts perfused with Krebs buffer experienced 30 min of regional ischaemia followed by 120 min of reperfusion. Infarct size was measured by tetrazolium staining. In control hearts infarction was 32.2(SEM 1.5)% of the risk zone. Preconditioning by 5 min ischaemia and 10 min reperfusion reduced infarct size to 8.8(2.3)%. Replacing the regional ischaemia with 5 min perfusion with 10 microM adenosine or 65 nM N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA), an adenosine A3 receptor agonist, was equally protective. The unselective antagonist 8-p-sulphophenyl theophylline at 100 microM abolished protection by preconditioning, adenosine, and APNEA, but 200 nM DPCPX did not block protection by any of the interventions. Likewise the potent but unselective A3 receptor antagonist 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine (BW A1433) completely blocked protection from ischaemic preconditioning. Because protection against infarction afforded by ischaemic preconditioning, adenosine, or the A3 receptor agonist APNEA could not be blocked by DPCPX and because the potent A3 receptor antagonist BW A1433 blocked protection from ischaemic preconditioning, these data indicate that the protection of preconditioning is not exclusively mediated by the adenosine A1 receptor in rabbit heart and could involve the A3 receptor.

  14. Computer-aided structure-affinity relationships in a set of piperazine and 3,8-diazabicyclo[3.2.1]octane derivatives binding to the μ-opioid receptor

    NASA Astrophysics Data System (ADS)

    Barlocco, Daniela; Cignarella, Giorgio; Greco, Giovanni; Novellino, Ettore

    1993-10-01

    Molecular modeling studies were carried out on a set of piperazine and 3,8-diazabicyclo[3.2.1]octane derivatives with the aim to highlight the main factors modulating their affinity for the μ-opioid receptor. Structure-affinity relationships were developed with the aid of molecular mechanics and semiempirical quantum-mechanics methods. According to our proposed pharmacodynamic model, the binding to the μ-receptor is promoted by the following physico-chemical features: the presence of hydrocarbon fragments on the nitrogen ring frame capable of interacting with one of two hypothesized hydrophobic receptor pockets; a `correct' orientation of an N-propionyl side chain so as to avoid a sterically hindered region of the receptor; the possibility of accepting a hydrogen bond from a receptor site complementary to the morphine phenol oxygen.

  15. Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

    PubMed Central

    1991-01-01

    Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

  16. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

  17. Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets

    PubMed Central

    Sun, Honglei; Pu, Juan; Wei, Yandi; Sun, Yipeng; Hu, Jiao; Liu, Litao; Xu, Guanlong; Gao, Weihua; Li, Chong; Zhang, Xuxiao; Huang, Yinhua; Chang, Kin-Chow; Liu, Xiufan

    2016-01-01

    ABSTRACT Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. IMPORTANCE Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored. PMID:27122581

  18. Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets.

    PubMed

    Sun, Honglei; Pu, Juan; Wei, Yandi; Sun, Yipeng; Hu, Jiao; Liu, Litao; Xu, Guanlong; Gao, Weihua; Li, Chong; Zhang, Xuxiao; Huang, Yinhua; Chang, Kin-Chow; Liu, Xiufan; Liu, Jinhua

    2016-07-15

    Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

    PubMed

    Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G M

    2017-03-01

    Human cannabinoid receptor CB 2 belongs to the class A of G protein-coupled receptor (GPCR). CB 2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB 2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB 2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB 2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB 2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB 2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB 2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification

  20. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.

    PubMed

    Xu, Qiang; Wang, Yanshu; Dabdoub, Alain; Smallwood, Philip M; Williams, John; Woods, Chad; Kelley, Matthew W; Jiang, Li; Tasman, William; Zhang, Kang; Nathans, Jeremy

    2004-03-19

    Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.

  1. Measurement of density and affinity for dopamine D2 receptors by a single positron emission tomography scan with multiple injections of [11C]raclopride

    PubMed Central

    Ikoma, Yoko; Watabe, Hiroshi; Hayashi, Takuya; Miyake, Yoshinori; Teramoto, Noboru; Minato, Kotaro; Iida, Hidehiro

    2010-01-01

    Positron emission tomography (PET) with [11C]raclopride has been used to investigate the density (Bmax) and affinity (Kd) of dopamine D2 receptors related to several neurological and psychiatric disorders. However, in assessing the Bmax and Kd, multiple PET scans are necessary under variable specific activities of administered [11C]raclopride, resulting in a long study period and unexpected physiological variations. In this paper, we have developed a method of multiple-injection graphical analysis (MI-GA) that provides the Bmax and Kd values from a single PET scan with three sequential injections of [11C]raclopride, and we validated the proposed method by performing numerous simulations and PET studies on monkeys. In the simulations, the three-injection protocol was designed according to prior knowledge of the receptor kinetics, and the errors of Bmax and Kd estimated by MI-GA were analyzed. Simulations showed that our method could support the calculation of Bmax and Kd, despite a slight overestimation compared with the true magnitudes. In monkey studies, we could calculate the Bmax and Kd of diseased or normal striatum in a 150 mins scan with the three-injection protocol of [11C]raclopride. Estimated Bmax and Kd values of D2 receptors in normal or partially dopamine-depleted striatum were comparable to the previously reported values. PMID:19904285

  2. Glioblastoma chemotherapy adjunct via potent serotonin receptor-7 inhibition using currently marketed high-affinity antipsychotic medicines

    PubMed Central

    Kast, RE

    2010-01-01

    Glioblastoma treatment as now constituted offers increased survival measured in months over untreated patients. Because glioblastomas are active in synthesizing a bewildering variety of growth factors, a systematic approach to inhibiting these is being undertaken as treatment adjunct. The serotonin 7 receptor is commonly overexpressed in glioblastoma. Research documentation showing agonists at serotonin receptor 7 cause increased extracellular regulated kinase 1/2 activation, increased interleukin-6 synthesis, increased signal transducer and activator of transcription-3 activation, increased resistance to apoptosis and other growth enhancing changes in glioblastoma is reviewed in this paper. Because three drugs in wide use to treat thought disorders – paliperidone, pimozide and risperidone – are also potent and well-tolerated inhibitors at serotonin receptor 7, these drugs should be studied for growth factor deprivation in an adjunctive role in glioblastoma treatment. PMID:20880389

  3. Adenosine A(3) receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Vacek, Antonín; Streitová, Denisa

    2007-07-01

    The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions.

  4. Nonconventional three-finger toxin BMLCL from krait Bungarus multicinctus venom with high affinity interacts with nicotinic acetylcholine receptors.

    PubMed

    Utkin, Yu N; Kasheverov, I E; Kudryavtsev, D S; Andreeva, T V; Starkov, V G; Ziganshin, R H; Kuznetsov, D V; Anh, Hoang Ngoc; Thao, Nguyen Thi Thanh; Khoa, Nguyen Cuu; Tsetlin, V I

    2015-01-01

    Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

  5. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

    PubMed

    Soheilypour, M; Mofrad, M R K

    2016-11-02

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

  6. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

    PubMed Central

    Soheilypour, M.; Mofrad, M. R. K.

    2016-01-01

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

  7. N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity.

    PubMed

    Weiss, Stefan; Keller, Max; Bernhardt, Günther; Buschauer, Armin; König, Burkhard

    2010-09-01

    N(G)-Acylated argininamides, covering a broad range of lipophilicity (calculated logD values: -1.8-12.5), were synthesized and investigated for NPY Y(1) receptor (Y(1)R) antagonism, Y(1)R affinity and stability in buffer (N(G)-deacylation, yielding BIBP 3226). Broad structural variation of substituents was tolerated. The K(i) (binding) and K(b) values (Y(1)R antagonism) varied from low nM to one-digit muM. Most of the compounds proved to be sufficiently stable at pH 7.4 over 90min to determine reliable pharmacological data in vitro. Exceptionally high instability was detected when a succinyl moiety was attached to the guanidine, probably, due to an intramolecular cleavage mechanism. Copyright 2010 Elsevier Ltd. All rights reserved.

  8. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-11-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml(-1) of antigen. This assay was modified from previous assays used to study human and canine allergic responses. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease.

  9. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

    PubMed Central

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3. PMID:25406512

  10. Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as potent and selective CB2 receptor ligands: The structural elements controlling binding affinity, selectivity and functionality.

    PubMed

    Osman, Noha A; Ligresti, Alessia; Klein, Christian D; Allarà, Marco; Rabbito, Alessandro; Di Marzo, Vincenzo; Abouzid, Khaled A; Abadi, Ashraf H

    2016-10-21

    CB2-based therapeutics show strong potential in the treatment of diverse diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this reason, research activities are currently directed towards the development of CB2 selective ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported. A set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity. Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (Ki = 7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity (∼70 and ∼200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series presented the most potent and selective CB2 ligand in this series (Ki = 2.15 nM and CB2 subtype selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2 receptor activity modulators. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. High-affinity binding of (/sup 3/H)estradiol-17 beta by an estrogen receptor in the liver of the turtle

    SciTech Connect

    Ho, S.M.; Fehrer, S.; Yu, M.

    1988-06-01

    Specific (3H)estradiol-17 beta ((3H)E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds (3H)E2 with highmore » affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of (3H)E2 binding activity in both cytosolic and nuclear fractions. The exchange between (3H)E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species.« less

  12. Identification and functional characterization of hemorphins VV-H-7 and LVV-H-7 as low-affinity agonists for the orphan bombesin receptor subtype 3

    PubMed Central

    Lammerich, Hans-Peter; Busmann, Annette; Kutzleb, Christian; Wendland, Martin; Seiler, Petra; Berger, Claudia; Eickelmann, Peter; Meyer, Markus; Forssmann, Wolf-Georg; Maronde, Erik

    2003-01-01

    The human orphan G-protein coupled receptor bombesin receptor subtype 3 (hBRS-3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV-hemorphin-7 (VV-H-7) and LVV-hemorphin-7 (LVV-H-7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS-3 compared to other bombesin receptors. VV-H-7 and LVV-H-7 induced a dose-dependent response in hBRS-3 overexpressing CHO cells, as well as in NCI-N417 cells expressing the hBRS-3 endogenously. The affinity of VV-H-7 was higher in NCI-N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45±15 μM for VV-H-7 and 183±60 μM for LVV-H-7 in CHO cells, and 19±6 μM for VV-H-7 and 38±18 μM for LVV-H-7 in NCI-N417 cells. Other hemorphins had no effect. Gastrin-releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13–20 μM (GRP) and of 1–2 μM (NMB) on both cell lines. Structure-function analysis revealed that both the N-terminal valine and the C-terminal phenylalanine residues of VV-H-7 are critical for the ligand-receptor interaction. Endogenous hBRS-3 in NCI-N417 activated by VV-H-7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)-sensitive store followed by a capacitive calcium entry from extracellular space. VV-H-7-induced hBRS-3 activation led to phosphorylation of p42/p44-MAP kinase in NCI-N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125FAK) was not observed. PMID:12721098

  13. Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

    PubMed

    Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

    2007-05-01

    Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

  15. Affinity chromatography for purification of the modular protein growth factor receptor-bound protein 2 and development of a screening test for growth factor receptor-bound protein 2 Src homology 3 domain inhibitor using peroxidase-linked ligand.

    PubMed

    Gril, B; Liu, W Q; Lenoir, C; Garbay, C; Vidal, M

    2006-04-01

    Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.

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

    PubMed

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

    2010-09-01

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

  17. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    PubMed

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

    PubMed Central

    Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

    1992-01-01

    Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

  19. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    SciTech Connect

    Lazarovici, P.; Yavin, E.

    1986-11-04

    The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme couldmore » not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.« less

  20. Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor.

    PubMed

    López Ordieres, María Graciela; Rodríguez de Lores Arnaiz, Georgina

    2002-11-01

    We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

  1. Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor

    NASA Astrophysics Data System (ADS)

    Rush, Michael D.; Walker, Elisabeth M.; Prehna, Gerd; Burton, Tristesse; van Breemen, Richard B.

    2017-03-01

    To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts.

  2. Additional lesions detected in therapeutic scans with 177Lu-DOTATATE reflect higher affinity of 177Lu-DOTATATE for somatostatin receptors.

    PubMed

    Mirzaei, Siroos; Bastati, Brigitte; Lipp, Rainer W; Knoll, Peter; Zojer, Niklas; Ludwig, Heinz

    2011-01-01

    Peptide receptor-targeted radionuclide therapy (PRRT) of somatostatin receptor (SR)-expressing neuroendocrine tumors (NETs) has become an established therapeutic option in patients with advanced NETs. The aim of this study was to compare the lesion detection rate of (99m)Tc-EDDA/HYNIC-TOC, a newly developed tracer for NET imaging, with (177)Lu-DOTATATE used for PRRT. 8 patients (4 women, 4 men, age range 46-76 years) with histologically proven NETs, who showed high SR loads by (99m)Tc-EDDA/HYNIC-TOC scintigraphy, were treated with (177)Lu-DOTATATE. After treatment, all patients were subjected to whole-body scintigraphy with additional low-dose single-photon emission computed tomography (SPECT-CT) of the chest and abdomen. All patients demonstrated (177)Lu-DOTATATE accumulation in all lesions previously detected by (99m)Tc- EDDA/HYNIC-TOC scintigraphy. Three patients showed additional lesions in the liver and lungs. SPECT-CT after (177)Lu-DOTATATE therapy may be helpful in detecting additional lesions not seen using (99m)Tc-EDDA/HYNIC-TOC. This could reflect the broader affinity of (177)Lu-DOTATATE for SRs compared with (99m)Tc-EDDA/HYNIC-TOC. Copyright © 2011 S. Karger AG, Basel.

  3. Humoral Immunity Provides Resident Intestinal Eosinophils Access to Luminal Antigen via Eosinophil-Expressed Low-Affinity Fcγ Receptors.

    PubMed

    Smith, Kalmia M; Rahman, Raiann S; Spencer, Lisa A

    2016-11-01

    Eosinophils are native to the healthy gastrointestinal tract and are associated with inflammatory diseases likely triggered by exposure to food allergens (e.g., food allergies and eosinophilic gastrointestinal disorders). In models of allergic respiratory diseases and in vitro studies, direct Ag engagement elicits eosinophil effector functions, including degranulation and Ag presentation. However, it was not known whether intestinal tissue eosinophils that are separated from luminal food Ags by a columnar epithelium might similarly engage food Ags. Using an intestinal ligated loop model in mice, in this study we determined that resident intestinal eosinophils acquire Ag from the lumen of Ag-sensitized but not naive mice in vivo. Ag acquisition was Ig-dependent; intestinal eosinophils were unable to acquire Ag in sensitized Ig-deficient mice, and passive immunization with immune serum or Ag-specific IgG was sufficient to enable intestinal eosinophils in otherwise naive mice to acquire Ag in vivo. Intestinal eosinophils expressed low-affinity IgG receptors, and the activating receptor FcγRIII was necessary for Ig-mediated acquisition of Ags by isolated intestinal eosinophils in vitro. Our combined data suggest that intestinal eosinophils acquire lumen-derived food Ags in sensitized mice via FcγRIII Ag focusing and that they may therefore participate in Ag-driven secondary immune responses to oral Ags. Copyright © 2016 by The American Association of Immunologists, Inc.

  4. Ligand-Binding Affinity at the Insulin Receptor Isoform-A and Subsequent IR-A Tyrosine Phosphorylation Kinetics are Important Determinants of Mitogenic Biological Outcomes

    PubMed Central

    Rajapaksha, Harinda; Forbes, Briony E.

    2015-01-01

    The insulin receptor (IR) is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A) arises from alternative splicing of exon 11 and has different ligand binding and signaling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II) with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival, and migration by activating some unique signaling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signaling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signaling (MAPK and Akt) and receptor internalization rates (related to mitogenic signaling). We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic [(His4, Tyr15, Thr49, Ile51) IGF-I, qIGF-I] or metabolic (S597 peptide) biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signaling through the IR-A. The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I. PMID:26217307

  5. Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): effects on binding affinity and selectivity for sigma receptors and monoamine transporters.

    PubMed

    Xu, Rong; Lord, Sarah A; Peterson, Ryan M; Fergason-Cantrell, Emily A; Lever, John R; Lever, Susan Z

    2015-01-01

    Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75-4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13-21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1Ki=20.8 nM; σ2Ki=16.4 nM) showed over 100-fold higher DAT affinity (Ki=121 nM) and 6-fold higher SERT affinity (Ki=128nM) than the parent SA4503 (DAT Ki=12650 nM; SERT Ki=760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

    PubMed

    Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

    2015-02-01

    One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

  7. BI-09EphA3 RECEPTOR IS A MOLECULAR TARGET EXPRESSED IN MULTIPLE COMPARTMENTS OF GBM

    PubMed Central

    Ferluga, Sara; Gibo, Denise; Debinski, Waldemar

    2014-01-01

    Eph receptor A3 belongs to the Eph family of receptor tyrosine kinases playing critical roles in cancer. We and others found this receptor to be over-expressed in Glioblastoma (GBM), but not in normal brain. EphA3 is a plasma membrane receptor, which is internalized upon ligand binding making it as an attractive target for specific drug delivery. EphA3 overexpression was found in tumor cells and tumor-initiating cells in GBM. However, we noted that EphA3-positive cells localize around the neovasculature, being consistent with tumor-infiltrating cells. Therefore, we decided to analyze EphA3 in relation to microglia/macrophages, as these cells highly infiltrate GBM favoring tumor progression. It has been demonstrated that glioma-infiltrating microglia acquire the M2 phenotype expressing CD163 and CD204 markers. Co-localization studies using immunofluorescence on tumor-derived primary cells showed that EphA3 co-localizes with CD163 on a sub-population of cells. The two markers also highly co-localize in snap-frozen sections of human GBM specimens, mainly in the perivascular region, as well as on cells within the bulk of the tumor and in the invasive ring, but not on the contralateral side of the diseased brain. EphA3 on snap-frozen specimens co-localized also with CD68, a more general macrophages marker, confirming the presence of EphA3 on these bone marrow-derived cells. Microglia/ macrophages have been shown also around tumor necrotic areas. We cultured GBM cells under normoxia, hypoxia and anoxia conditions and found that the levels of EphA3 receptor increased under anoxia compared to hypoxia, following the same pattern seen with CD163 and CD204. We have already generated a novel and specific cytotoxin capable of activating and internalizing the receptor and potently killing EphA3-overexpressing cells. In this study we demonstrate that by utilizing the EphA3 receptor, we will target not only tumor and tumor-initiating cells, but also infiltrating cells active in

  8. Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

    PubMed

    Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

    2006-01-01

    An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

  9. Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme

    NASA Astrophysics Data System (ADS)

    Leong, Max K.; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

    2017-01-01

    The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r2 = 0.928-0.988,  = 0.894-0.954, RMSE = 0.002-0.412, s = 0.001-0.214), and the predicted pKi values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r2 = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q2 = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery.

  10. Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme

    PubMed Central

    Leong, Max K.; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

    2017-01-01

    The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r2 = 0.928–0.988,  = 0.894–0.954, RMSE = 0.002–0.412, s = 0.001–0.214), and the predicted pKi values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r2 = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q2 = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery. PMID:28059133

  11. Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme.

    PubMed

    Leong, Max K; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

    2017-01-06

    The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r 2  = 0.928-0.988,  = 0.894-0.954, RMSE = 0.002-0.412, s = 0.001-0.214), and the predicted pK i values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r 2  = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q 2  = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery.

  12. Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

    PubMed Central

    Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

    2018-01-01

    Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783

  13. N-(4-(4-(2,3-Dichloro- or 2-methoxyphenyl)piperazin-1-yl)-butyl)-heterobiarylcarboxamides with Functionalized Linking Chains as High Affinity and Enantioselective D3 Receptor Antagonistsγ

    PubMed Central

    Newman, Amy Hauck; Grundt, Peter; Cyriac, George; Deschamps, Jeffrey R.; Taylor, Michelle; Kumar, Rakesh; Ho, David; Luedtke, Robert R.

