Antagonist Affinity Research Articles

SB-616234-A (1-[6-( cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2′methyl-4′-(5-methyl-1,2,3-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): A novel, potent and selective 5-HT 1B receptor antagonist

SB-616234-A possesses high affinity for human 5-HT 1B receptors stably expressed in Chinese hamster ovary (CHO) cells (p K i 8.3 ± 0.2), and is over 100-fold selective for a range of molecular targets except h5-HT 1D receptors (p K i 6.6 ± 0.1). Similarly, affinity (p K i) for rat and guinea pig striatal 5-HT 1B receptors is 9.2 ± 0.1. In [ 35S]-GTPγS binding studies in the human recombinant cell line, SB-616234-A acted as a high affinity antagonist with a pA 2 value of 8.6 ± 0.2 whilst providing no evidence of agonist activity in this system. In [ 35S]-GTPγS binding studies in rat striatal membranes, SB-616234-A acted as a high affinity antagonist with an apparent p K B of 8.4 ± 0.5, again whilst providing no evidence of agonist activity in this system. SB-616234-A (1 μM) potentiated electrically stimulated [ 3H]-5-HT release from guinea pig and rat cortical slices (S 2/S 1 ratios of 1.8 and 1.6, respectively). SB-616234-A (0.3–30 mg kg −1 p.o.) caused a dose-dependent inhibition of ex vivo [ 3H]-GR125743 binding to rat striatal 5-HT 1B receptors with an ED 50 of 2.83 ± 0.39 mg kg −1 p.o. Taken together these data suggest that SB-616234-A is a potent and selective 5-HT 1B autoreceptor antagonist that occupies central 5-HT 1B receptors in vivo following oral administration.

Increased Potency of Muscarinic Agonists at Muscarinic M3 Receptor Following Co‐expression with b2 Adrenoceptor in CHO‐K1 Cells

Both muscarinic (M2 and M3 subtypes) and b2 adrenoceptors (b2 AR) are known to co-localize and interact in many contractile tissues. We investigated the signaling properties of CHO cells co-expressing the human M3 receptor and b2AR. These cells (M3/b2) exhibited significantly increased (20–30 fold) potency in [Ca2+]i release for various muscarinic agonists; EC50 (nM)- 0.8, 0.6, 4.8, 134, 0.2 and 4.4 nM for carbachol, oxotremorine, pilocarpine, McN-A-343, 5-mehtylfurmethiodide, and aceclidine) compared with control cells expressing a comparable level of M3 receptor alone. However, M3 receptor binding affinity ([3H]NMS) for both agonists and antagonists was similar between M3/b2 and M3 clones. In addition, in cells co-expressing the human M2 subtype with b2 adrenoceptors, muscarinic agonist activity was unaltered when tested in cAMP inhibition assays, suggesting that the change of agonist potency in the presence of b2 adrenoceptors is more likely M3 specific. Furthermore, this enhanced agonist potency at M3 receptors was not affected by pretreatment with either the b2AR antagonist alprenolol (10 mM) or agonist (1 mM isoproterenol). Our in vitro findings of enhanced muscarinic agonist potency in M3/b2 receptor co-expressed cells compared with cells expressing the M3 receptor alone recapitulate published findings in transgenic mice over-expressing b2 adrenoceptors in airway smooth muscle [McGraw et al. 2003], where the constrictor response to muscarinic receptors was enhanced due to an increase in PLCb activity and expression by increased b2 adrenoceptor expression. These results suggest that the expression level of the b2 adrenoceptor in a cell, rather than its stimulation by agonist, is a requisite for enhancement of M3 receptor agonist potency.

Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

Pharmacological Characterization of Novel High Affinity CGRP Receptor Antagonists that are Selective for Human CGRP Receptors

Human-α-calcitonin gene-related peptide (CGRP) is a 37 amino acid residue peptide produced from alternative splicing of the calcitonin gene. CGRP mediates vascular relaxation and other effects through CGRP receptors, which activate adenylyl cyclase. We found that benzoylation of the N-terminus and benzylation of the His10 side-chain of the antagonist CGRP(8-37) produced a high affinity antagonist with selectivity for human CGRP receptors. CGRP(8-37) was synthesized on a p-MBHA resin by rapid assembly methods using Boc-chemistry and in situ neutralization solid phase peptide synthesis. His10 benzylation was accomplished by reaction with benzyl bromide and N-terminal acylation was accomplished by reaction with benzoic anhydride. The peptide was cleaved form the resin using TFMSA, purified by reversed-phase HPLC to > 98% and characterized by amino acid analysis and mass spectrometry. CGRP(8-37), N-benzoyl-CGRP(8-37) and N-benzoyl-(His10-benzyl)-CGRP(8-37) produced concentration dependent inhibition of CGRP-mediated vasodilation of mouse thoracic aortas studied in vitro, with KB values of 1000.3, 88.9 and 22.9 nM, respectively. These antagonists also produced concentration dependent inhibition of CGRP-mediated cAMP production in human SK-MC cells with KB values of 797.0, 15.4 and 0.65 nM, respectively. N-terminus benzoylation combined with His10 benzylation significantly increased the affinity and selectivity for human SK-N-MC cells. Thus, this antagonist inhibited CGRP-mediated responses in vitro and may be a useful tool to investigate CGRP-mediated functional responses in human tissue.