    2009-01-01

    In the present report, the D3 receptor pharmacophore is modified in the 2,3-diCl-and 2-OCH3-phenyl piperazine class of compounds with the goal to improve D3 receptor affinity and selectivity. This extension of structure-activity relationships (SAR) has resulted in the identification of the first enantioselective D3 antagonists (R- and S-22) to be reported, wherein enantioselectivity is more pronounced at D3 than at D2, and that a binding region on the second extracellular loop (E2) may play a role in both enantioselectivity and D3 receptor selectivity. Moreover, we have discovered some of the most D3-selective compounds reported to date that show high affinity (Ki =1 nM) for D3 and ∼400-fold selectivity over the D2 receptor subtype. Several of these analogues showed exquisite selectivity for D3 receptors over >60 other receptors further underscoring their value as in vivo research tools. These lead compounds also have appropriate physical characteristics for in vivo exploration and therefore will be useful in determining how intrinsic activity at D3 receptors tested in vitro is related to behaviors in animal models of addiction and other neuropsychiatric disorders. PMID:19331412

  14. Predicting the affinity of Farnesoid X Receptor ligands through a hierarchical ranking protocol: a D3R Grand Challenge 2 case study

    NASA Astrophysics Data System (ADS)

    Réau, Manon; Langenfeld, Florent; Zagury, Jean-François; Montes, Matthieu

    2018-01-01

    The Drug Design Data Resource (D3R) Grand Challenges are blind contests organized to assess the state-of-the-art methods accuracy in predicting binding modes and relative binding free energies of experimentally validated ligands for a given target. The second stage of the D3R Grand Challenge 2 (GC2) was focused on ranking 102 compounds according to their predicted affinity for Farnesoid X Receptor. In this task, our workflow was ranked 5th out of the 77 submissions in the structure-based category. Our strategy consisted in (1) a combination of molecular docking using AutoDock 4.2 and manual edition of available structures for binding poses generation using SeeSAR, (2) the use of HYDE scoring for pose selection, and (3) a hierarchical ranking using HYDE and MM/GBSA. In this report, we detail our pose generation and ligands ranking protocols and provide guidelines to be used in a prospective computer aided drug design program.

  15. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    PubMed

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor

    PubMed Central

    2014-01-01

    Background Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. Methods The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. Results Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNβ in the RAW264.7 challenged with LPS. Conclusion Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action. PMID:24602493

  17. An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

    PubMed

    Ferguson, A D; Breed, J; Diederichs, K; Welte, W; Coulton, J W

    1998-07-01

    FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

  18. Antinociceptive action of DBO 17 and DBO 11 in mice: two 3,8 diazabicyclo (3.2.1.) octane derivates with selective mu opioid receptor affinity.

    PubMed

    Fadda, P; Barlocco, D; Tronci, S; Cignarella, G; Fratta, W

    1997-11-01

    Two 3,8 diazabicyclo (3.2.1.) octane derivates, namely DBO 17 and DBO 11, were studied for the opioid-like activity. In the rat brain membrane preparation binding studies, DBO 17 and DBO 11 showed a high affinity and selectivity for the mu opioid receptor (Ki's: 5.1 and 25 nM, respectively). DBO 17 and DBO 11 inhibited the nociceptive response in the hot-plate test of mice with ED50 values of 0.16 mg/kg and 0.44 mg/kg, respectively. The antinociceptive action of both DBO 17 and DBO 11 was blocked by naloxone. Tolerance to the antinociceptive action of DBO 17 and DBO 11 was present after 13 and 7 days of repeated treatment, respectively. Both DBO 17 and DBO 11 were ineffective in morphine-tolerant mice and vice versa. Chronic treatments (three times daily for seven consecutive days) of DBO 17 and DBO 11 induced a naloxone-precipitated withdrawal syndrome in DBO 17 treated mice similar to that in morphine treated mice, whereas in DBO 11 treated mice abstinence signs were virtually absent. These results indicate an interesting pharmacological profile that suggests these compounds as possible new candidates for the clinical treatment of pain.

  19. Hypothermia in mouse is caused by adenosine A1 and A3 receptor agonists and AMP via three distinct mechanisms.

    PubMed

    Carlin, Jesse Lea; Jain, Shalini; Gizewski, Elizabeth; Wan, Tina C; Tosh, Dilip K; Xiao, Cuiying; Auchampach, John A; Jacobson, Kenneth A; Gavrilova, Oksana; Reitman, Marc L

    2017-03-01

    Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1 -/- , Adora3 -/- ) mice. Confirming prior data, stimulation of the A 3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H 1 receptors. In contrast, A 1 AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A 1 AR agonists, including N 6 -cyclopentyladenosine (CPA), N 6 -cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A 1 AR and A 3 AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N 6 -endo-norbornyladenosine], or using Adora3 -/- mice allowed selective stimulation of A 1 AR. AMP-stimulated hypothermia can occur independently of A 1 AR, A 3 AR, and mast cells. A 1 AR and A 3 AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A 1 AR nor A 3 AR was required for fasting-induced torpor. A 1 AR and A 3 AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms. Published by Elsevier Ltd.

  20. Structure-5-HT/D2 Receptor Affinity Relationship in a New Group of 1-Arylpiperazynylalkyl Derivatives of 8-Dialkylamino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione.

    PubMed

    Żmudzki, Paweł; Satała, Grzegorz; Chłoń-Rzepa, Grażyna; Bojarski, Andrzej J; Kazek, Grzegorz; Siwek, Agata; Gryboś, Anna; Głuch-Lutwin, Monika; Wesołowska, Anna; Pawłowski, Maciej

    2016-10-01

    In our previous papers, we have reported that some 8-amino-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione derivatives possessed high affinity and displayed agonistic, partial agonistic, or antagonistic activity for serotonin 5-HT 1A and dopamine D 2 receptors. In order to examine further the influence of the substituent in the position 8 of the purine moiety and the influence of the xanthine core on the affinity for serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors, two series of 1-arylpiperazynylalkyl derivatives of 8-amino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione were synthesized. All the final compounds were investigated in in vitro competition binding experiments for the serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors. The structure-affinity relationships for this group of compounds were discussed. For selected compounds, the functional assays for the 5-HT 1A and D 2 receptors were carried out. The results of the assays indicated that these groups of derivatives possessed antagonistic activity for 5-HT 1A receptors and agonistic, partial agonistic, or antagonistic activity for D 2 receptors. In total, 26 new compounds were synthesized, 20 of which were tested in in vitro binding experiments and 5 were tested in in vitro functional assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Receptor Surface Models in the Classroom: Introducing Molecular Modeling to Students in a 3-D World

    ERIC Educational Resources Information Center

    Geldenhuys, Werner J.; Hayes, Michael; Van der Schyf, Cornelis J.; Allen, David D.; Malan, Sarel F.

    2007-01-01

    A simple, novel and generally applicable method to demonstrate structure-activity associations of a group of biologically interesting compounds in relation to receptor binding is described. This method is useful for undergraduates and graduate students in medicinal chemistry and computer modeling programs.

  2. N-Substituted cis-4a-(3-Hydroxyphenyl)-8a-methyloctahydroisoquinolines Are Opioid Receptor Pure Antagonists

    PubMed Central

    Carroll, F. Ivy; Chaudhari, Sachin; Thomas, James B.; Mascarella, S. Wayne; Gigstad, Kenneth M.; Deschamps, Jeffrey; Navarro, Hernán A.

    2008-01-01

    N-Substituted cis-4a-(3-hydroxyphenyl)-8a-methyloctahydroisoquinolines (6a–g) were designed and synthesized as conformationally constrained analogues of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (4) class of opioid receptor pure antagonists. The methyloctahydroisoquinolines 6a–g can exist in conformations where the 3-hydroxyphenyl substituent is either axial or equatorial similar to the (3-hydroxyphenyl)piperidines 4. The 3-hydroxyphenyl equatorial conformation is responsible for the antagonist activity observed in the (3-hydroxyphenyl)piperidine antagonists. Single crystal X-ray analysis of 6a shows that the 3-hydroxyphenyl equatorial conformation is favored in the solid state. Molecular modeling studies also suggest that the equatorial conformation has the lower potential energy relative to the axial conformation. Evaluation of compounds 6a–g in the [35S]GTP-γ-S in vitro functional assay showed that they were opioid receptor pure antagonists. N-[4a-(3-Hydroxyphenyl)-8a-methyl-2-(3-phenylpropyl)octahydroisoquinoline-6-yl]-3-(piperidin-1-yl)propionamide (6d) with a Ke of 0.27 nM at the κ opioid receptor with 154- and 46-fold selectively relative to the μ and δ receptors, respectively, possessed the best combination of κ potency and selectivity. PMID:16366600

  3. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    PubMed

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

  4. Blockade of the High-Affinity Interleukin-2 Receptors with Daclizumab High-Yield Process: Pharmacokinetic/Pharmacodynamic Analysis of Single- and Multiple-Dose Phase I Trials.

    PubMed

    Minocha, Mukul; Tran, Jonathan Q; Sheridan, James P; Othman, Ahmed A

    2016-01-01

    Daclizumab high-yield process (DAC HYP) is a humanized monoclonal antibody that selectively blocks the α-subunit (CD25) of the high-affinity interleukin-2 receptors, and has shown robust efficacy as a treatment for multiple sclerosis (MS). This work quantitatively characterized the relationship between DAC HYP serum concentrations and saturation of CD25 expressed on antigen-rich target T cells in blood. Serial pharmacokinetic and 968 CD25 measurements from three double-blind, randomized, placebo-controlled, phase I studies of DAC HYP (50-300 mg subcutaneous and 200-400 mg intravenous doses or placebo) in healthy volunteers (n = 95) were analyzed using nonlinear mixed-effects modeling. CD25 occupancy was determined using flow cytometry and a fluorescently-labeled DAC HYP-competing antibody. CD25 occupancy was described using a direct inhibitory sigmoidal maximum effect (E max) model (where DAC HYP fully inhibited CD25 labeling with competing antibody). Two IC50 (serum concentration corresponding to 50 % of maximal inhibition) parameters were used to describe rapid CD25 saturation at initiation of dosing and apparently slower desaturation during DAC HYP washout. Parameter estimates (95 % bootstrap confidence intervals) were: baseline CD25 labeling, 47 % (45-48); DAC HYP IC50(saturation), 0.023 µg/mL (0.005-0.073); IC50(desaturation) 0.86 µg/mL (0.74-0.98); Hill coefficient 5.6 (4.3-6.8). Based on the developed model, the 150 mg monthly subcutaneous regimen of DAC HYP in subjects with MS is predicted to saturate CD25 on target effector T cells within a few hours of dosing and maintain CD25 saturation during the entire dosing interval. Free CD25 levels return to baseline within 4-6 months of the last DAC HYP dose.

  5. Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its low-affinity receptor in cerebellum.

    PubMed

    Figueiredo, B C; Otten, U; Strauss, S; Volk, B; Maysinger, D

    1993-04-16

    Deficits or excesses of thyroid hormones during critical periods of mammalian cerebellar development can lead to profound biochemical and morphological abnormalities in this system. The goal of this study was to investigate the effects of perinatal hypo- and hyperthyroidism on the ontogeny of nerve growth factor (NGF) and its low-affinity receptor (p75NGFR) in the rat cerebellum. The concentration of NGF and of p75NGFR immunoreactivity (IR) were measured, several days after birth, in cerebella of rats which had received propylthiouracil (PTU) or thyroxine. NGF concentration was markedly enhanced only on postnatal day 2 (P2) in hyperthyroid rats, whereas in hypothyroid (PTU-treated) rats NGF values were similar to age-matched controls. These observations suggest that thyroid hormone affects NGF synthesis during early periods of cerebellar development. In Purkinje cells of control animals, p75NGFR IR peaked at P10. In hypothyroid rats, the expression of p75NGFR was retarded, peaking at P15, whereas in hyperthyroid rats it was advanced, peaking at P8. The increased p75NGFR IR found in Purkinje cell bodies and the delayed disappearance of p75NGFR IR from the external granular layer of hypothyroid rats suggest different roles for thyroid hormone in the developing cerebellum. We conclude that p75NGFR and NGF are independently regulated by thyroid hormone during critical periods of cerebellar development. The effect of thyroid hormone deficiency on p75NGFR content in Purkinje cells may involve complex mechanisms such as impaired efficiency of axonal transport.

  6. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

    PubMed Central

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  7. The Duration of Nicotine Withdrawal-Associated Deficits in Contextual Fear Conditioning Parallels Changes in Hippocampal High Affinity Nicotinic Acetylcholine Receptor Upregulation

    PubMed Central

    Gould, Thomas J.; Portugal, George S.; André, Jessica M.; Tadman, Matthew P.; Marks, Michael J.; Kenney, Justin W.; Yildirim, Emre; Adoff, Michael

    2012-01-01

    A predominant symptom of nicotine withdrawal is cognitive deficits, yet understanding of the neural basis for these deficits is limited. Withdrawal from chronic nicotine disrupts contextual learning in mice and this deficit is mediated by direct effects of nicotine in the hippocampus. Chronic nicotine treatment upregulates nicotinic acetylcholine receptors (nAChR); however, it is unknown whether upregulation is related to the observed withdawal-induced cognitive deficits. If a relationship between altered learning and nAChR levels exists, changes in nAChR levels after cessation of nicotine treatment should match the duration of learning deficits. To test this hypothesis, mice were chronically administered 6.3 mg/kg/day (freebase) nicotine for 12 days and trained in contextual fear conditioning on day 11 or between 1 to 16 days after withdrawal of treatment. Changes in [125I]-epibatidine binding at cytisine-sensitive and cytisine-resistant nAChRs and chronic nicotine-related changes in α4, α7, and β2 nAChR subunit mRNA expression were assessed. Chronic nicotine had no behavioral effect but withdrawal produced deficits in contextual fear conditioning that lasted 4 days. Nicotine withdrawal did not disrupt cued fear conditioning. Chronic nicotine upregulated hippocampal cytisine-sensitive nAChR binding; upregulation continued after cessation of nicotine administration and the duration of upregulation during withdrawal paralleled the duration of behavioral changes. Changes in binding in cortex and cerebellum did not match behavioral changes. No changes in α4, α7, and β2 subunit mRNA expression were seen with chronic nicotine. Thus, nicotine withdrawal-related deficits in contextual learning are time-limited changes that are associated with temporal changes in upregulation of high-affinity nAChR binding. PMID:22285742

  8. Inhibitory effect of carotenoids on the degranulation of mast cells via suppression of antigen-induced aggregation of high affinity IgE receptors.

    PubMed

    Sakai, Shota; Sugawara, Tatsuya; Matsubara, Kiminori; Hirata, Takashi

    2009-10-09

    Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and beta-carotene significantly inhibited the antigen-induced release of beta-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (Fc epsilonRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited Fc epsilonRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of Fc epsilonRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of Fc epsilonRI to lipid rafts, which are known as platforms of the aggregation of Fc epsilonRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of Fc epsilonRI to lipid rafts. This is the first report that focused on the aggregation of Fc epsilonRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

  9. Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors*

    PubMed Central

    Sakai, Shota; Sugawara, Tatsuya; Matsubara, Kiminori; Hirata, Takashi

    2009-01-01

    Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts. PMID:19700409

  10. Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study

    PubMed Central

    Jamalapuram, Seshulatha; Vuppala, Pradeep K.; Mesangeau, Christophe; McCurdy, Christopher R.; Avery, Bonnie A.

    2014-01-01

    A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLCTM BEH HILIC column (1.7 µm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats. PMID:22406103

  11. 2-Position base-modified analogues of adenophostin A as high-affinity agonists of the D-myo-inositol trisphosphate receptor: in vitro evaluation and molecular modeling.

    PubMed

    Sureshan, Kana M; Trusselle, Melanie; Tovey, Stephen C; Taylor, Colin W; Potter, Barry V L

    2008-03-07

    Adenophostin A (AdA) is a potent agonist of the d-myo-inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P3R). Various 2-aminopurine analogues of AdA were synthesized, all of which (guanophostin 5, 2,6-diaminopurinophostin 6, 2-aminopurinophostin 7, and chlorophostin 8) are more potent than 2-methoxy-N6-methyl AdA, the only benchmark of this class. The 2-amino-6-chloropurine nucleoside 11, from Vorbrüggen condensation of 2-amino-6-chloropurine with appropriately protected disaccharide, served as the advanced common precursor for all the analogues. Alcoholysis provided the precursor for 5, ammonolysis at high temperature the precursor for 6, and ammonolysis under mild conditions the precursor for synthesis of 7 and 8. For 8, the debenzylation of precursor leaving the chlorine untouched was achieved by judicious use of BCl3. The reduced potency of chlorophostin 8 and higher potency of guanophostin 5 in assays of Ca2+ release via recombinant Ins(1,4,5)P3R are in agreement with our model suggesting a cation-pi interaction between AdA and Ins(1,4,5)P3R. The similar potencies of 2,6-diaminopurinophostin (6) and 2-aminopurinophostin (7) concur with previous reports that the 6-NH2 moiety contributes negligibly to the potency of AdA. Molecular modeling of the 2-amino derivatives suggests an interaction between the carboxylate side chain of Glu505 of the receptor and the 2-NH2 of the ligand, but for 2-methoxy-N6-methyl AdA the carboxylate group of Glu505 is deflected away from the methoxy group. A helix-dipole interaction between the 1-phosphate of Ins(1,4,5)P3 and the 2'-phosphate of AdA with alpha-helix 6 of Ins(1,4,5)P3R is postulated. The results support a proposed model for high-affinity binding of AdA to Ins(1,4,5)P3R.

  12. N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.

    PubMed

    Gourni, Eleni; Mansi, Rosalba; Jamous, Mazen; Waser, Beatrice; Smerling, Christiane; Burian, Antje; Buchegger, Franz; Reubi, Jean Claude; Maecke, Helmut R

    2014-10-01

    Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated

  13. Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors.

    PubMed

    Gabe, Maria Buur Nordskov; Sparre-Ulrich, Alexander Hovard; Pedersen, Mie Fabricius; Gasbjerg, Lærke Smidt; Inoue, Asuka; Bräuner-Osborne, Hans; Hartmann, Bolette; Rosenkilde, Mette Marie

    2018-04-01

    GIP(3-30)NH 2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH 2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH 2 among other human and animal GPCRs. cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125 I-GIP(3-30)NH 2 . The selectivity of human GIP(3-30)NH 2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH 2 inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA 2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH 2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (K d values of 2.0, 2.5, 31

  14. Derivation of a 3D pharmacophore model for the angiotensin-II site one receptor

    NASA Astrophysics Data System (ADS)

    Prendergast, Kristine; Adams, Kym; Greenlee, William J.; Nachbar, Robert B.; Patchett, Arthur A.; Underwood, Dennis J.

    1994-10-01

    A systematic search has been used to derive a hypothesis for the receptor-bound conformation of A-II antagonists at the AT1 receptor. The validity of the pharmacophore hypothesis has been tested using CoMFA, which included 50 diverse A-II antagonists, spanning four orders of magnitude in activity. The resulting cross-validated R2 of 0.64 (conventional R2 of 0.76) is indicative of a good predictive model of activity, and has been used to estimate potency for a variety of non-peptidyl antagonists. The structural model for the non-peptide has been compared with respect to the natural substrate, A-II, by generating peptide to non-peptide overlays.

  15. Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

    PubMed

    Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

    2017-01-01

    CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

  16. Probes for narcotic receptor mediated phenomena. 43. Synthesis of the ortho-a and para-a, and improved synthesis and optical resolution of the ortho-b and para–b oxide-bridged phenylmorphans: Compounds with moderate to low opioid-receptor affinity

    PubMed Central

    Li, Feng; Folk, John E.; Cheng, Kejun; Kurimura, Muneaki; Deck, Jason A.; Deschamps, Jeffrey R.; Rothman, Richard B.; Dersch, Christina M.; Jacobson, Arthur E.; Rice, Kenner C.

    2011-01-01

    N-Phenethyl-substituted ortho-a and para-a oxide-bridged phenylmorphans have been obtained through an improved synthesis and their binding affinity examined at the various opioid receptors. Although the N-phenethyl substituent showed much greater affinity for μ- and κ-opioid receptors than their N-methyl relatives (e.g., Ki = 167 nM and 171 nM at μ- and κ-receptors vs >2800 and 7500 nM for the N-methyl ortho-a oxide-bridged phenylmorphan), the a-isomers were not examined further because of their relatively low affinity. The N-phenethyl substituted ortho-b and para-b oxide-bridged phenylmorphans were also synthesized and their enantiomers were obtained using supercritical fluid chromatography. Of the four enantiomers, only the (+)-ortho-b isomer had moderate affinity for μ- and κ-receptors (Ki = 49 and 42 nM, respectively, and it was found to also have moderate μ- and κ-opioid antagonist activity in the [35S]GTP-γ-S assay (Ke = 31 and 26 nM). PMID:21684752

  17. Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

    PubMed

    Ferry, D R; Goll, A; Glossmann, H

    1987-04-01

    The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

  18. The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

    PubMed

    Ochaion, A; Bar-Yehuda, S; Cohen, S; Amital, H; Jacobson, K A; Joshi, B V; Gao, Z G; Barer, F; Patoka, R; Del Valle, L; Perez-Liz, G; Fishman, P

    2008-08-15

    The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

  19. Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson

    PubMed Central

    Barer, Faina; Itzhak, Inbal; Silverman, Michael H.

    2018-01-01

    Interleukin-17 and interleukin-23 play major roles in the inflammatory process in psoriasis. The Gi protein-associated A3 adenosine receptor (A3AR) is known to be overexpressed in inflammatory cells and in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory conditions. Piclidenoson, a selective agonist at the A3AR, induces robust anti-inflammatory effect in psoriasis patients. In this study, we aimed to explore A3AR expression levels in psoriasis patients and its role in mediating the anti-inflammatory effect of piclidenoson in human keratinocyte cells. A3AR expression levels were evaluated in skin tissue and PBMCs derived from psoriasis patients and healthy subjects. Proliferation assay and the expression of signaling proteins were used to evaluate piclidenoson effect on human keratinocytes (HaCat). High A3AR expression levels were found in a skin biopsy and in PBMCs from psoriasis patients in comparison to healthy subjects. Piclidenoson inhibited the proliferation of HaCat cells through deregulation of the NF-κB signaling pathway, leading to a decrease in interleukin-17 and interleukin-23 expression levels. This effect was counteracted by the specific antagonist MRS 1523. A3AR overexpression in skin and PBMCs of psoriasis patients may be used as a target to inhibit pathological cell proliferation and the production of interleukin-17 and interleukin-23. PMID:29862305

  20. Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

    SciTech Connect

    Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.

    2009-06-24

    A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from micromolar to low nanomolar.

  1. Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors.

    PubMed

    Shi, B; Narayanan, T K; Yang, Z Y; Christian, B T; Mukherjee, J

    1999-10-01

    We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (+/-)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (+/-)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (+/-)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites (IC50 = 0.010 nM), PPHT was somewhat weaker (IC50 = 0.65 nM), and 5-OH-DPAT exhibited the weakest affinity (IC50 = 2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-5-OH-DPAT), 2-(N-phenethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-PPHT), and 2-(N-cyclohexylethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-ZYY-339) was achieved by first synthesizing 11C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11CO2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11C-5-OH-DPAT, 11C-PPHT, and 11C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata.

  2. Understanding allosteric interactions in G protein-coupled receptors using Supervised Molecular Dynamics: A prototype study analysing the human A3 adenosine receptor positive allosteric modulator LUF6000.

    PubMed

    Deganutti, Giuseppe; Cuzzolin, Alberto; Ciancetta, Antonella; Moro, Stefano

    2015-07-15

    The search for G protein-coupled receptors (GPCRs) allosteric modulators represents an active research field in medicinal chemistry. Allosteric modulators usually exert their activity only in the presence of the orthosteric ligand by binding to protein sites topographically different from the orthosteric cleft. They therefore offer potentially therapeutic advantages by selectively influencing tissue responses only when the endogenous agonist is present. The prediction of putative allosteric site location, however, is a challenging task. In facts, they are usually located in regions showing more structural variation among the family members. In the present work, we applied the recently developed Supervised Molecular Dynamics (SuMD) methodology to interpret at the molecular level the positive allosteric modulation mediated by LUF6000 toward the human adenosine A3 receptor (hA3 AR). Our data suggest at least two possible mechanisms to explain the experimental data available. This study represent, to the best of our knowledge, the first case reported of an allosteric recognition mechanism depicted by means of molecular dynamics simulations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Streitová, Denisa; Vacek, Antonín

    2008-07-01

    Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression.

  4. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  5. Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

    PubMed

    Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

    2017-04-01

    Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The Differential Expression of Calcitonin Gene Related Peptide, alpha CGRP mRNA, Choline Acetyltransferase, and Low Affinity Nerve Growth Factor Receptor in Cranial Motoneurons After Hypoglossal Nerve Injury During Postnatal Development

    DTIC Science & Technology

    1996-08-21

    maintenance. Since the classic neurotrophic effects ofNGF are not observed when it is bound to the low affinity receptor and since the high affmity type...is not present in motoneurons after injury this may explain why the classic NGF response is not observed (Wood et aI., 1990) and suggests that perhaps...Heumann et aI., 1987; Meyer et aI., 1992; Funakoshi et aI., 1993; Friedman et aI., 1995a). This sequencing ofneurotrophic changes may be related to the

  7. High Specific Activity Tritium-Labeled N-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine (INBMeO): A High Affinity 5-HT2A Receptor-Selective Agonist Radioligand

    PubMed Central

    Nichols, David E.; Frescas, Stewart P.; Chemel, Benjamin R.; Rehder, Kenneth S.; Zhong, Desong; Lewin, Anita H.

    2009-01-01

    The title compound ([3H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic precursor using no carrier added tritiated iodomethane in DMF with K2CO3. Removal of the t-BOC protecting group and purification by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164 Ci/mmol. The new radioligand was suitable for labeling human 5-HT2A receptors in two heterologous cell lines and had about 20-fold higher affinity than [3H]ketanserin. PMID:18468904

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

    PubMed

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

    2015-02-15

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

  9. Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

    PubMed

    Newman-Tancredi, A; Verrièle, L; Touzard, M; Millan, M J

    2001-10-05

    5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.

  10. Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

    PubMed

    Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

    2010-11-30

    Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

    PubMed

    Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

    1994-02-25

    Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

  12. Renoprotective Effects of a Highly Selective A3 Adenosine Receptor Antagonist in a Mouse Model of Adriamycin-induced Nephropathy.

    PubMed

    Min, Hye Sook; Cha, Jin Joo; Kim, Kitae; Kim, Jung Eun; Ghee, Jung Yeon; Kim, Hyunwook; Lee, Ji Eun; Han, Jee Young; Jeong, Lak Shin; Cha, Dae Ryong; Kang, Young Sun

    2016-09-01

    The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-β1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1β, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

  13. Novel 4-Substituted-N,N-dimethyltetrahydronaphthalen-2-amines: Synthesis, Affinity, and In Silico Docking Studies at Serotonin 5-HT2-type and Histamine H1 G Protein-Coupled Receptors

    PubMed Central

    Sakhuja, Rajeev; Kondabolu, Krishnakanth; Córdova-Sintjago, Tania; Travers, Sean; Vincek, Adam S.; Kim, Myong Sang; Abboud, Khalil A.; Fang, Lijuan; Sun, Zhuming; Canal, Clinton E.; Booth, Raymond G.

    2015-01-01

    Syntheses were undertaken of derivatives of (2S, 4R)-(−)-trans-4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (4-phenyl-2-dimethylaminotetralin, PAT), a stereospecific agonist at the serotonin 5-HT2C G protein-coupled receptor (GPCR), with inverse agonist activity at 5-HT2A and 5-HT2B GPCRs. Molecular changes were made at the PAT C(4)-position, while preserving N, N-dimethyl substitution at the 2-position as well as trans-stereochemistry, structural features previously shown to be optimal for 5-HT2 binding. Affinities of analogs were determined at recombinant human 5-HT2 GPCRs in comparison to the phylogenetically closely-related histamine H1 GPCR, and in silico ligand docking studies were conducted at receptor molecular models to help interpret pharmacological results and guide future ligand design. In most cases, C(4)-substituted PAT analogs exhibited the same stereoselectivity ([−]-trans > [+]-trans) as the parent PAT across 5-HT2 and H1 GPCRs, albeit, with variable receptor selectivity. 4-(4′-substituted)-PAT analogs, however, demonstrated reversed stereoselectivity ([2S, 4R]-[+]-trans > [2S, 4R]-[−]-trans), with absolute configuration confirmed by single X-ray crystallographic data for the 4-(4′-Cl)-PAT analog. Pharmacological affinity results and computational results herein support further PAT drug development studies and provide a basis for predicting and interpreting translational results, including, for (+)-trans-4-(4′-Cl)-PAT and (−)-trans-4-(3′-Br)-PAT that were previously shown to be more potent and efficacious than their corresponding enantiomers in rodent models of psychoses, psychostimulant-induced behaviors, and compulsive feeding (‘binge-eating’). PMID:25703249

  14. Cutting Edge: The murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis.

    PubMed

    Mancardi, David A; Jönsson, Friederike; Iannascoli, Bruno; Khun, Huot; Van Rooijen, Nico; Huerre, Michel; Daëron, Marc; Bruhns, Pierre

    2011-02-15

    K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

  15. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors.

    PubMed

    García-Garayoa, E; Allemann-Tannahill, L; Bläuenstein, P; Willmann, M; Carrel-Rémy, N; Tourwé, D; Iterbeke, K; Conrath, P; Schubiger, P A

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  16. A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

    PubMed Central

    Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai

    2016-01-01

    ABSTRACT H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues

  17. A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

    PubMed

    Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai; Ma, Wenjun; Li, Zejun

    2016-11-01

    H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse

  18. in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence, structure analysis of crystal proteins for stability.

    PubMed

    Ebenezer, King Solomon; Nachimuthu, Ramesh; Thiagarajan, Prabha; Velu, Rajesh Kannan

    2013-01-01

    Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125(th) amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.

  19. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    PubMed

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  20. Serum Levels of Two Immunological Markers, the Soluble Low Affinity Receptor for IGE (SFCERII, SCD23) and their Correlation with Age, Gender and the Onset of Childhood Atopy

    DTIC Science & Technology

    1993-01-01

    immunoglobulin receptor because it is the only known Fc Receptor which does not belong to the same gene I 0 superfamily as its ligand. (9, 14, 46). This FC...type) animal lectins. (2, 3, 4, 6, 8, 9, 13, 14, 20, 46). This homology domain spanning from cysteine 163 through cysteine 282, contAins four highly...substantial amount of CD23a and CD23b. (12) The expression of CD23 is lost after immunoglobulin isotype switching and during the differentiation of B cells into

  1. Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

    PubMed

    Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

    2009-08-15

    In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

  2. An Efficient Metadynamics-Based Protocol To Model the Binding Affinity and the Transition State Ensemble of G-Protein-Coupled Receptor Ligands.

    PubMed

    Saleh, Noureldin; Ibrahim, Passainte; Saladino, Giorgio; Gervasio, Francesco Luigi; Clark, Timothy

    2017-05-22

    A generally applicable metadynamics scheme for predicting the free energy profile of ligand binding to G-protein-coupled receptors (GPCRs) is described. A common and effective collective variable (CV) has been defined using the ideally placed and highly conserved Trp6.48 as a reference point for ligand-GPCR distance measurement and the common orientation of GPCRs in the cell membrane. Using this single CV together with well-tempered multiple-walker metadynamics with a funnel-like boundary allows an efficient exploration of the entire ligand binding path from the extracellular medium to the orthosteric binding site, including vestibule and intermediate sites. The protocol can be used with X-ray structures or high-quality homology models (based on a high-quality template and after thorough refinement) for the receptor and is universally applicable to agonists, antagonists, and partial and reverse agonists. The root-mean-square error (RMSE) in predicted binding free energies for 12 diverse ligands in five receptors (a total of 23 data points) is surprisingly small (less than 1 kcal mol -1 ). The RMSEs for simulations that use receptor X-ray structures and homology models are very similar.

  3. Low molecular weight glycosaminoglycan C3 attenuates AF64A-stimulated, low-affinity nerve growth factor receptor-immunoreactive axonal varicosities in the rat septum.

    PubMed

    Dudas, Bertalan; Rose, Michael; Cornelli, Umberto; Hanin, Israel

    2005-02-01

    Glycosaminoglycans (GAGs) play a pivotal role in the pathogenesis of Alzheimer's disease (AD). Although, as we have shown earlier, a low molecular weight GAG, C3, protects against ethylcholine aziridinium (AF64A)-induced cholinergic damage, and against A(beta)-induced tau-2-immunoreactivity (IR), the mechanism of the neuroprotective effect of GAGs is not yet known. Several clues exist. Previous studies in rats revealed that continuous NGF infusion (icv) after AF64A injection increases septal ChAT and AChE activities. Moreover, C3 increases axonal outgrowth in the rat hippocampus, raising the possibility of a NGF-receptor mediated neuroprotection. Furthermore, it has been reported that NGF expression is increased in the septum following AF64A administration. To study the question regarding the mechanism of neuroprotective action of GAGs, AF64A, a selective cholinotoxin, was administered stereotaxically, bilaterally, into the lateral ventricles of Fischer albino male rats (1 nmol/2 microl/side). In order to establish the effect of C3 on the expression of the NGF receptor-IR elements, C3 was administered orally (25 mg/kg, once a day), by gavage, 7 days before, and 7 days after the AF64A injection. NGF receptor immunohistochemistry revealed that AF64A induced the appearance of NGF-receptor-IR axonal varicosities in the rat medial septum. These varicose fibers were attenuated by 14 days' administration of C3. The possible explanation of our data may be that C3 increases NGF synthesis in the lateral septum. The increased level of NGF could suppress the increased, AF64A-induced NGF receptor expression in the medial septal nucleus. These results further accentuate our earlier observations that C3 may have potential as a therapeutic agent in AD and other neurodegenerative disorders.

  4. Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135.

    PubMed

    De Luca, Maria Antonietta; Castelli, M Paola; Loi, Barbara; Porcu, Alessandra; Martorelli, Mariella; Miliano, Cristina; Kellett, Kathryn; Davidson, Colin; Stair, Jacqueline L; Schifano, Fabrizio; Di Chiara, Gaetano

    2016-06-01

    In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [(35)S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018. The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 μM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003-0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose-response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. In vitro binding affinities of a series of flavonoids for μ-opioid receptors. Antinociceptive effect of the synthetic flavonoid 3,3-dibromoflavanone in mice.

    PubMed

    Higgs, Josefina; Wasowski, Cristina; Loscalzo, Leonardo M; Marder, Mariel

    2013-09-01

    The pharmacotherapy for the treatment of pain is an active area of investigation. There are effective drugs to treat this problem, but there is also a need to find alternative treatments free of undesirable side effects. In the present work the capacity of a series of flavonoids to bind to the μ opioid receptor was evaluated. The most active compound, 3,3-dibromoflavanone (31), a synthetic flavonoid, presented a significant inhibition of the binding of the selective μ opioid ligand [(3)H]DAMGO, with a Ki of 0.846 ± 0.263 μM. Flavanone 31 was further synthesized using a simple and cheap procedure with good yield. Its in vivo effects in mice, after acute treatments, were studied using antinociceptive and behavioral assays. It showed no sedative, anxiolytic, motor incoordination effects or inhibition of the gastrointestinal transit in mice at the doses tested. It evidenced antinociceptive activity on the acetic acid-induced nociception, hot plate and formalin tests (at 10 mg/kg and 30 mg/kg). The results showed that the 5-HT2 receptor and the adrenoceptors seem unlikely to be involved in its antinociceptive effects. Naltrexone, a nonselective opioid receptors antagonist, totally blocked compound 31 antinociceptive effects on the hot plate test, but naltrindole (δ opioid antagonist) and nor-binaltorphimine (κ opioid antagonist) did not. These findings demonstrated that 3,3-dibromoflavanone (31), at doses that did not interfere with the motor performance, exerted clear dose dependent antinociception when assessed in the chemical and thermal models of nociception in mice and it seems that its action is related to the activation of the μ opioid receptor. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Studies of the Mu-Opioid Receptor/G-protein Complex Affinity Co-Purified and Membrane Preparations from 7315c Cells

    DTIC Science & Technology

    1989-06-09

    centrifugational sedimentation rates of the GTPys- activated and inactivated purified Gs (Birnbaumer et al., 1985). Recently, different antibodies raised against...and Membrane Preparations from 7315c Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e...were shown to be fused with the 1 membranes from turkey erythrocytes that have ~-adrenergic receptors but inactive adenylyl cyclase activity due to

  7. In Vitro Mouse and Human Serum Stability of a Heterobivalent Dual-Target Probe That Has Strong Affinity to Gastrin-Releasing Peptide and Neuropeptide Y1 Receptors on Tumor Cells.

    PubMed

    Ghosh, Arijit; Raju, Natarajan; Tweedle, Michael; Kumar, Krishan

    2017-02-01

    Receptor-targeting radiolabeled molecular probes with high affinity and specificity are useful in studying and monitoring biological processes and responses. Dual- or multiple-targeting probes, using radiolabeled metal chelates conjugated to peptides, have potential advantages over single-targeting probes as they can recognize multiple targets leading to better sensitivity for imaging and radiotherapy when target heterogeneity is present. Two natural hormone peptide receptors, gastrin-releasing peptide (GRP) and Y1, are specifically interesting as their expression is upregulated in most breast and prostate cancers. One of our goals has been to develop a dual-target probe that can bind both GRP and Y1 receptors. Consequently, a heterobivalent dual-target probe, t-BBN/BVD15-DO3A (where a GRP targeting ligand J-G-Abz4-QWAVGHLM-NH 2 and Y1 targeting ligand INP-K [ɛ-J-(α-DO3A-ɛ-DGa)-K] YRLRY-NH 2 were coupled), that recognizes both GRP and Y1 receptors was synthesized, purified, and characterized in the past. Competitive displacement cell binding assay studies with the probe demonstrated strong affinity (IC 50 values given in parentheses) for GRP receptors in T-47D cells (18 ± 0.7 nM) and for Y1 receptors in MCF7 cells (80 ± 11 nM). As a further evaluation of the heterobivalent dual-target probe t-BBN/BVD15-DO3A, the objective of this study was to determine its mouse and human serum stability at 37°C. The in vitro metabolic degradation of the dual-target probe in mouse and human serum was studied by using a 153 Gd-labeled t-BBN/BVD15-DO3A and a high-performance liquid chromatography/radioisotope detector analytical method. The half-life (t 1/2 ) of degradation of the dual-target probe in mouse serum was calculated as 7 hours and only ∼20% degradation was seen after 6 hours incubation in human serum. The slow in vitro metabolic degradation of the dual-target probe can be compared with the degradation t 1/2 of the corresponding monomeric probes, BVD15

  8. The decoy Fcγ receptor encoded by the cytomegalovirus UL119-UL118 gene has differential affinity to IgG proteins expressing different GM allotypes.

    PubMed

    Pandey, Janardan P; Namboodiri, Aryan M; Radwan, Faisal F; Nietert, Paul J

    2015-08-01

    Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in many diseases. However, there is significant divergence between HCMV seroprevalence and the prevalence of HCMV-associated diseases, implying the presence of host genetic factors that might modulate immunity to this virus. HCMV deploys many sophisticated strategies to evade host immunosurveillance. One strategy involves encoding for proteins that have functional properties of the Fcγ receptor (FcγR). The aim of the present investigation was to determine whether the UL119-UL118-encoded recombinant FcγR ectodomain binds differentially to genetically disparate IgG1 proteins. Results show that mean absorbance values for binding of HCMV UL119-UL118-encoded Fcγ receptor to the immunoglobulin GM (γ marker) 1,17-expressing IgG1 were significantly higher than to the IgG1 expressing the allelic GM 3 allotype (0.225 vs. 0.151; p=0.039). These findings suggest possible mechanisms underlying the maintenance of immunoglobulin GM gene polymorphism and its putative role in the etiology of HCMV-associated diseases. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  9. The β subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin

    PubMed Central

    Hanner, Markus; Schmalhofer, William A.; Munujos, Petraki; Knaus, Hans-Günther; Kaczorowski, Gregory J.; Garcia, Maria L.

    1997-01-01

    Transient expression of either α or α+β subunits of the high-conductance Ca2+-activated K+ (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the α subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the β subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the β subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the β subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of β attract the positively charged 125I-ChTX to its binding site on α through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of β must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the α subunit. Certain regions in the extracellular loop of the β subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function. PMID:9096310

  10. Phosphorylation of hepatocyte growth factor receptor and epidermal growth factor receptor of human hepatocytes can be maintained in a (3D) collagen sandwich culture system.

    PubMed

    Engl, Tobias; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Jonas, Dietger; Oppermann, Elsie; Auth, Marcus K H; Schaudt, André; Bechstein, Wolf-Otto; Blaheta, Roman A

    2004-08-01

    In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three-dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c-met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two-dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.

  11. β-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.

    PubMed

    Reynolds, Merrick S; Hancock, Chad R; Ray, Jason D; Kener, Kyle B; Draney, Carrie; Garland, Kevin; Hardman, Jeremy; Bikman, Benjamin T; Tessem, Jeffery S

    2016-07-01

    β-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study, we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here, we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion. Copyright © 2016 the American Physiological Society.

  12. Binding affinities of the farnesoid X receptor in the D3R Grand Challenge 2 estimated by free-energy perturbation and docking

    NASA Astrophysics Data System (ADS)

    Olsson, Martin A.; García-Sosa, Alfonso T.; Ryde, Ulf

    2018-01-01

    We have studied the binding of 102 ligands to the farnesoid X receptor within the D3R Grand Challenge 2016 blind-prediction competition. First, we employed docking with five different docking software and scoring functions. The selected docked poses gave an average root-mean-squared deviation of 4.2 Å. Consensus scoring gave decent results with a Kendall's τ of 0.26 ± 0.06 and a Spearman's ρ of 0.41 ± 0.08. For a subset of 33 ligands, we calculated relative binding free energies with free-energy perturbation. Five transformations between the ligands involved a change of the net charge and we implemented and benchmarked a semi-analytic correction (Rocklin et al., J Chem Phys 139:184103, 2013) for artifacts caused by the periodic boundary conditions and Ewald summation. The results gave a mean absolute deviation of 7.5 kJ/mol compared to the experimental estimates and a correlation coefficient of R 2 = 0.1. These results were among the four best in this competition out of 22 submissions. The charge corrections were significant (7-8 kJ/mol) and always improved the results. By employing 23 intermediate states in the free-energy perturbation, there was a proper overlap between all states and the precision was 0.1-0.7 kJ/mol. However, thermodynamic cycles indicate that the sampling was insufficient in some of the perturbations.

  13. The Arabidopsis ROP-activated receptor-like cytoplasmic kinase RLCK VI_A3 is involved in control of basal resistance to powdery mildew and trichome branching.

    PubMed

    Reiner, Tina; Hoefle, Caroline; Huesmann, Christina; Ménesi, Dalma; Fehér, Attila; Hückelhoven, Ralph

    2015-03-01

    The Arabidopsis receptor-like cytoplasmic kinase AtRLCK VI_A3 is activated by AtROPs and is involved in trichome branching and pathogen interaction. Receptor-like cytoplasmic kinases (RLCKs) belong to the large superfamily of receptor-like kinases, which are involved in a variety of cellular processes like plant growth, development and immune responses. Recent studies suggest that RLCKs of the VI_A subfamily are possible downstream effectors of the small monomeric G proteins of the plant-specific Rho family, called 'Rho of plants' (RAC/ROPs). Here, we describe Arabidopsis thaliana AtRLCK VI_A3 as a molecular interactor of AtROPs. In Arabidopsis epidermal cells, transient co-expression of plasma membrane located constitutively activated (CA) AtROP4 or CA AtROP6 resulting in the recruitment of green fluorescent protein-tagged AtRLCK VI_A3 to the cell periphery. Intrinsic kinase activity of AtRLCK VI_A3 was enhanced in the presence of CA AtROP6 in vitro and further suggested a functional interaction between the proteins. In the interaction of the biotrophic powdery mildew fungus Erysiphe cruciferarum (E. cruciferarum) and its host plant Arabidopsis, Atrlck VI_A3 mutant lines supported enhanced fungal reproduction. Furthermore Atrlck VI_A3 mutant lines showed slightly reduced size and an increase in trichome branch number compared to wild-type plants. In summary, our data suggest a role of the AtROP-regulated AtRLCK VI_A3 in basal resistance to E. cruciferarum as well as in plant growth and cellular differentiation during trichome morphogenesis. Results are discussed in the context of literature suggesting a function of RAC/ROPs in both resistance and susceptibility to pathogen infection.

  14. Silver Nanoparticle-Directed Mast Cell Degranulation Is Mediated through Calcium and PI3K Signaling Independent of the High Affinity IgE Receptor

    PubMed Central

    Alsaleh, Nasser B.; Persaud, Indushekhar; Brown, Jared M.

    2016-01-01

    Engineered nanomaterial (ENM)-mediated toxicity often involves triggering immune responses. Mast cells can regulate both innate and adaptive immune responses and are key effectors in allergic diseases and inflammation. Silver nanoparticles (AgNPs) are one of the most prevalent nanomaterials used in consumer products due to their antimicrobial properties. We have previously shown that AgNPs induce mast cell degranulation that was dependent on nanoparticle physicochemical properties. Furthermore, we identified a role for scavenger receptor B1 (SR-B1) in AgNP-mediated mast cell degranulation. However, it is completely unknown how SR-B1 mediates mast cell degranulation and the intracellular signaling pathways involved. In the current study, we hypothesized that SR-B1 interaction with AgNPs directs mast cell degranulation through activation of signal transduction pathways that culminate in an increase in intracellular calcium signal leading to mast cell degranulation. For these studies, we utilized bone marrow-derived mast cells (BMMC) isolated from C57Bl/6 mice and RBL-2H3 cells (rat basophilic leukemia cell line). Our data support our hypothesis and show that AgNP-directed mast cell degranulation involves activation of PI3K, PLCγ and an increase in intracellular calcium levels. Moreover, we found that influx of extracellular calcium is required for the cells to degranulate in response to AgNP exposure and is mediated at least partially via the CRAC channels. Taken together, our results provide new insights into AgNP-induced mast cell activation that are key for designing novel ENMs that are devoid of immune system activation. PMID:27907088

  15. 6-Mercaptopurine augments glucose transport activity in skeletal muscle cells in part via a mechanism dependent upon orphan nuclear receptor NR4A3

    PubMed Central

    Zhu, Xiaolin; Xu, Lusheng; Fu, Yuchang; Garvey, W. Timothy

    2013-01-01

    The purine anti-metabolite 6-mercaptopurine (6-MP) is widely used for the treatment of leukemia and inflammatory diseases. The cellular effects of 6-MP on metabolism remain unknown; however, 6-MP was recently found to activate the orphan nuclear receptor NR4A3 in skeletal muscle cell lines. We have reported previously that NR4A3 (also known as NOR-1, MINOR) is a positive regulator of insulin sensitivity in adipocytes. To further explore the role of NR4A3 activation in insulin action, we explored whether 6-MP activation of NR4A3 could modulate glucose transport system activity in L6 skeletal muscle cells. We found that 6-MP increased both NR4A3 expression and NR4A3 transcriptional activity and enhanced glucose transport activity via increasing GLUT4 translocation in both basal and insulin-stimulated L6 cells in an NR4A3-dependent manner. Furthermore, 6-MP increased levels of phospho-AS160, although this effect was not modulated by NR4A3 overexpression or knockdown. These primary findings provide a novel proof of principle that 6-MP, a small molecule NR4A3 agonist, can augment glucose uptake in insulin target cells, although this occurs via both NR4A3-dependent and -independent actions; the latter is related to an increase in phospho-AS160. These results establish a novel target for development of new treatments for insulin resistance. PMID:24022864

  16. 6-Mercaptopurine augments glucose transport activity in skeletal muscle cells in part via a mechanism dependent upon orphan nuclear receptor NR4A3.

    PubMed

    Liu, Qinglan; Zhu, Xiaolin; Xu, Lusheng; Fu, Yuchang; Garvey, W Timothy

    2013-11-01

    The purine anti-metabolite 6-mercaptopurine (6-MP) is widely used for the treatment of leukemia and inflammatory diseases. The cellular effects of 6-MP on metabolism remain unknown; however, 6-MP was recently found to activate the orphan nuclear receptor NR4A3 in skeletal muscle cell lines. We have reported previously that NR4A3 (also known as NOR-1, MINOR) is a positive regulator of insulin sensitivity in adipocytes. To further explore the role of NR4A3 activation in insulin action, we explored whether 6-MP activation of NR4A3 could modulate glucose transport system activity in L6 skeletal muscle cells. We found that 6-MP increased both NR4A3 expression and NR4A3 transcriptional activity and enhanced glucose transport activity via increasing GLUT4 translocation in both basal and insulin-stimulated L6 cells in an NR4A3-dependent manner. Furthermore, 6-MP increased levels of phospho-AS160, although this effect was not modulated by NR4A3 overexpression or knockdown. These primary findings provide a novel proof of principle that 6-MP, a small molecule NR4A3 agonist, can augment glucose uptake in insulin target cells, although this occurs via both NR4A3-dependent and -independent actions; the latter is related to an increase in phospho-AS160. These results establish a novel target for development of new treatments for insulin resistance.

  17. Pre-activation with IL-12, IL-15, and IL-18 induces CD25 and a functional high affinity IL-2 receptor on human cytokine-induced memory-like NK cells

    PubMed Central

    Leong, Jeffrey W.; Chase, Julie M.; Romee, Rizwan; Schneider, Stephanie E.; Sullivan, Ryan P.; Cooper, Megan A.; Fehniger, Todd A.

    2014-01-01

    NK cells are effector lymphocytes that are under clinical investigation for the adoptive immunotherapy of hematologic malignancies, especially acute myeloid leukemia. Recent work in mice has identified innate memory-like properties of NK cells. Human NK cells also exhibit memory-like properties, and cytokine-induced memory-like (CIML) NK cells are generated via brief pre-activation with IL-12, IL-15, and IL-18, which later exhibit enhanced functionality upon restimulation. However, investigation of the optimal cytokine receptors and signals for maintenance of enhanced function and homeostasis following pre-activation remains unclear. Here, we show that IL-12, IL-15, and IL-18 pre-activation induces a rapid and prolonged expression of CD25, resulting in a functional high affinity IL-2 receptor (IL-2Rαβγ) that confers responsiveness to picomolar concentrations of IL-2. The expression of CD25 correlated with STAT5 phosphorylation in response to picomolar concentrations of IL-2, indicating the presence of a signal-competent IL-2Rαβγ. Furthermore, picomolar concentrations of IL-2 acted synergistically with IL-12 to co-stimulate IFN-γ production by pre-activated NK cells, an effect that was CD25-dependent. Picomolar concentrations of IL-2 also enhanced NK cell proliferation and cytotoxicity via the IL-2Rαβγ. Further, following adoptive transfer into immunodeficient NOD-SCID-γc−/− mice, human cytokine pre-activated NK cells expand preferentially in response to exogenous IL-2. Collectively, these data demonstrate that human CIML NK cells respond to IL-2 via IL-2Rαβγ with enhanced survival and functionality, and provide additional rationale for immunotherapeutic strategies that include brief cytokine pre-activation prior to adoptive NK cell transfer, followed by low dose IL-2 therapy. PMID:24434782

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

    PubMed

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

    2012-03-14

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

  19. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  20. Investigation of various N-heterocyclic substituted piperazine versions of 5/ 7-{[2-(4-Aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol: Effect on affinity and selectivity for dopamine D3 receptor

    PubMed Central

    Brown, Dennis A.; Mishra, Manoj; Zhang, Suhong; Biswas, Swati; Parrington, Ingrid; Antonio, Tamara; Reith, Maarten E. A.; Dutta, Aloke K.

    2009-01-01

    Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/ 7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol series of molecules. Compounds were subjected to [3H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (Ki) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (-)-10e (Ki; D2 = 47.5 nM, D3 = 0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (Ki; D2 = 113 nM, D3 = 3.73 nM). Additionally, compound (-)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPγS binding study, one of the lead molecules, (-)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential activity at D3. PMID:19427222

  1. Identification of the Beer Component Hordenine as Food-Derived Dopamine D2 Receptor Agonist by Virtual Screening a 3D Compound Database

    NASA Astrophysics Data System (ADS)

    Sommer, Thomas; Hübner, Harald; El Kerdawy, Ahmed; Gmeiner, Peter; Pischetsrieder, Monika; Clark, Timothy

    2017-03-01

    The dopamine D2 receptor (D2R) is involved in food reward and compulsive food intake. The present study developed a virtual screening (VS) method to identify food components, which may modulate D2R signalling. In contrast to their common applications in drug discovery, VS methods are rarely applied for the discovery of bioactive food compounds. Here, databases were created that exclusively contain substances occurring in food and natural sources (about 13,000 different compounds in total) as the basis for combined pharmacophore searching, hit-list clustering and molecular docking into D2R homology models. From 17 compounds finally tested in radioligand assays to determine their binding affinities, seven were classified as hits (hit rate = 41%). Functional properties of the five most active compounds were further examined in β-arrestin recruitment and cAMP inhibition experiments. D2R-promoted G-protein activation was observed for hordenine, a constituent of barley and beer, with approximately identical ligand efficacy as dopamine (76%) and a Ki value of 13 μM. Moreover, hordenine antagonised D2-mediated β-arrestin recruitment indicating functional selectivity. Application of our databases provides new perspectives for the discovery of bioactive food constituents using VS methods. Based on its presence in beer, we suggest that hordenine significantly contributes to mood-elevating effects of beer.

  2. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  3. Notch and affinity boundaries in Drosophila.

    PubMed

    Herranz, Héctor; Milán, Marco

    2006-02-01

    Cells in multicellular organisms often do not intermingle freely with each other. Differential cell affinities can contribute to organizing cells into different tissues. Drosophila limbs and the vertebrate central nervous system are subdivided into compartments. Cells in adjacent compartments do not mix. Cell interactions mediated by Notch-family receptors have been implicated in the specification of these compartment boundaries. Two recent reports analyze the role of the Notch signaling pathway in the generation of an affinity boundary in the Drosophila wing. The first report analyzes the connection between Notch and the actin cytoskeleton. The second report analyzes the differential requirements of Notch and the transcription factor Suppressor of Hairless in generating the affinity boundary.

  4. Positive transcriptional regulation of the human micro opioid receptor gene by poly(ADP-ribose) polymerase-1 and increase of its DNA binding affinity based on polymorphism of G-172 -> T.

    PubMed

    Ono, Takeshi; Kaneda, Toshio; Muto, Akihiro; Yoshida, Tadashi

    2009-07-24

    Micro opioid receptor (MOR) agonists such as morphine are applied widely in clinical practice as pain therapy. The effects of morphine through MOR, such as analgesia and development of tolerance and dependence, are influenced by individual specificity. Recently, we analyzed single nucleotide polymorphisms on the human MOR gene to investigate the factors that contribute to individual specificity. In process of single nucleotide polymorphisms analysis, we found that specific nuclear proteins bound to G(-172) --> T region in exon 1 in MOR gene, and its affinity to DNA was increased by base substitution from G(-172) to T(-172). The isolated protein was identified by mass spectrometry and was confirmed by Western blotting to be poly(ADP-ribose) polymerase-1 (PARP-1). The overexpressed PARP-1 bound to G(-172) --> T and enhanced the transcription of reporter vectors containing G(-172) and T(-172). Furthermore, PARP-1 inhibitor (benzamide) decreased PARP-1 binding to G(-172) --> T without affecting mRNA or protein expression level of PARP-1 and down-regulated the subsequent MOR gene expression in SH-SY5Y cells. Moreover, we found that tumor necrosis factor-alpha enhanced MOR gene expression as well as increased PARP-1 binding to the G(-172) --> T region and G(-172) --> T-dependent transcription in SH-SY5Y cells. These effects were also inhibited by benzamide. In this study, our data suggest that PARP-1 positively regulates MOR gene transcription via G(-172) --> T, which might influence individual specificity in therapeutic opioid effects.

  5. Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells

    PubMed Central

    Torres, Angelo; Vargas, Yosselyn; Uribe, Daniel; Jaramillo, Catherine; Gleisner, Alejandra; Salazar-Onfray, Flavio; López, Mercedes N.; Melo, Rómulo; Oyarzún, Carlos; Martín, Rody San; Quezada, Claudia

    2016-01-01

    MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs. PMID:27634913

  6. Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

    PubMed

    Torres, Angelo; Vargas, Yosselyn; Uribe, Daniel; Jaramillo, Catherine; Gleisner, Alejandra; Salazar-Onfray, Flavio; López, Mercedes N; Melo, Rómulo; Oyarzún, Carlos; San Martín, Rody; Quezada, Claudia

    2016-10-11

    MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.

  7. Choline+ is a low-affinity ligand for alpha 1-adrenoceptors.

    PubMed

    Unelius, L; Cannon, B; Nedergaard, J

    1994-10-07

    The effect of choline+, a commonly used Na+ substitute, on ligand binding to alpha 1-adrenoceptors was investigated. It was found that replacement of 25% of the Na+ in a Krebs-Ringer bicarbonate buffer with choline+ led to a 3-fold decrease in the apparent affinity of [3H]prazosin for its binding site (i.e. the alpha 1-receptor) in a membrane preparation from brown adipose tissue, while no decrease in the total number of binding sites was observed. Similar effects were seen in membrane preparations from liver and brain. In competition experiments, it was found that choline+ could inhibit [3H]prazosin binding; from the inhibition curve, an affinity (Ki) of 31 mM choline+ for the [3H]prazosin-binding site could be calculated. In fully choline(+)-substituted buffers, where the level of [3H]prazosin binding was substantially reduced, both phentolamine and norepinephrine could still compete with [3H]prazosin for its binding site, with virtually unaltered affinity; thus choline+ did not substantially affect the characteristics of those receptors to which it did not bind. Choline+ did not affect the binding characteristics of the beta 1/beta 2 radioligand [3H]CGP-12177; thus, the effect on alpha 1-receptors was not due to general, unspecific effects on the membrane preparations. It is concluded that choline+ possesses characteristics similar to those of a competitive ligand for the alpha 1-adrenoceptor; it has a low affinity but the competitive type of interaction of choline may nonetheless under experimental conditions interfere with agonist interaction with the alpha 1-receptor.

  8. Human neuronal acetylcholine receptor A5-A3-B4 haplotypes are associated with multiple nicotine dependence phenotypes

    PubMed Central

    Weiss, Robert B.; Bolt, Daniel; von Niederhausern, Andrew; Fiore, Michael C.; Dunn, Diane M.; Piper, Megan E.; Matsunami, Nori; Smith, Stevens S.; Coon, Hilary; McMahon, William M.; Scholand, Mary B.; Singh, Nanda; Hoidal, John R.; Kim, Su-Young; Leppert, Mark F.; Cannon, Dale S.

    2009-01-01

    Introduction: Previous research revealed significant associations between haplotypes in the CHRNA5-A3-B4 subunit cluster and scores on the Fagerström Test for Nicotine Dependence among individuals reporting daily smoking by age 17. The present study used subsamples of participants from that study to investigate associations between the CHRNA5-A3-B4 haplotypes and an array of phenotypes not analyzed previously (i.e., withdrawal severity, ability to stop smoking, and specific scales on the Wisconsin Inventory of Smoking Dependence Motives (WISDM-68) that reflect loss of control, strong craving, and heavy smoking. Methods: Two cohorts of current or former smokers (N = 886) provided both self-report data and DNA samples. One sample (Wisconsin) comprised smokers making a quit smoking attempt, which permitted the assessment of withdrawal and relapse during the attempt. The other sample (Utah) comprised participants studied for risk factors for nicotine dependence and chronic obstructive pulmonary disease and included individuals originally recruited in the Lung Health Study. Results: The CHRNA5-A3-B4 haplotypes were significantly associated with the targeted WISDM-68 scales (Tolerance, Craving, Loss of Control) in both samples of participants but only among individuals who began smoking early in life. The haplotypes were significantly associated with relapse likelihood and withdrawal severity, but these associations showed no evidence of an interaction with age at daily smoking. Discussion: The CHRNA5-A3-B4 haplotypes are associated with a broad range of nicotine dependence phenotypes, but these associations are not consistently moderated by age at initial smoking. PMID:19436041

  9. An agonist to the A3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3 beta and NF-kappa B.

    PubMed

    Fishman, Pnina; Bar-Yehuda, Sara; Ohana, Gil; Barer, Faina; Ochaion, Avivit; Erlanger, Abigail; Madi, Lea

    2004-04-01

    A(3) adenosine receptor (A(3)AR) activation with the specific agonist CF101 has been shown to inhibit the development of colon carcinoma growth in syngeneic and xenograft murine models. In the present study, we looked into the effect of CF101 on the molecular mechanisms involved in the inhibition of HCT-116 colon carcinoma in mice. In tumor lesions derived from CF101-treated mice, a decrease in the expression level of protein kinase A (PKA) and an increase in glycogen synthase kinase-3 beta (GSK-3 beta) was observed. This gave rise to downregulation of beta-catenin and its transcriptional gene products cyclin D1 and c-Myc. Further mechanistic studies in vitro revealed that these responses were counteracted by the selective A(3)AR antagonist MRS 1523 and by the GSK-3 beta inhibitors lithium and SB216763, confirming that the observed effects were A(3)AR and GSK-3 beta mediated. CF101 downregulated PKB/Akt expression level, resulting in a decrease in the level and DNA-binding capacity of NF-kappa B, both in vivo and in vitro. Furthermore, the PKA and PKB/Akt inhibitors H89 and Worthmannin mimicked the effect of CF101, supporting their involvement in mediating the response to the agonist. This is the first demonstration that A(3)AR activation induces colon carcinoma growth inhibition via the modulation of the key proteins GSK-3 beta and NF-kappa B.

  10. Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.

    PubMed

    Yoshikawa, Noriko; Yamada, Shizuo; Takeuchi, Chihiro; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru; Nakamura, Kazuki

    2008-06-01

    Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.

  11. Synthesis, molecular modeling, and opioid receptor affinity of 9, 10-diazatricyclo[4.2.1.1(2,5)]decanes and 2,7-diazatricyclo[4.4.0. 0(3,8)]decanes structurally related to 3,8-diazabicyclo[3.2. 1]octanes.

    PubMed

    Vianello, P; Albinati, A; Pinna, G A; Lavecchia, A; Marinelli, L; Borea, P A; Gessi, S; Fadda, P; Tronci, S; Cignarella, G

    2000-06-01

    Various lines of evidence, including molecular modeling studies, imply that the endoethylenic bridge of 3,8-diazabicyclo[3.2. 1]octanes (DBO, 1) plays an essential role in modulating affinity toward mu opioid receptors. This hypothesis, together with the remarkable analgesic properties observed for N(3) propionyl, N(8) arylpropenyl derivatives (2) and of the reverted isomers (3), has prompted us to insert an additional endoethylenic bridge on the piperazine moiety in order to identify derivatives with increased potency toward this receptor class. In the present report, we describe the synthesis of the novel compounds 9,10-diazatricyclo[4.2. 1.1(2,5)]decane (4) and 2,7-diazatricyclo[4.4.0.0(3,8)]decane (5), as well as the representative derivatives functionalized at the two nitrogen atoms by propionyl and arylpropenyl groups (6a-e, 7a-d). Opioid receptor binding assays revealed that, among the compounds tested, the N-propionyl-N-cinnamyl derivatives 6a and 7a exhibited the highest mu-receptor affinity, and remarkably, compound 7a displayed in vivo (mice) an analgesic potency 6-fold that of morphine.

  12. PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL II

    EPA Science Inventory

    The training set used to derive a common reactivity pattern (COREPA) model for estrogen receptor (ER) binding affinity in Model I (see Abstract I in this series) was extended to include 47 rat estrogen receptor (rER) relative binding affinity (RBA) measurements in addition to the...

  13. Translocation of arrestin induced by human A(3) adenosine receptor ligands in an engineered cell line: comparison with G protein-dependent pathways.

    PubMed

    Gao, Zhan-Guo; Jacobson, Kenneth A

    2008-04-01

    Structurally diverse ligands were studied in A(3) adenosine receptor (AR)-mediated beta-arrestin translocation in engineered CHO cells. The agonist potency and efficacy were similar, although not identical, to their G protein signaling. However, differences have also been found. MRS542, MRS1760, and other adenosine derivatives, A(3)AR antagonists in cyclic AMP assays, were partial agonists in beta-arrestin translocation, indicating possible biased agonism. The xanthine 7-riboside DBXRM, a full agonist, was only partially efficacious in beta-arrestin translocation. DBXRM was shown to induce a lesser extent of desensitization compared with IB-MECA. In kinetic studies, MRS3558, a potent and selective A(3)AR agonist, induced beta-arrestin translocation significantly faster than IB-MECA and Cl-IB-MECA. Non-nucleoside antagonists showed similar inhibitory potencies as previously reported. PTX pretreatment completely abolished ERK1/2 activation, but not arrestin translocation. Thus, lead candidates for biased agonists at the A(3)AR have been identified with this arrestin-translocation assay, which promises to be an effective tool for ligand screening.

  14. IgE low affinity receptor (CD23) expression, Plasmodium falciparum specific IgE and tumor necrosis factor-alpha production in Thai uncomplicated and severe falciparum malaria patients.

    PubMed

    Kumsiri, Ratchanok; Troye-Blomberg, Marita; Pattanapanyasat, Kovit; Krudsood, Srivicha; Maneerat, Yaowapa

    2016-02-01

    Previous studies have suggested that Plasmodium falciparum (P. falciparum) specific IgE in the form of immune complexes crosslinking the low-affinity receptor (CD23) on monocyte results in tumor necrosis factor (TNF)-α and nitric oxide (NO) production. However, the roles of these parameters in severity and immune protection are still unclear. This study aimed to determine the association between CD23 expression on monocytes, plasma soluble CD23 (sCD23), total IgE, malaria-specific IgE and IgG, and TNF-α levels in P. falciparum infected patients. We evaluated 64 uncomplicated (UC) and 25 severe patients (S), admitted at the Hospital for Tropical Diseases, Mahidol University, and 34 healthy controls (C) enrolled in 2001. Flow cytometry and enzyme linked immunosorbent assays (ELISA) demonstrated that trends of the CD23 expression, levels of sCD23 and specific IgE were higher in the S group as compared to those in the UC and C groups. Plasma levels of P. falciparum specific IgE in the UC (p=0.011) and S groups (p=0.025) were significantly higher than those in C group. In contrast the TNF-α levels tended to be higher in the UC than those in the S (p=0.343) and significantly higher than those in C (p=0.004) groups. The specific IgG levels in UC were significantly higher than those in S and C (p<0.001) groups. At admission, a strong significant negative correlation was found between specific IgG and sCD23 (r=-0.762, p=0.028), and TNF-α and IgE-IgG complexes (r=-0.715, p=0.002). Significant positive correlations between levels of specific IgE and TNF-α (r=0.575, p=0.010); and sCD23 (r=0.597, p=0.000) were also observed. In conclusion, our data suggest that CD23 expression and malaria-specific IgE levels may be involved in the severity of the disease while TNF-α and the malaria-specific IgG may correlate with protection against falciparum malaria. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Effects of dopamine D2 receptor (DRD2) and transporter (SLC6A3) polymorphisms on smoking cue-induced cigarette craving among African-American smokers.

    PubMed

    Erblich, J; Lerman, C; Self, D W; Diaz, G A; Bovbjerg, D H

    2005-04-01

    Cue-induced craving for addictive substances has long been known to contribute to the problem of persistent addiction in humans. Research in animals over the past decade has solidly established the central role of dopamine in cue-induced craving for addictive substances, including nicotine. Analogous studies in humans, however, are lacking, especially among African-American smokers, who have lower quit rates than Caucasian smokers. Based on the animal literature, the study's objective was to test the hypothesis that smokers carrying specific variants in dopamine-related genes previously associated with risk for addictive behaviors would exhibit heightened levels of cigarette craving following laboratory exposure to cues. To this end, cigarette craving was induced in healthy African-American smokers (n=88) through laboratory exposure to smoking cues. Smokers carrying either the DRD2 (D2 dopamine receptor gene) TaqI A1 RFLP or the SLC6A3 (dopamine transporter gene) 9-repeat VNTR polymorphisms had stronger cue-induced cravings than noncarriers (Ps <0.05 and 0.01, respectively). Consistent with the separate biological pathways involved (receptor, transporter), carriers of both polymorphisms had markedly higher craving responses compared to those with neither (P<0.0006), reflecting additive effects. Findings provide support for the role of dopamine in cue-induced craving in humans, and suggest a possible genetic risk factor for persistent smoking behavior in African-American smokers.

  16. Affine theory of gravitation

    NASA Astrophysics Data System (ADS)

    Popławski, Nikodem

    2014-01-01

    We propose a theory of gravitation, in which the affine connection is the only dynamical variable describing the gravitational field. We construct a simple dynamical Lagrangian density that is entirely composed from the connection, via its curvature and torsion, and is a polynomial function of its derivatives. It is given by the contraction of the Ricci tensor with a tensor which is inverse to the symmetric, contracted square of the torsion tensor, . We vary the total action for the gravitational field and matter with respect to the affine connection, assuming that the matter fields couple to the connection only through . We derive the resulting field equations and show that they are identical with the Einstein equations of general relativity with a nonzero cosmological constant if the tensor is regarded as proportional to the metric tensor. The cosmological constant is simply a constant of proportionality between the two tensors, which together with and provides a natural system of units in gravitational physics. This theory therefore provides a physical construction of the metric as a polynomial function of the connection, and explains dark energy as an intrinsic property of spacetime.

  17. Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

    PubMed

    Gysler, Stefan M; Mulla, Melissa J; Guerra, Marta; Brosens, Jan J; Salmon, Jane E; Chamley, Lawrence W; Abrahams, Vikki M

    2016-07-01

    What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was

  18. High oxygen affinity hemoglobins.

    PubMed

    Mangin, O

    2017-02-01

    High oxygen affinity hemoglobins are responsible for rare and heterogeneous autosomic dominant genetic diseases. They cause pure erythrocytosis, sometimes accountable for hyperviscosity and thrombosis, or hemolysis. Differential diagnoses must be first ruled out. The diagnosis is based on the identification of a decreased P50, and their possible characterization by cation exchange-high performance liquid chromatography and capillary electrophoresis. Finally, genetic studies of the responsible globin chain gene will confirm the mutation. The prognosis mainly relies on the P50 decrease rate and on the hemoglobin cooperativity impairment. Disease management should be personalized, and it should primarily depend on smoking cessation and physical activity. Phlebotomy and platelet aggregation inhibitors' prescriptions can be discussed. There is no contraindication to flights, high-altitude conditions, or pregnancy. Nevertheless, blood donation must be prohibited. Copyright © 2016 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier SAS. All rights reserved.

  19. Adjoint affine fusion and tadpoles

    SciTech Connect

    Urichuk, Andrew, E-mail: andrew.urichuk@uleth.ca; Walton, Mark A., E-mail: walton@uleth.ca; International School for Advanced Studies

    2016-06-15

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are writtenmore » for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.« less

  20. Substitution of lysine-181 to aspartic acid in the third transmembrane region of the endothelin (ET) type B receptor selectively reduces its high-affinity binding with ET-3 peptide.

    PubMed

    Mauzy, C; Wu, L H; Egloff, A M; Mirzadegan, T; Chung, F Z

    1992-01-01

    In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.

  1. Activation of adenosine A(3) receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice.

    PubMed

    Hofer, Michal; Pospísil, Milan; Sefc, Ludek; Dusek, Ladislav; Vacek, Antonín; Holá, Jirina; Hoferová, Zuzana; Streitová, Denisa

    2010-08-01

    Research areas of 'post-exposure treatment' and 'cytokines and growth factors' have top priority among studies aimed at radiological nuclear threat countermeasures. The experiments were aimed at testing the ability of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A(3) receptor agonist, to modulate hematopoiesis in sublethally irradiated mice, when administered alone or in a combination with granulocyte colony-stimulating factor (G-CSF) in a two-day post-irradiation treatment regimen. A complete analysis of hematopoiesis including determination of numbers of bone marrow hematopoietic progenitor and precursor cells, as well as of numbers of peripheral blood cells, was performed. The outcomes of the treatment were assessed at days 3 to 22 after irradiation. IB-MECA alone has been found to induce a significant elevation of numbers of bone marrow granulocyte-macrophage progenitor cells (GM-CFC) and peripheral blood neutrophils. IB-MECA given concomitantly with G-CSF increased significantly bone marrow GM-CFC and erythroid progenitor cells (BFU-E) in comparison with the controls and with animals administered each of the drugs alone. The findings suggest the ability of IB-MECA to stimulate hematopoiesis and to support the hematopoiesis-stimulating effects of G-CSF in sublethally irradiated mice.

  2. Fundamentals of affinity cell separations.

    PubMed

    Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

    2018-03-01

    Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: A COMPARISON OF THREE MODELS

    EPA Science Inventory

    A comparative analysis of how three COREPA models for ER binding affinity performed when used to predict potential estrogen receptor (ER) ligands is presented. Models I and II were developed based on training sets of 232 and 279 rat ER binding affinity measurements, respectively....

  4. PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL I

    EPA Science Inventory

    A Common Reactivity Pattern (COREPA) model, based on consideration of multiple energetically reasonable conformations of flexible chemicals was developed using a training set of 232 rat estrogen receptor (rER) relative binding affinity (RBA) measurements. The training set include...

  5. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

    PubMed Central

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

  6. Lp-dual affine surface area

    NASA Astrophysics Data System (ADS)

    Wei, Wang; Binwu, He

    2008-12-01

    According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

  7. Probes for Narcotic Receptor Mediated Phenomena. 37.1 Synthesis and Opioid Binding Affinity of the Final Pair of Oxide-Bridged Phenylmorphans, the ortho- and para-b Isomers and Their N-Phenethyl Analogues, and the Synthesis of the N-Phenethyl Analogues of the ortho- and para-d Isomers

    PubMed Central

    Kurimura, Muneaki; Liu, Hehua; Sulima, Agnieszka; Hashimoto, Akihiro; Przybyl, Anna K.; Ohshima, Etsuo; Kodato, Shinichi; Deschamps, Jeffrey R.; Dersch, Christina M.; Rothman, Richard B.; Lee, Yong Sok; Jacobson, Arthur E.; Rice, Kenner C.

    2008-01-01

    In the isomeric series of 12 racemic topologically rigid N-methyl analogues of oxide-bridged phenylmorphans, all but two of the racemates, the ortho- and para-b-oxide-bridged phenylmorphansa 20 and 12, have remained to be synthesized. The b-isomers were very difficult to synthesize because of the highly strained 5,6-trans-fused ring junction that had to be formed. Our successful strategy required functionalization of the position para (or ortho) to a fluorine atom on the aromatic ring using an electron-withdrawing nitro group to activate that fluorine. The racemic N-phenethyl analogues 24 and 16 were moderately potent κ-receptor antagonists in the [35S]GTPγS assay. We synthesized the N-phenethyl-substituted oxide-bridged phenylmorphans in the ortho- and para-d oxide-bridged phenylmorphana series (51 and 52) which had not been previously evaluated using contemporary receptor binding assays to see whether they also have higher affinity for opioid receptors than their N-methyl relatives 46 and 47. PMID:19053757

  8. 2017 Guralp Affinity Digitizer Evaluation.

    SciTech Connect

    Merchant, Bion J.

    Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

  9. The molecular mechanism of flop-selectivity and subsite recognition for an AMPA receptor allosteric modulator: Structures of GluA2 and GluA3 complexed with PEPA

    PubMed Central

    Ahmed, Ahmed H.; Ptak, Christopher P.; Oswald, Robert E.

    2011-01-01

    Glutamate receptors are important potential drug targets for cognitive enhancement and the treatment of schizophrenia in part because they are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. One approach to the application of therapeutic agents to the AMPA subtype of glutamate receptors is the use of allosteric modulators, which promote dimerization by binding to a dimer interface thereby reducing desensitization and deactivation. AMPA receptors exist in two alternatively spliced variants (flip and flop) that differ in desensitization and receptor activation profiles. Most of the structural information on modulators of the AMPA receptor target the flip subtype. We report here the crystal structure of the flop-selective allosteric modulator, PEPA, bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors. Specific hydrogen bonding patterns can explain the preference for the flop isoform. This includes a bidentate hydrogen bonding pattern between PEPA and N754 of the flop isoforms of GluA2 and GluA3 (the corresponding position in the flip isoform is S754). Comparison with other allosteric modulators provides a framework for the development of new allosteric modulators with preferences for either the flip or flop isoforms. In addition to interactions with N/S754, specific interactions of the sulfonamide with conserved residues in the binding site are characteristics of a number of allosteric modulators. These, in combination, with variable interactions with five subsites on the binding surface lead to different stoichiometries, orientations within the binding pockets, and functional outcomes. PMID:20199107

  10. Aberrant antibody affinity selection in SHIP-deficient B cells.

    PubMed

    Leung, Wai-Hang; Tarasenko, Tatiana; Biesova, Zuzana; Kole, Hemanta; Walsh, Elizabeth R; Bolland, Silvia

    2013-02-01

    The strength of the Ag receptor signal influences development and negative selection of B cells, and it might also affect B-cell survival and selection in the GC. Here, we have used mice with B-cell-specific deletion of the 5'-inositol phosphatase SHIP as a model to study affinity selection in cells that are hyperresponsive to Ag and cytokine receptor stimulation. In the absence of SHIP, B cells have lower thresholds for Ag- and interferon (IFN)-induced activation, resulting in augmented negative selection in the BM and enhanced B-cell maturation in the periphery. Despite a tendency to spontaneously downregulate surface IgM expression, SHIP deficiency does not alter anergy induction in response to soluble hen-egg lysozyme Ag in the MDA4 transgenic model. SHIP-deficient B cells spontaneously produce isotype-switched antibodies; however, they are poor responders in immunization and infection models. While SHIP-deficient B cells form GCs and undergo mutation, they are not properly selected for high-affinity antibodies. These results illustrate the importance of negative regulation of B-cell responses, as lower thresholds for B-cell activation promote survival of low affinity and deleterious receptors to the detriment of optimal Ab affinity maturation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

    PubMed Central

    Liñán-Rico, A.; Wunderlich, JE.; Enneking, JT.; Tso, DR.; Grants, I.; Williams, KC.; Otey, A.; Michel, K.; Schemann, M.; Needleman, B.; Harzman, A.; Christofi, FL.

    2015-01-01

    Rationale The role of purinergic signaling in the human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. Experimental Approach LSCM-Fluo-4-(Ca2+)-imaging of postsynaptic Ca2+ transients (PSCaTs) was used as a reporter of neural activity. Synaptic transmission was evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons from 234 separate ganglia 107 patients; immunochemical labeling for P2XRs of neurons in ganglia from 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging was used to test effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Results Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines. Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50=25μM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50=111nM) in neurons without stimulatory ADPβS responses (EC50=960nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca2+ transients or Ca2+ oscillations (EC50=400μM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20nM) or high (5μM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-ir follow the order P2X2>P2X1≫P2X3; P2X1+ P2X2 and P2X3+P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7,P2Y1,2,12-14R. Responsive neurons were also identified by HuC/D-ir. Conclusions Purines are critical regulators of neurotransmission in the human enteric nervous system. Purinergic signaling involves

  12. Vaccine Efficacy and Affinity Maturation

    NASA Astrophysics Data System (ADS)

    Lee, Hayoun; Deem, Michael W.

    2002-03-01

    We propose macroscopic equations to describe variable vaccine efficacy between repeated vaccinee and first time vaccinee. The main ingredients are antigenic distance between epidemic strain and vaccne strain, and affinity maturation dynamics which differs in primary and second response. Increase of affinity by repeated vaccine leads to localization in immune space. This localization decreases the ability of the immune system to response to distant, but related epidemic strains.

  13. RECEPTOR AFFINITY AND PHOSPHODIESTERASES 4B AND 10A ACTIVITY OF OCTAHYDRO- AND 6,7-DIMETHOXY-3,4-DIHYDRO- ISOQUINOLIN-2(1H)-YL-ALKYL DERIVATIVES OF IMIDAZO- AND PYRIMIDINO[2,1-f]PURINES.

    PubMed

    Zagórska, Agnieszka; Gryzło, Beata; Satała, Grzegorz; Bojarski, Andrzej J; Głuch-Lutwin, Monika; Mordyl, Barbara; Kazek, Grzegorz; Pawłowski, Maciej

    2016-01-01

    A series of octahydro- and 6,7-dimethoxy-3,4-dihydro- isoquinolin-2(1H)-yl-alkyl derivatives of imidazo- and pyrimidino[2,1-f]purines were synthesized and biologically evaluated in in vitro competition binding experiments for serotonin 5-HT(1A), 5-HT(6), 5-HT(7), and dopamine D2 receptors and inhibitory potencies for phosphodiesterases - PDE4B1 and PDE10A. The structure-activity relationships allowed to determine the structural features responsible for receptor and enzyme activity. Compound 5 (8-(4-(6,7-dimethoxy-3,4-dihydroiso- quinolin-2(1H)butyl)1,3-dimethyl-H-imidazo[2,1-f]purine-2,4(3H,8H)-dione) could be regarded as promising structure for further modification and detailed mechanistic study for obtained hybrid ligands.

  14. Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

    PubMed

    Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

    1994-10-20

    By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy

  15. Dye-ligand affinity systems.

    PubMed

    Denizli, A; Pişkin, E

    2001-10-30

    Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

  16. Alternative Affinity Ligands for Immunoglobulins.

    PubMed

    Kruljec, Nika; Bratkovič, Tomaž

    2017-08-16

    The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

  17. Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

    NASA Astrophysics Data System (ADS)

    Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

    2008-05-01

    Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

  18. Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

    PubMed

    Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

    1994-09-01

    Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

  19. High affinity dopamine D2 receptor radioligands. 3. [[sup 123]I] and [[sup 125]I]epidepride: In vivo studies in rhesus monkey brain and comparison with in vitro pharmacokinetics in rat brain

    SciTech Connect

    Kessler, R.M.; Votaw, J.R.; Schmidt, D.E.

    1993-01-01

    Studies of [[sup 123]I]epidepride uptake in rhesus monkey brain were performed using single photon tomography. Striatal uptake peaked at 0.85% of administered dose/g at 107 min post-injection, then declined slowly to 0.70% of administered dose/g at 6 h. Striatal:posterior brain ratios rose from 2 at 25 min to 6.8 at 105 min, to 15 at 4 h and to 58 at 6.4 h. [[sup 123]I]Epidepride was displaced by haloperidol (0.1 and 1 mg/kg) with a half-life of washout of 55 min. Little displacement of [[sup 123]I]epidepride was observed following administration of 1 or 2 mg/kg d-amphetamine, respectively, indicating [[sup 123]I]epidepridemore » is not easily displaced by endogenous dopamine. In vitro equilibrium binding studies with [[sup 125]I]epidepride using rat striatum revealed a K[sub D] of 46 pM and B[sub max] of 33 pmol/g tissue at 37[degrees]C, while at 25[degrees]C the K[sub D] was 25 pM and the B[sub max] 32 pmol/g tissue. In vitro kinetic analysis of association and dissociation curves revealed a half-life for receptor dissociation at 37[degrees]C of 15 min and 79--90 min at 25[degrees]C. Allowing for the temperature difference, there is good correspondence between in vivo and in vitro dissociation kinetics at 25[degrees]C. Increasing in vitro incubation temperature from 25 to 37[degrees]C caused a 6-fold increase in the dissociation rate, suggesting that there is a change in binding kinetics at the dopamine D2 receptor at 37[degrees]C compared to in vivo binding. The results of this study indicate that [[sup 123]I]epidepride is an excellent radioligand for SPECT studies of the dopamine D2 receptor in man. 34 refs., 4 figs.« less

  20. First-In-Human Study Demonstrating Tumor-Angiogenesis by PET/CT Imaging with 68Ga-NODAGA-THERANOST, a High-Affinity Peptidomimetic for αvβ3 Integrin Receptor Targeting

    PubMed Central

    Baum, Richard P.; Kulkarni, Harshad R.; Müller, Dirk; Danthi, Narasimhan; Kim, Young-Seung; Brechbiel, Martin W.

    2015-01-01

    Abstract 68Ga-NODAGA-THERANOST™ is an αvβ3 integrin antagonist and the first radiolabeled peptidomimetic to reach clinical development for targeting integrin receptors. In this first-in-human study, the feasibility of integrin receptor peptidomimetic positron emission tomography/computed tomography (PET/CT) imaging was confirmed in patients with non-small-cell lung cancer and breast cancer. Methods: Patients underwent PET/CT imaging with 68Ga NODAGA-THERANOST. PET images were analyzed qualitatively and quantitatively and compared to 2-deoxy-2-(18F) fluoro-d-glucose (18F-FDG) findings. Images were obtained 60 minutes postinjection of 300–500 MBq of 68Ga-NODAGA-THERANOST. Results: 68Ga-NODAGA-THERANOST revealed high tumor-to-background ratios (SUVmax=4.8) and uptake at neoangiogenesis sites. Reconstructed fused images distinguished cancers with high malignancy potential and enabled enhanced bone metastasis detection. 18F-FDG-positive lung and lymph node metastases did not show uptake, indicating the absence of neovascularization. Conclusions: 68Ga-NODAGA-THERANOST was found to be safe and effective, exhibiting in this study rapid blood clearance, stability, rapid renal excretion, favorable biodistribution and PK/PD, low irradiation burden (μSv/MBq/μg), and convenient radiolabeling. This radioligand might enable theranostics, that is, a combination of diagnostics followed by the appropriate therapeutics, namely antiangiogenic therapy, image-guided presurgical assessment, treatment response evaluation, prediction of pathologic response, neoadjuvant-peptidomimetic-radiochemotherapy, and personalized medicine strategies. Further clinical trials evaluating 68Ga-NODAGA-THERANOST are warranted. PMID:25945808

  1. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  2. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  3. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  4. The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse*

    PubMed Central

    Sullivan, Chelsea S.; Kümper, Maike; Temple, Brenda S.; Maness, Patricia F.

    2016-01-01

    Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo. PMID:27803162

  5. Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

    PubMed Central

    Azimi, Mohammad; Mofrad, Mohammad R. K.

    2013-01-01

    Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

  6. Modification of the Fc Region of a Human Anti-oncostatin M Monoclonal Antibody for Higher Affinity to FcRn Receptor and Extension of Half-life in Cynomolgus Monkeys.

    PubMed

    Nnane, Ivo P; Han, Chao; Jiao, Qun; Tam, Susan H; Davis, Hugh M; Xu, Zhenhua

    2017-07-01

    The purpose of this study was to evaluate the pharmacokinetics (PK) of anti-oncostatin M (OSM) IgG1 monoclonal antibodies, CNTO 1119 and its Fc variant (CNTO 8212), which incorporates the LS(Xtend) mutation to extend terminal half-life (T 1/2 ), after a single intravenous (IV) or subcutaneous (SC) administration in cynomolgus monkeys, and to predict human PK. In study 1, single doses of CNTO 1119 and CNTO 8212 were administered IV or SC at 3 mg/kg to cynomolgus monkeys (n = 3 per group). In study 2, single doses of CNTO 8212 were administered IV at 1, 5 or 20 mg/kg, or SC at 5 mg/kg to cynomolgus monkeys (n = 5 per group). Serial blood samples were collected for assessment of serum concentrations of CNTO 1119 and/or CNTO 8212. A two-compartment population PK model with first-order elimination was utilized to simultaneously describe the serum concentrations of CNTO 1119 and CNTO 8212 over time after IV and SC administration in cynomolgus monkeys. The typical population PK parameter estimates for CNTO 1119 in cynomolgus monkeys were clearance (CL) = 2.81 mL/day/kg, volume of distribution of central compartment (V 1 ) = 31.3 mL/kg, volume of distribution of peripheral compartment (V 2 ) = 23.3 mL/kg, absolute bioavailability (F) = 0.84 and T 1/2 = 13.4 days. In comparison, the typical population PK parameter estimates for CNTO 8212 in cynomolgus monkeys were CL = 1.41 mL/day/kg, V 1 = 39.8 mL/kg, V 2 = 32.6 mL/kg, F = 0.75 and T 1/2 = 35.7 days. The mean CL of CNTO 8212 was ~50% lower compared with that for CNTO 1119 in cynomolgus monkeys. The overall volume of distribution (V 1 +V 2 ) for CNTO 8212 was about 32% larger compared with that for CNTO 1119, but generally similar to the vascular volume in cynomolgus monkeys. The T 1/2 of CNTO 8212 was significantly (p < 0.05) longer by about 2.7-fold than that for CNTO 1119 in cynomolgus monkeys. Thus, the modification of the Fc portion of an anti-OSM IgG1 mAb for higher FcRn binding affinity resulted in lower systemic

  7. The Importance of the Presence of a 5′-Ribonucleotide and the Contribution of the T1R1 + T1R3 Heterodimer and an Additional Low-Affinity Receptor in the Taste Detection of l-Glutamate as Assessed Psychophysically

    PubMed Central

    Smith, Kimberly R.

    2014-01-01

    The molecular receptors underlying the purported “umami” taste quality commonly associated with l-glutamate have been controversial. Evidence supports the involvement of the T1R1 + T1R3 heterodimer, a GPCR broadly tuned to l-amino acids, but variants of two mGluRs expressed in taste buds have also been implicated. Using a rigorous psychophysical taste-testing paradigm, we demonstrated impaired, if not eliminated, detection of MSG in WT and T1R1, T1R2, T1R3, and T1R2 + T1R3 KO mice when the contribution of sodium was minimized by the epithelial sodium channel blocker amiloride. When inosine 5′-monophosphate (IMP), a ribonucleotide that potentiates the l-glutamate signal through the T1R1 + T1R3 heterodimer, was added, the WT and T1R2 KO mice were able to detect the compound stimulus across all MSG (+amiloride) concentrations due, in part, to the taste of IMP. In contrast, mice lacking T1R1 or T1R3 could not detect IMP alone, yet some were able to detect MSG + amiloride + IMP, but only at the higher MSG concentrations. Interestingly, the sensitivity of T1R1 KO mice to another l-amino acid, lysine, was unimpaired, suggesting that some l-amino acids can be detected through T1R1 + T1R3-independent receptors without sensitivity loss. Given that IMP is not thought to affect mGluRs, behavioral detection of l-glutamate appears to require the contribution of the T1R1 + T1R3 receptor. However, the partial competence observed in some T1R1 and T1R3 KO mice when MSG + amiloride + IMP was tested suggests that a T1R1 or T1R3 homodimer or an unidentified protein, perhaps in conjunction with T1R1 or T1R3, can serve as a low-affinity taste receptor for l-glutamate in the presence of IMP. PMID:25253867

  8. The importance of the presence of a 5'-ribonucleotide and the contribution of the T1R1 + T1R3 heterodimer and an additional low-affinity receptor in the taste detection of L-glutamate as assessed psychophysically.

    PubMed

    Smith, Kimberly R; Spector, Alan C

    2014-09-24

    The molecular receptors underlying the purported "umami" taste quality commonly associated with l-glutamate have been controversial. Evidence supports the involvement of the T1R1 + T1R3 heterodimer, a GPCR broadly tuned to l-amino acids, but variants of two mGluRs expressed in taste buds have also been implicated. Using a rigorous psychophysical taste-testing paradigm, we demonstrated impaired, if not eliminated, detection of MSG in WT and T1R1, T1R2, T1R3, and T1R2 + T1R3 KO mice when the contribution of sodium was minimized by the epithelial sodium channel blocker amiloride. When inosine 5'-monophosphate (IMP), a ribonucleotide that potentiates the l-glutamate signal through the T1R1 + T1R3 heterodimer, was added, the WT and T1R2 KO mice were able to detect the compound stimulus across all MSG (+amiloride) concentrations due, in part, to the taste of IMP. In contrast, mice lacking T1R1 or T1R3 could not detect IMP alone, yet some were able to detect MSG + amiloride + IMP, but only at the higher MSG concentrations. Interestingly, the sensitivity of T1R1 KO mice to another l-amino acid, lysine, was unimpaired, suggesting that some l-amino acids can be detected through T1R1 + T1R3-independent receptors without sensitivity loss. Given that IMP is not thought to affect mGluRs, behavioral detection of l-glutamate appears to require the contribution of the T1R1 + T1R3 receptor. However, the partial competence observed in some T1R1 and T1R3 KO mice when MSG + amiloride + IMP was tested suggests that a T1R1 or T1R3 homodimer or an unidentified protein, perhaps in conjunction with T1R1 or T1R3, can serve as a low-affinity taste receptor for l-glutamate in the presence of IMP. Copyright © 2014 the authors 0270-6474/14/3413234-12$15.00/0.

  9. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

    PubMed

    Rubio, María E; Matsui, Ko; Fukazawa, Yugo; Kamasawa, Naomi; Harada, Harumi; Itakura, Makoto; Molnár, Elek; Abe, Manabu; Sakimura, Kenji; Shigemoto, Ryuichi

    2017-11-01

    The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

  10. Affine invariants of convex polygons.

    PubMed

    Flusser, Jan

    2002-01-01

    In this correspondence, we prove that the affine invariants, for image registration and object recognition, proposed recently by Yang and Cohen (see ibid., vol.8, no.7, p.934-46, July 1999) are algebraically dependent. We show how to select an independent and complete set of the invariants. The use of this new set leads to a significant reduction of the computing complexity without decreasing the discrimination power.

  11. Electron affinity of liquid water

    DOE PAGES

    Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; ...

    2018-01-16

    Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

  12. Electron affinity of liquid water

    SciTech Connect

    Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco

    Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

  13. Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the {beta} subunit of the high-affinity IgE receptor: Evidence for a family of proteins with four membrane-spanning regions

    SciTech Connect

    Adra, C.N.; Morrison, P.; Lim, B.

    1994-10-11

    The authors report the cloning of the cDNA for a human gene whose mRNA is expressed specifically in hematopoietic cells. A long open reading frame in the 1.7-kb mRNA encodes a 214-aa protein of 25 kDa with four hydrophobic regions consistent with a protein that traverses the membrane four times. To reflect the structure and expression of this gene in diverse hematopoietic lineages of lymphoid and myeloid origin, the authors named the gene HTm{sub 4}. The protein is about 20% homologous to two other {open_quotes}four-transmembrane{close_quotes} proteins; the B-cell-specific antigen CD20 and the {beta} subunit of the high-affinity receptor for IgE,more » Fc{sub {epsilon}}RI{beta}. The highest homologies among the three proteins are found in the transmembrane domains, but conserved residues are also recognized in the inter-transmembrane domains and in the N and C termini. Using fluorescence in situ hybridization, they localized HTm{sub 4} to human chromosome 11q12-13.1, where the CD20 and Fc{sub {epsilon}}RI{beta} genes are also located. Both the murine homologue for CD20, Ly-44, and the murine Fc{sub {epsilon}}RI{beta} gene map to the same region in murine chromosome 19. The authors propose that the HTm{sub 4}, CD20, and Fc{sub {epsilon}}RI{beta} genes evolved from the same ancestral gene to form a family of four-transmembrane proteins. It is possible that other related members exist. Similar to CD20 and Fc{sub {epsilon}}RI{beta}, it is likely that Htm{sub 4} has a role in signal transduction and, like Fc{sub {epsilon}}RI{beta}, might be a subunit associated with receptor complexes.« less

  14. Memantine, a Low-Affinity NMDA Receptor Antagonist, Protects against Methylmercury-Induced Cytotoxicity of Rat Primary Cultured Cortical Neurons, Involvement of Ca2+ Dyshomeostasis Antagonism, and Indirect Antioxidation Effects.

    PubMed

    Liu, Wei; Xu, Zhaofa; Yang, Tianyao; Xu, Bin; Deng, Yu; Feng, Shu

    2017-09-01

    Methylmercury (MeHg) is an extremely dangerous environmental pollutant that induces severe toxic effects in the central nervous system. Neuronal damage plays critical roles mediating MeHg-induced loss of brain function and neurotoxicity. The molecular mechanisms of MeHg neurotoxicity are incompletely understood. The objective of the study is to explore mechanisms that contribute to MeHg-induced neurocyte injuries focusing on neuronal Ca 2+ dyshomeostasis and alteration of N-methyl-D-aspartate receptors (NMDARs) expression, as well as oxidative stress in primary cultured cortical neurons. In addition, the neuroprotective effects of memantine against MeHg cytotoxicity were also investigated. The cortical neurons were exposed to 0, 0.01, 0.1, 1, or 2 μM methylmercury chloride (MeHgCl) for 0.5-12 h, or pre-treated with 2.5, 5, 10, or 20 μM memantine for 0.5-6 h, respectively; cell viability and LDH release were then quantified. For further experiments, 2.5, 5, and 10 μM of memantine pre-treatment for 3 h followed by 1 μM MeHgCl for 6 h were performed for evaluation of neuronal injuries, specifically addressing apoptosis; intracellular free Ca 2+ concentrations; ATPase activities; calpain activities; expressions of NMDAR subunits (NR1, NR2A, NR2B); NPSH levels; and ROS formation. Exposure of MeHgCl resulted in toxicity of cortical neurons, which were shown as a loss of cell viability, high levels of LDH release, morphological changes, and cell apoptosis. Moreover, intracellular Ca 2+ dyshomeostasis, ATPase activities inhibition, calpain activities, and NMDARs expression alteration were observed with 1 μM MeHgCl administration. Last but not least, NPSH depletion and reactive oxygen species (ROS) overproduction showed an obvious oxidative stress in neurons. However, memantine pre-treatment dose-dependently antagonized MeHg-induced neuronal toxic effects, apoptosis, Ca 2+ dyshomeostasis, NMDARs expression alteration, and oxidative stress. In conclusion, the

  15. Lp-mixed affine surface area

    NASA Astrophysics Data System (ADS)

    Wang, Weidong; Leng, Gangsong

    2007-11-01

    According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

  16. Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

    PubMed

    Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S

    2016-10-01

    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. © 2016 Society for Laboratory Automation and Screening.

  17. Effect of rs6923761 gene variant of glucagon-like peptide 1 receptor on metabolic response and weight loss after a 3-month intervention with a hypocaloric diet.

    PubMed

    de Luis, Daniel Antonio; Aller, Rocío; Izaola, Olatz; Lopez, J J; Gomez, E; Torres, B; Soto, G Diaz

    2014-10-01

    Studies of the GLP-1 receptor (GLP-1 R) have been directed at identifying polymorphisms in the GLP-1 R gene that may be a contributing factor in the pathogenesis of obesity and cardiovascular risk factors. Nevertheless, the role of GLP-1 R variants on body weight response after dietary intervention has not been evaluated. We decided to analyze the effects of the rs6923761 GLP-1 R polymorphism on body weight changes and metabolic parameters after 3 months of a hypocaloric diet. A sample of 91 obese subjects was analyzed in a prospective way. The hypocaloric diet had 1,520 calories per day; 52 % of carbohydrates, 25 % of lipids and 23 % of proteins. Distribution of fats was: 50.7 % of monounsaturated fats, 38.5 % of saturated fats and 11.8 % of polyunsaturated fats. In both genotype groups (GG vs. GA + AA), weight, body mass index, fat mass, waist circumference, systolic blood pressure, total cholesterol, LDL cholesterol, leptin, insulin and HOMA levels decreased. No statistical differences were detected in these changes between genotypes. In wild group (GG genotype) (pretreatment and posttreatment), BMI, weight, fat mass, waist circumference and triglyceride levels were higher than (GA + AA) group. Our data showed better anthropometric parameters and triglyceride levels in obese subjects with the mutant allele (A) of rs6923761 GLP-1R polymorphism. A lack of association of this polymorphism with weight loss or biochemical changes after a hypocaloric diet was observed.

  18. MicroRNA-210 Modulates Endothelial Cell Response to Hypoxia and Inhibits the Receptor Tyrosine Kinase Ligand Ephrin-A3*S⃞

    PubMed Central

    Fasanaro, Pasquale; D'Alessandra, Yuri; Di Stefano, Valeria; Melchionna, Roberta; Romani, Sveva; Pompilio, Giulio; Capogrossi, Maurizio C.; Martelli, Fabio

    2008-01-01

    MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation. PMID:18417479

  19. Affinity Spaces and 21st Century Learning

    ERIC Educational Resources Information Center

    Gee, James Paul

    2017-01-01

    This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

  20. Exploring new scaffolds for angiotensin II receptor antagonism.

    PubMed

    Kritsi, Eftichia; Matsoukas, Minos-Timotheos; Potamitis, Constantinos; Karageorgos, Vlasios; Detsi, Anastasia; Magafa, Vasilliki; Liapakis, George; Mavromoustakos, Thomas; Zoumpoulakis, Panagiotis

    2016-09-15

    Nowadays, AT1 receptor (AT1R) antagonists (ARBs) constitute the one of the most prevalent classes of antihypertensive drugs that modulate the renin-angiotensin system (RAS). Their main uses include also treatment of diabetic nephropathy (kidney damage due to diabetes) and congestive heart failure. Towards this direction, our study has been focused on the discovery of novel agents bearing different scaffolds which may evolve as a new class of AT1 receptor antagonists. To fulfill this aim, a combination of computational approaches and biological assays were implemented. Particularly, a pharmacophore model was established and served as a 3D search query to screen the ChEMBL15 database. The reliability and accuracy of virtual screening results were improved by using molecular docking studies. In total, 4 compounds with completely diverse chemical scaffolds from potential ARBs, were picked and tested for their binding affinity to AT1 receptor. Results revealed high nanomolar to micromolar affinity (IC50) for all the compounds. Especially, compound 4 exhibited a binding affinity of 199nM. Molecular dynamics simulations were utilized in an effort to provide a molecular basis of their binding to AT1R in accordance to their biological activities. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    NASA Astrophysics Data System (ADS)

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-06-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

  2. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

    PubMed

    Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

    2010-02-05

    Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

  4. Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

    PubMed

    Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

    2014-02-01

    Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  5. Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

    PubMed

    Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

    1999-12-01

    Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

  6. Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

    PubMed Central

    Zhao, Ming; Wan, Zhu-li; Whittaker, Linda; Xu, Bin; Phillips, Nelson B.; Katsoyannis, Panayotis G.; Ismail-Beigi, Faramarz; Whittaker, Jonathan; Weiss, Michael A.

    2009-01-01

    Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition α-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of GlyA1 by d-Ala or d-Leu, and (ii) substitution of ThrA8 by diaminobutyric acid (Dab). The crystal structure of [d-AlaA1,DabA8]insulin, as determined within a T6 zinc hexamer to a resolution of 1.35 Å, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer. PMID:19773552

  7. Prediction of kinase-inhibitor binding affinity using energetic parameters

    PubMed Central

    Usha, Singaravelu; Selvaraj, Samuel

    2016-01-01

    The combination of physicochemical properties and energetic parameters derived from protein-ligand complexes play a vital role in determining the biological activity of a molecule. In the present work, protein-ligand interaction energy along with logP values was used to predict the experimental log (IC50) values of 25 different kinase-inhibitors using multiple regressions which gave a correlation coefficient of 0.93. The regression equation obtained was tested on 93 kinase-inhibitor complexes and an average deviation of 0.92 from the experimental log IC50 values was shown. The same set of descriptors was used to predict binding affinities for a test set of five individual kinase families, with correlation values > 0.9. We show that the protein-ligand interaction energies and partition coefficient values form the major deterministic factors for binding affinity of the ligand for its receptor. PMID:28149052

  8. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  9. Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

    PubMed

    Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

    2006-07-01

    High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

  10. Multi-exemplar affinity propagation.

    PubMed

    Wang, Chang-Dong; Lai, Jian-Huang; Suen, Ching Y; Zhu, Jun-Yong

    2013-09-01

    The affinity propagation (AP) clustering algorithm has received much attention in the past few years. AP is appealing because it is efficient, insensitive to initialization, and it produces clusters at a lower error rate than other exemplar-based methods. However, its single-exemplar model becomes inadequate when applied to model multisubclasses in some situations such as scene analysis and character recognition. To remedy this deficiency, we have extended the single-exemplar model to a multi-exemplar one to create a new multi-exemplar affinity propagation (MEAP) algorithm. This new model automatically determines the number of exemplars in each cluster associated with a super exemplar to approximate the subclasses in the category. Solving the model is NP-hard and we tackle it with the max-sum belief propagation to produce neighborhood maximum clusters, with no need to specify beforehand the number of clusters, multi-exemplars, and superexemplars. Also, utilizing the sparsity in the data, we are able to reduce substantially the computational time and storage. Experimental studies have shown MEAP's significant improvements over other algorithms on unsupervised image categorization and the clustering of handwritten digits.

  11. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

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

    EPA Pesticide Factsheets

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

  13. CGRP Receptor Biology: Is There More Than One Receptor?

    PubMed

    Hay, Debbie L

    2018-05-25

    Calcitonin gene-related peptide (CGRP) has many reported pharmacological actions. Can a single receptor explain all of these? This chapter outlines the molecular nature of reported CGRP binding proteins and their pharmacology. Consideration of whether CGRP has only one or has more receptors is important because of the key role that this peptide plays in migraine. It is widely thought that the calcitonin receptor-like receptor together with receptor activity-modifying protein 1 (RAMP1) is the only relevant receptor for CGRP. However, some closely related receptors also have high affinity for CGRP and it is still plausible that these play a role in CGRP biology, and in migraine. The calcitonin receptor/RAMP1 complex, which is currently called the AMY 1 receptor, seems to be the most likely candidate but more investigation is needed to determine its role.

  14. Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor.

    PubMed

    Armour, S L; Foord, S; Kenakin, T; Chen, W J

    1999-12-01

    Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.

  15. Proton affinities of hydrated molecules

    NASA Astrophysics Data System (ADS)

    Valadbeigi, Younes

    2016-09-01

    Proton affinities (PA) of non-hydrated, M, and hydrated forms, M(H2O)1,2,3, of 20 organic molecules including alcohols, ethers, aldehydes, ketones and amines were calculated by the B3LYP/6-311++G(d,p) method. For homogeneous families, linear correlations were observed between PAs of the M(H2O)1,2,3 and the PAs of the non-hydrated molecules. Also, the absolute values of the hydration enthalpies of the protonated molecules decreased linearly with the PAs. The correlation functions predicted that for an amine with PA < 1100 kJ/mol the PA(M(H2O)) is larger than the corresponding PA, while for an amine with PA > 1100 kJ/mol the PA(M(H2O)) is smaller than the PA.

  16. Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy.

    PubMed

    Raman, Marine C C; Rizkallah, Pierre J; Simmons, Ruth; Donnellan, Zoe; Dukes, Joseph; Bossi, Giovanna; Le Provost, Gabrielle S; Todorov, Penio; Baston, Emma; Hickman, Emma; Mahon, Tara; Hassan, Namir; Vuidepot, Annelise; Sami, Malkit; Cole, David K; Jakobsen, Bent K

    2016-01-13

    Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.

  17. Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy

    PubMed Central

    Raman, Marine C C; Rizkallah, Pierre J; Simmons, Ruth; Donnellan, Zoe; Dukes, Joseph; Bossi, Giovanna; Le Provost, Gabrielle S; Todorov, Penio; Baston, Emma; Hickman, Emma; Mahon, Tara; Hassan, Namir; Vuidepot, Annelise; Sami, Malkit; Cole, David K; Jakobsen, Bent K.

    2016-01-01

    Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics. PMID:26758806

  18. Antisymmetric tensor generalizations of affine vector fields.

    PubMed

    Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-02-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

  19. Dopamine receptor D2 (DRD2), dopamine transporter solute carrier family C6, member 4 (SLC6A3), and catechol-O-methyltransferase (COMT) genes as moderators of the relation between maternal history of maltreatment and infant emotion regulation.

    PubMed

    Villani, Vanessa; Ludmer, Jaclyn; Gonzalez, Andrea; Levitan, Robert; Kennedy, James; Masellis, Mario; Basile, Vincenzo S; Wekerle, Christine; Atkinson, Leslie

    2018-05-01

    Although infants less than 18 months old are capable of engaging in self-regulatory behavior (e.g., avoidance, withdrawal, and orienting to other aspects of their environment), the use of self-regulatory strategies at this age (as opposed to relying on caregivers) is associated with elevated behavioral and physiological distress. This study investigated infant dopamine-related genotypes (dopamine receptor D2 [DRD2], dopamine transporter solute carrier family C6, member 4 [SLC6A3], and catechol-O-methyltransferase [COMT]) as they interact with maternal self-reported history of maltreatment to predict observed infant independent emotion regulation behavior. A community sample (N = 193) of mother-infant dyads participated in a toy frustration challenge at infant age 15 months, and infant emotion regulation behavior was coded. Buccal cells were collected for genotyping. Maternal maltreatment history significantly interacted with infant SLC6A3 and COMT genotypes, such that infants with more 10-repeat and valine alleles of SLC6A3 and COMT, respectively, relative to infants with fewer or no 10-repeat and valine alleles, utilized more independent (i.e., maladaptive) regulatory behavior if mother reported a more extensive maltreatment history, as opposed to less. The findings indicate that child genetic factors moderate the intergenerational impact of maternal maltreatment history. The results are discussed in terms of potential mechanism of Gene × Environment interaction.

  20. Personality Types and Affinity for Computers

    DTIC Science & Technology

    1991-03-01

    differences on personality dimensions between the respondents, and to explore the relationship between these differences and computer affinity. The results...between the respondents, and to explore the relationship between these differences and computer affinity. The results revealed no significant differences...type to this measure of computer affinity. 2 II. LITERATURZ REVIEW The interest of this study was the relationship between a person’s psychological

  1. Selection of imprinted nanoparticles by affinity chromatography.

    PubMed

    Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

    2009-04-15

    Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

  2. Ecdysteroid receptors in Drosophila melanogaster adult females

    USDA-ARS?s Scientific Manuscript database

    Ecdysteroid receptors were identified and partially characterized from total cell extracts of whole animals and dissected tissues from Drosophila melanogaster adult females. Binding studies indicated the presence of two ecdysteroid binding components having high affinity and specificity consistent w...

  3. Non-affine deformations in polymer hydrogels

    PubMed Central

    Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

    2012-01-01

    Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

  4. Concepts in receptor optimization: targeting the RGD peptide.

    PubMed

    Chen, Wei; Chang, Chia-en; Gilson, Michael K

    2006-04-12

    Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

  5. Affinity chemiresistor sensor for sugars.

    PubMed

    Tlili, Chaker; Badhulika, Sushmee; Tran, Thien-Toan; Lee, Ilkeun; Mulchandani, Ashok

    2014-10-01

    In this work, a non-enzymatic chemiresistive sugar sensor has been developed by combining a synthetic receptor with aligned single-walled carbon nanotubes (SWNTs) device. Briefly, boronic acid as a multivalent sugar receptor was immobilized on carbon nanotubes through amide bond formation. The interaction between three common sugars (d-glucose, d-fructose and sucrose) and boronic acid modified SWNTs device was studied. The effect of pH on the receptor-ligand binding was examined and highest response was observed at pH 9. The chemiresistive sensor exhibited specific and reproducible detection with sensitivity over the concentration range of 1-20mM, 1-25 mM, and 1-30 mM for fructose, glucose, and sucrose, respectively. The sensor showed no interference from common electroactive compounds such as citric acid, uric acid, and ascorbic acid. Furthermore, the sensor retained 97.4% of the initial value after five regeneration cycles with an acidic buffer at pH 5, thus ensuring good reusability. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    PubMed

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

  7. EFFECTS OF CYTOSOLIC CONVERSION OF ESTRONE TO ESTRADIOL ON RAINBOW TROUT ER BINDING AFFINITY

    EPA Science Inventory

    Relative binding affinity (RBA) for estrone (E1) to the rainbow trout (Oncorhynchus mykiss) estrogen receptor (rtER) was measured as part of a larger effort to determine chemical structural features predictive of chemical estrogenicity in fish. Estrone RBA was found to vary consi...

  8. Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

    PubMed

    Fonseca, L P; Cabral, J M

    1996-01-01

    Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

  9. Electron affinity and excited states of methylglyoxal

    NASA Astrophysics Data System (ADS)

    Dauletyarov, Yerbolat; Dixon, Andrew R.; Wallace, Adam A.; Sanov, Andrei

    2017-07-01

    Using photoelectron imaging spectroscopy, we characterized the anion of methylglyoxal (X2A″ electronic state) and three lowest electronic states of the neutral methylglyoxal molecule: the closed-shell singlet ground state (X1A'), the lowest triplet state (a3A″), and the open-shell singlet state (A1A″). The adiabatic electron affinity (EA) of the ground state, EA(X1A') = 0.87(1) eV, spectroscopically determined for the first time, compares to 1.10(2) eV for unsubstituted glyoxal. The EAs (adiabatic attachment energies) of two excited states of methylglyoxal were also determined: EA(a3A″) = 3.27(2) eV and EA(A1A″) = 3.614(9) eV. The photodetachment of the anion to each of these two states produces the neutral species near the respective structural equilibria; hence, the a3A″ ← X2A″ and A1A″ ← X2A″ photodetachment transitions are dominated by intense peaks at their respective origins. The lowest-energy photodetachment transition, on the other hand, involves significant geometry relaxation in the X1A' state, which corresponds to a 60° internal rotation of the methyl group, compared to the anion structure. Accordingly, the X1A' ← X2A″ transition is characterized as a broad, congested band, whose vertical detachment energy, VDE = 1.20(4) eV, significantly exceeds the adiabatic EA. The experimental results are in excellent agreement with the ab initio predictions using several equation-of-motion methodologies, combined with coupled-cluster theory.

  10. KB004, a first in class monoclonal antibody targeting the receptor tyrosine kinase EphA3, in patients with advanced hematologic malignancies: Results from a phase 1 study.

    PubMed

    Swords, Ronan T; Greenberg, Peter L; Wei, Andrew H; Durrant, Simon; Advani, Anjali S; Hertzberg, Mark S; Jonas, Brian A; Lewis, Ian D; Rivera, Gabriel; Gratzinger, Dita; Fan, Alice C; Felsher, Dean W; Cortes, Jorge E; Watts, Justin M; Yarranton, Geoff T; Walling, Jackie M; Lancet, Jeffrey E

    2016-11-01

    EphA3 is an Ephrin receptor tyrosine kinase that is overexpressed in most hematologic malignancies. We performed a first-in-human multicenter phase I study of the anti-EphA3 monoclonal antibody KB004 in refractory hematologic malignancies in order to determine safety and tolerability, along with the secondary objectives of pharmacokinetics (PK) and pharmacodynamics (PD) assessments, as well as preliminary assessment of efficacy. Patients were enrolled on a dose escalation phase (DEP) initially, followed by a cohort expansion phase (CEP). KB004 was administered by intravenous infusion on days 1, 8, and 15 of each 21-day cycle in escalating doses. A total of 50 patients (AML 39, MDS/MPN 3, MDS 4, DLBCL 1, MF 3) received KB004 in the DEP; an additional 14 patients were treated on the CEP (AML 8, MDS 6). The most common toxicities were transient grade 1 and grade 2 infusion reactions (IRs) in 79% of patients. IRs were dose limiting above 250mg. Sustained exposure exceeding the predicted effective concentration (1ug/mL) and covering the 7-day interval between doses was achieved above 190mg. Responses were observed in patients with AML, MF, MDS/MPN and MDS. In this study, KB004 was well tolerated and clinically active when given as a weekly infusion. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-10-08

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

  13. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  14. Mobile Technology Affinity in Renal Transplant Recipients.

    PubMed

    Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

    Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

    PubMed

    Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

    1992-05-01

    Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

  16. In search of selective P2 receptor ligands: interaction of dihydropyridine derivatives at recombinant rat P2X(2) receptors.

    PubMed

    Jacobson, K A; Kim, Y C; King, B F

    2000-07-03

    1,4-Dihydropyridines are regarded as privileged structures for drug design, i.e. they tend to bind to a wide variety of receptor sites. We have shown that upon appropriate manipulation of the substituent groups on a 1,4-dihydropyridine template, high affinity and selectivity for the A(3) subtype of adenosine receptors ('P1 receptors') may be attained. In the present study we have begun to extend this approach to P2 receptors which are activated by ATP and other nucleotides. Nicardipine, a representative dihydropyridine, used otherwise as an L-type calcium channel blocker, was shown to be an antagonist at recombinant rat P2X(2) (IC(50)=25 microM) and P2X(4) (IC(50) approximately 220 microM) receptors expressed in Xenopus oocytes. Thus, this class of compounds represents a suitable lead for enhancement of affinity through chemical synthesis. In an attempt to modify the 1,4-dihydropyridine structure with a predicted P2 receptor recognition moiety, we have replaced one of the ester groups with a negatively charged phosphonate group. Several 4-phenyl-5-phosphonato-1,4-dihydropyridine derivatives, MRS 2154 (2, 6-dimethyl), MRS 2155 (6-methyl-2-phenyl), and MRS 2156 (2-methyl-6-phenyl), were synthesized through three component condensation reactions. These derivatives were not pure antagonists of the effects of ATP at P2X(2) receptors, rather were either inactive (MRS 2156) or potentiated the effects of ATP in a concentration-dependent manner (MRS 2154 in the 0.3-10 microM range and MRS 2155 at >1 microM). Antagonism of the effects of ATP at P2X(2) receptor superimposed on the potentiation was also observed at >10 microM (MRS 2154) or 0.3-1 microM (MRS 2155). Thus, while a conventional dihydropyridine, nicardipine, was found to antagonize rat P2X(2) receptors ninefold more potently than P2X(4) receptors, the effects of novel, anionic 5-phosphonate analogues at the receptor were more complex.

  17. Affinity of antigen encounter and other early B-cell signals determine B-cell fate

    PubMed Central

    Benson, Micah J; Erickson, Loren D; Gleeson, Michael W; Noelle, Randolph J

    2010-01-01

    Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway. PMID:17433651

  18. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  19. Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning*

    PubMed Central

    van Rosmalen, Martijn; Janssen, Brian M. G.; Hendrikse, Natalie M.; van der Linden, Ardjan J.; Pieters, Pascal A.; Wanders, Dave; de Greef, Tom F. A.; Merkx, Maarten

    2017-01-01

    Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nm. Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies. PMID:27974464

  20. Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel

    PubMed Central

    Dainiak, Maria B.; Kumar, Ashok; Galaev, Igor Yu.; Mattiasson, Bo

    2006-01-01

    Adsorption of bioparticles to affinity surfaces involves polyvalent interactions, complicating greatly the recovery of the adsorbed material. A unique system for the efficient binding and release of different cells and particles is described. Affinity-bound bioparticles and synthetic particles are detached from the macroporous hydrogel matrix, a so-called cryogel, when the cryogel undergoes elastic deformation. The particle detachment upon elastic deformation is believed to be due to breaking of many of the multipoint attachments between the particles and the affinity matrix and the change in the distance between affinity ligands when the matrix is deformed. However, no release of affinity-bound protein occurred upon elastic deformation. The phenomenon of particle detachment upon elastic deformation is believed to be of a generic nature, because it was demonstrated for a variety of bioparticles of different sizes and for synthetic particles, for different ligand–receptor pairs (IgG–protein A, sugar–ConA, metal ion–chelating ligand), and when the deformation was caused by either external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive, macroporous hydrogel upon an increase in temperature). The elasticity of cryogel monoliths ensures high recovery of captured cells under mild conditions, with highly retained viability. This property, along with their continuous porous structure makes cryogel monoliths very attractive for applications in affinity cell separation. PMID:16418282

  1. Motion planning in velocity affine mechanical systems

    NASA Astrophysics Data System (ADS)

    Jakubiak, Janusz; Tchoń, Krzysztof; Magiera, Władysław

    2010-09-01

    We address the motion planning problem in specific mechanical systems whose linear and angular velocities depend affinely on control. The configuration space of these systems encompasses the rotation group, and the motion planning involves the system orientation. Derivation of the motion planning algorithm for velocity affine systems has been inspired by the continuation method. Performance of this algorithm is illustrated with examples of the kinematics of a serial nonholonomic manipulator, the plate-ball kinematics and the attitude control of a rigid body.

  2. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  3. 5-Functionalized indazoles as glucocorticoid receptor agonists.

    PubMed

    Bai, Mei; Carr, Grant; Deorazio, Russell J; Friedrich, Thomas D; Dobritsa, Svetlana; Fitzpatrick, Kevin; Guzzo, Peter R; Kitchen, Douglas B; Lynch, Michael A; Peace, Denise; Sajad, Mohammed; Usyatinsky, Alexander; Wolf, Mark A

    2010-05-15

    An indazole based series of glucocorticoid receptor agonists is reported. The SAR exploration of this scaffold yielded compounds with nanomolar affinity for the glucocorticoid receptor with indications of selectivity for the preferred transrepression mechanism; in vivo efficacy was observed in the mouse LPS induced TNFalpha model for compound 28. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Adrenergic receptors in frontal cortex in human brain.

    PubMed

    Cash, R; Raisman, R; Ruberg, M; Agid, Y

    1985-02-05

    The binding of three adrenergic ligands ([3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol) was studied in the frontal cortex of human brain. alpha 1-Receptors, labeled by [3H]prazosin, predominated. [3H]Clonidine bound to two classes of sites, one of high affinity and one of low affinity. Guanosine triphosphate appeared to lower the affinity of [3H]clonidine for its receptor. [3H]Dihydroalprenolol bound to three classes of sites: the beta 1-receptor, the beta 2-receptor and a receptor with low affinity which represented about 40% of the total binding, but which was probably a non-specific site; the beta 1/beta 2 ratio was 1/2.

  5. Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

    PubMed Central

    Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

    2016-01-01

    Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents. PMID:27094554

  6. Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

    NASA Astrophysics Data System (ADS)

    Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

    2016-04-01

    Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1-3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

  7. Cross species comparisons of chemical interaction with recombinant steroid receptors in vitro.

    EPA Science Inventory

    Typically, mammalian receptors are used for in vitro hazard identification and screening for endocrine disrupting compounds. There is concern, however, that differences may exist in the affinities of environmental compounds for steroid receptors from other vertebrate classes. S...

  8. An affine model of the dynamics of astrophysical discs

    NASA Astrophysics Data System (ADS)

    Ogilvie, Gordon I.

    2018-06-01

    Thin astrophysical discs are very often modelled using the equations of 2D hydrodynamics. We derive an extension of this model that describes more accurately the behaviour of a thin disc in the absence of self-gravity, magnetic fields, and complex internal motions. The ideal fluid theory is derived directly from Hamilton's Principle for a 3D fluid after making a specific approximation to the deformation gradient tensor. We express the equations in Eulerian form after projection on to a reference plane. The disc is thought of as a set of fluid columns, each of which is capable of a time-dependent affine transformation, consisting of a translation together with a linear transformation in three dimensions. Therefore, in addition to the usual 2D hydrodynamics in the reference plane, the theory allows for a deformation of the mid-plane (as occurs in warped discs) and for the internal shearing motions that accompany such deformations. It also allows for the vertical expansions driven in non-circular discs by a variation of the vertical gravitational field around the horizontal streamlines, or by a divergence of the horizontal velocity. The equations of the affine model embody conservation laws for energy and potential vorticity, even for non-planar discs. We verify that they reproduce exactly the linear theories of 3D warped and eccentric discs in a secular approximation. However, the affine model does not rely on any secular or small-amplitude assumptions and should be useful in more general circumstances.

  9. Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

    PubMed Central

    Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

    2016-01-01

    The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

  10. Serum Leukocyte Immunoglobulin-Like Receptor A3 (LILRA3) Is Increased in Patients with Multiple Sclerosis and Is a Strong Independent Indicator of Disease Severity; 6.7kbp LILRA3 Gene Deletion Is Not Associated with Diseases Susceptibility.

    PubMed

    An, Hongyan; Lim, Chai; Guillemin, Gilles J; Vollmer-Conna, Ute; Rawlinson, William; Bryant, Katherine; Tedla, Nicodemus

    2016-01-01

    Leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble immune regulatory molecule primarily expressed by monocytes and macrophages. A homozygous 6.7kbp LILRA3 gene deletion that removes the first seven of its eight exons is predicted to lead to lack of LILRA3 protein, although this has not been experimentally confirmed. Moreover, there are conflicting results with regards to the link between the LILRA3 homozygous genetic deletion and susceptibility to multiple sclerosis (MS) in different European populations. The aim of this study was to investigate whether LILRA3 gene deletion is associated with MS susceptibility in a North American cohort of European ancestry and assess if serum LILRA3 protein level is a marker of clinical subtype and/or disease severity in MS. A total of 456 patients with MS and 99 unrelated healthy controls were genotyped for the 6.7kbp LILRA3 gene deletion and levels of LILRA3 protein in sera determined by in-house sandwich ELISA. We showed that LILRA3 gene deletion was not associated with MS susceptibility and did not affect the age of disease onset, clinical subtype or disease severity. However, we discovered for the first time that homozygous LILRA3 gene deletion results in lack of production of LILRA3 protein. Importantly, LILRA3 protein level was significantly increased in sera of patients with MS when compared with control subjects, particularly in more severe type primary progressive MS. Multiple regression analysis showed that LILRA3 level in serum was one of the strongest independent markers of disease severity in MS, which potentially can be used as a diagnostic marker.

  11. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    NASA Astrophysics Data System (ADS)

    Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

  12. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    PubMed

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  13. Affinity of C-Reactive Protein toward FcγRI Is Strongly Enhanced by the γ-Chain

    PubMed Central

    Röcker, Carlheinz; Manolov, Dimitar E.; Kuzmenkina, Elza V.; Tron, Kyrylo; Slatosch, Holger; Torzewski, Jan; Nienhaus, G. Ulrich

    2007-01-01

    C-reactive protein (CRP), the prototype human acute phase protein, is widely regarded as a key player in cardiovascular disease, but the identity of its cellular receptor is still under debate. By using ultrasensitive confocal imaging analysis, we have studied CRP binding to transfected COS-7 cells expressing the high-affinity IgG receptor FcγRI. Here we show that CRP binds to FcγRI on intact cells, with a kd of 10 ± 3 μmol/L. Transfection of COS-7 cells with a plasmid coding for both FcγRI and its functional counterpart, the γ-chain, markedly increases CRP affinity to FcγRI, resulting in a kd of 0.35 ± 0.10 μmol/L. The affinity increase results from an ∼30-fold enhanced association rate coefficient. The pronounced enhancement of affinity by the γ-chain suggests its crucial involvement in the CRP receptor interaction, possibly by mediating interactions between the transmembrane moieties of the receptors. Dissociation of CRP from the cell surfaces cannot be detected throughout the time course of several hours and is thus extremely slow. Considering the pentameric structure of CRP, this result indicates that multivalent binding and receptor clustering are crucially involved in the interaction of CRP with nucleated cells. PMID:17255341

  14. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  15. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

  16. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Identification of 2-arylbenzimidazoles as potent human histamine H4 receptor ligands.

    PubMed

    Lee-Dutra, Alice; Arienti, Kristen L; Buzard, Daniel J; Hack, Michael D; Khatuya, Haripada; Desai, Pragnya J; Nguyen, Steven; Thurmond, Robin L; Karlsson, Lars; Edwards, James P; Breitenbucher, J Guy

    2006-12-01

    A series of 2-arylbenzimidazoles was synthesized and found to bind with high affinity to the human histamine H(4) receptor. Structure-activity relationships were investigated through library preparation and evaluation as well as traditional medicinal chemistry approaches, leading to the discovery of compounds with single-digit nanomolar affinity for the H(4) receptor.

  18. A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165)

    NASA Astrophysics Data System (ADS)

    Koide, Hiroyuki; Yoshimatsu, Keiichi; Hoshino, Yu; Lee, Shih-Hui; Okajima, Ai; Ariizumi, Saki; Narita, Yudai; Yonamine, Yusuke; Weisman, Adam C.; Nishimura, Yuri; Oku, Naoto; Miura, Yoshiko; Shea, Kenneth J.

    2017-07-01

    Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

  19. A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering.

    PubMed

    Kruziki, Max A; Bhatnagar, Sumit; Woldring, Daniel R; Duong, Vandon T; Hackel, Benjamin J

    2015-07-23

    Small protein ligands can provide superior physiological distribution compared with antibodies, and improved stability, production, and specific conjugation. Systematic evaluation of the PDB identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α helix opposite a β sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 10(8) mutants and directed evolution toward four targets yielded target-specific binders with affinities as strong as 200 ± 100 pM, Tms from 65 °C ± 3 °C to 80°C ± 1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ± 8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Solubilization and purification of melatonin receptors from lizard brain

    SciTech Connect

    Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

    Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

  1. Solubilization and purification of melatonin receptors from lizard brain.

    PubMed

    Rivkees, S A; Conron, R W; Reppert, S M

    1990-09-01

    Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

  2. Asymptotic Representations of Quantum Affine Superalgebras

    NASA Astrophysics Data System (ADS)

    Zhang, Huafeng

    2017-08-01

    We study representations of the quantum affine superalgebra associated with a general linear Lie superalgebra. In the spirit of Hernandez-Jimbo, we construct inductive systems of Kirillov-Reshetikhin modules based on a cyclicity result that we established previously on tensor products of these modules, and realize their inductive limits as modules over its Borel subalgebra, the so-called q-Yangian. A new generic asymptotic limit of the same inductive systems is proposed, resulting in modules over the full quantum affine superalgebra. We derive generalized Baxter's relations in the sense of Frenkel-Hernandez for representations of the full quantum group.

  3. Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

    PubMed

    Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

    2017-11-10

    Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Optimal affinity ranking for automated virtual screening validated in prospective D3R grand challenges

    NASA Astrophysics Data System (ADS)

    Wingert, Bentley M.; Oerlemans, Rick; Camacho, Carlos J.

    2018-01-01

    The goal of virtual screening is to generate a substantially reduced and enriched subset of compounds from a large virtual chemistry space. Critical in these efforts are methods to properly rank the binding affinity of compounds. Prospective evaluations of ranking strategies in the D3R grand challenges show that for targets with deep pockets the best correlations (Spearman ρ 0.5) were obtained by our submissions that docked compounds to the holo-receptors with the most chemically similar ligand. On the other hand, for targets with open pockets using multiple receptor structures is not a good strategy. Instead, docking to a single optimal receptor led to the best correlations (Spearman ρ 0.5), and overall performs better than any other method. Yet, choosing a suboptimal receptor for crossdocking can significantly undermine the affinity rankings. Our submissions that evaluated the free energy of congeneric compounds were also among the best in the community experiment. Error bars of around 1 kcal/mol are still too large to significantly improve the overall rankings. Collectively, our top of the line predictions show that automated virtual screening with rigid receptors perform better than flexible docking and other more complex methods.

  5. High affinity binding of 125I-neurotensin to dispersed cells from chicken liver and brain.

    PubMed

    Mitra, S P; Carraway, R E

    1997-01-01

    Dispersed cells from chicken brain and liver were found to possess cell surface binding sites for 125I-neurotensin (125I-NT). Scatchard analyses indicated the presence of high affinity (K4, 25-80 pM) and low affinity (Kd, 250-450 pM) components in adult tissues. Binding capacity was reduced 25-40% by incubation with pertussis toxin. Ontogenetic studies indicated that NT receptor capacity increased approximately 20-fold from the embryonic stage to adult. Cross-linking of 125I-NT to intact cells labeled one major band (52 kDa, > or = 90%) and two minor bands (40 and 90 kDa, < or = 10%) which could represent distinct NT-receptors or one receptor partly degraded or cross-linked to G-protein(s). The binding of 125I-NT to dispersed cells was enhanced by reduction with dithoithreitol and suppressed by alkylation with N-ethyl-maleimide (NEM), maleimidocaproic acid (MCA) and p-chloromercuribenzenesulfonate (PCMBS). Since MCA and PCMBS do not permeate cells, this suggests that the sulfhydryl group(s) critical to binding are located within the NT receptor itself. Preincubation of cells with NT prior to treatment with NEM diminished its inhibitory effect, suggesting that the critical SH-group(s) were within the NT binding pocket or were protected by an allosteric effect. These results suggest that one or more of the nine cysteine residues in the NT receptor is involved in the NT binding reaction.

  6. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  7. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Compound immobilization and drug-affinity chromatography.

    PubMed

    Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

    2012-01-01

    Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

  9. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  10. EGF receptor ligands: recent advances.

    PubMed

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

  11. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  12. Insulin receptors

    SciTech Connect

    Kahn, C.R.; Harrison, L.C.

    1988-01-01

    This book contains the procedure in insulin receptors. Part B: Clinical assessment, biological responses, and comparison to the IGF-1 receptor. Topics covered include: Insulin and IGF-1 receptors, Clinical assessment of receptor functions, and Biological responses.

  13. Evidence of paired M2 muscarinic receptors

    SciTech Connect

    Potter, L.T.; Ballesteros, L.A.; Bichajian, L.H.

    Binding assays involving various antagonists, including N-(3H) methylscopolamine, (3H)quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat brainstem (medulla, pons, and colliculi). However, competition curves between N-(3H)methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization withmore » digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of (3H)-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with (3H)quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.« less

  14. Negative Cooperativity in the EGF Receptor

    PubMed Central

    Pike, Linda J.

    2012-01-01

    Scatchard analyses of the binding of EGF to its receptor yield concave up Scatchard plots, indicative of some type of heterogenity in ligand binding affinity. This was typically interpreted as being due to the presence of two independent binding site–one of high affinity representing ≤10% of the receptor population and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGF receptor that show symmetric binding of the two ligands. A new approach to the analysis of 125I-EGF binding data combined with the structure of the singly-occupied Drosophila EGF receptor have now shown that this heterogeneity is due to the presence of negative cooperativity in the EGF receptor. Concerns that negative cooperativity precludes ligand-induced dimerization of the EGF receptor confuse the concepts of linkage cooperativity. Linkage refers to the effect of ligand on the assembly of dimers while cooperativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly but shows negative cooperativity within the dimer. PMID:22260659

  15. Docking model of the nicotinic acetylcholine receptor and nitromethylene neonicotinoid derivatives with a longer chiral substituent and their biological activities.

    PubMed

    Nagaoka, Hikaru; Nishiwaki, Hisashi; Kubo, Takuya; Akamatsu, Miki; Yamauchi, Satoshi; Shuto, Yoshihiro

    2015-02-15

    In the present study, nitromethylene neonicotinoid derivatives possessing substituents that contain a sulfur atom, oxygen atom or aromatic ring at position 5 on the imidazolidine ring were synthesized to evaluate their affinity for the nicotinic acetylcholine receptor (nAChR) and their insecticidal activity against adult female houseflies. Comparing the receptor affinity of the alkylated derivative with the receptor affinity of compounds possessing either ether or thioether groups revealed that conversion of the carbon atom to a sulfur atom did not influence the receptor affinity, whereas conversion to an oxygen atom was disadvantageous for the receptor affinity. The receptor affinity of compounds possessing a benzyl or phenyl group was lower than that of the unsubstituted compound. Analysis of the three-dimensional quantitative structure-activity relationship using comparative molecular field analysis demonstrated that steric hindrance of the receptor should exist around the C3 of an n-butyl group attached at position 5 on the imidazolidine ring. A docking study of the nAChR-ligand model suggested that the ligand-binding region expands as the length of the substituent increases by brushing against the amino acids that form the binding region. The insecticidal activity of the compounds was positively correlated with the receptor affinity by considering logP and the number of heteroatoms, including sulfur and oxygen atoms, in the substituents, suggesting that the insecticidal activity is influenced by the receptor affinity, hydrophobicity, and metabolic stability of the compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    1984-10-30

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

  17. 5D-QSAR for spirocyclic sigma1 receptor ligands by Quasar receptor surface modeling.

    PubMed

    Oberdorf, Christoph; Schmidt, Thomas J; Wünsch, Bernhard

    2010-07-01

    Based on a contiguous and structurally as well as biologically diverse set of 87 sigma(1) ligands, a 5D-QSAR study was conducted in which a quasi-atomistic receptor surface modeling approach (program package Quasar) was applied. The superposition of the ligands was performed with the tool Pharmacophore Elucidation (MOE-package), which takes all conformations of the ligands into account. This procedure led to four pharmacophoric structural elements with aromatic, hydrophobic, cationic and H-bond acceptor properties. Using the aligned structures a 3D-model of the ligand binding site of the sigma(1) receptor was obtained, whose general features are in good agreement with previous assumptions on the receptor structure, but revealed some novel insights since it represents the receptor surface in more detail. Thus, e.g., our model indicates the presence of an H-bond acceptor moiety in the binding site as counterpart to the ligands' cationic ammonium center, rather than a negatively charged carboxylate group. The presented QSAR model is statistically valid and represents the biological data of all tested compounds, including a test set of 21 ligands not used in the modeling process, with very good to excellent accuracy [q(2) (training set, n=66; leave 1/3 out) = 0.84, p(2) (test set, n=21)=0.64]. Moreover, the binding affinities of 13 further spirocyclic sigma(1) ligands were predicted with reasonable accuracy (mean deviation in pK(i) approximately 0.8). Thus, in addition to novel insights into the requirements for binding of spirocyclic piperidines to the sigma(1) receptor, the presented model can be used successfully in the rational design of new sigma(1) ligands. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

  18. Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

    PubMed

    Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

    2017-03-01

    Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

  19. Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

    PubMed

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

    1999-10-22

    Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

  20. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  1. Artificial Affinity Proteins as Ligands of Immunoglobulins

    PubMed Central

    Mouratou, Barbara; Béhar, Ghislaine; Pecorari, Frédéric

    2015-01-01

    A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized. PMID:25647098