Synthesis, antioxidant activity and structure–activity relationships for a new series of 2‐(N‐acylaminoethyl)indoles with melatonin‐like cytoprotective activity

5-Methoxy-2-(N-acetylaminoethyl)indole (5d), a melatonin analogue derived from the transposition of the acetylaminoethyl side chain from C3 to C2 of the indole nucleus, had been previously characterized as a low affinity antagonist at MT1 and MT2 membrane receptors; this molecule is endowed with good in vitro antioxidant and cytoprotective potency in rat cerebellar cell cultures, comparable to or better than those of melatonin. In order to further investigate the role of structure-antioxidant activity relationships in cytoprotection, the structure of 5d was systematically modulated to design a new series of compounds. The 5-methoxy group was replaced by substituents with different electronic and lipophilic properties and it was moved to a different position on the indole ring. Other modifications of the lead structure involved the methylation of the indole nitrogen or its replacement by a sulfur atom. The side chain was also modified either increasing its lipophilicity or introducing an ionisable acid group. The antioxidant activity of this set of compounds was evaluated by the ABTS and conjugated dienes (CD) assays, while their cytoprotection was evaluated against kainate-induced cytotoxicity in cultured cerebellar neurons. In both antioxidant assays, the shift of the 5-methoxy group to the 4-position of the indole nucleus led to the most active radical scavenger (9), more potent than the parent compound and melatonin in the antioxidant tests, but much less effective as a cytoprotectant. Sharp structure-activity relationships were registered for cytoprotection, where the maintenance of the 5-alkoxy-2-(N-acylaminoethyl)indole scaffold appeared as the key feature to confer both antioxidant and cytoprotective activity to the structure. Some derivatives of the set, however, together with the most potent 5d, maintained a significant antioxidant and cytoprotective effect and could be employed as tools for in vivo pharmacological investigations on neuroprotective efficacy of melatonin-related indoles.

RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists.

Prostacyclin (PGI2) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794).RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pKi) were 9.3 +/- 0.1 and 7.7 +/- 0.03, respectively; in a recombinant IP receptor system, pKi values were 8.7 +/- 0.06 and 6.9 +/- 0.1, respectively. Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinities (pKi) of RO1138452 and RO3244794 were 9.0 +/- 0.06 and 8.5 +/- 0.11, respectively. Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pKi values for EP1 (< 5), EP3 (5.38), EP4 (5.74) and TP (5.09). RO1138452 (1-10 mg kg(-1), i.v.) and RO3244794 (1-30 mg kg(-1), i.v.) significantly reduced acetic acid-induced abdominal constrictions. RO1138452 (3-100 mg kg(-1), p.o.) and RO3244794 (0.3-30 mg kg(-1), p.o.) significantly reduced carrageenan-induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg(-1), p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti-inflammatory potential.

Structure-affinity relationships of adenosine A2B receptor ligands

Many selective and high affinity agonists and antagonists have been developed for the adenosine A(1), A(2A), and A(3) receptors. Very recently such compounds have been identified for the adenosine A(2B) receptors. This review presents an overview of the structure-affinity relationships of antagonists and agonists for this receptor subtype as published in the scientific and patent literature. To date the most selective >370-fold, high affinity adenosine A(2B) receptor antagonist is the xanthine analog, compound 16 (8-(1-(3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-1H-pyrazol-4-yl)-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione). The pyrrolopyrimidine analog OSIP339391 (73) is slightly less selective, 70-fold, but has a higher affinity 0.41 nM compared to 1 nM for compound 16. Other promising classes of compounds with selectivities ranging from 10- to 160-fold and affinities ranging from 3 to 112 nM include triazolo, aminothiazole, quinazoline, and pyrimidin-2-amine analogs. Progress has also been achieved concerning the development of selective high affinity agonists for the adenosine A(2B) receptor. For years the most potent, albeit non-selective adenosine A(2B) receptor agonist was (S)PHPNECA (88). Last year, a new class of non-ribose ligands was reported. Several compounds displayed selectivity with respect to adenosine A(2A) and A(3) receptors. In addition, full and partial agonists for the adenosine A(2B) receptor were identified with EC(50) values of 10 nM (LUF5835, 103) and 9 nM (LUF5845, 105), respectively.

Effects of 138–355, a β3‐adrenoceptor selective agonist, on relaxation of the human detrusor muscle in vitro

Beta-adrenoceptors are the predominant beta-adrenoceptor subtype present in the bladder and urethra. This study investigates the effects of 138-355, an active-metabolite of TT-138 and beta(3)-adrenoceptor agonist, on relaxation of the human detrusor in vitro. Tumor-free tissue samples of human bladder muscle from 39 patients undergoing total cystectomy due to bladder cancer were obtained, and the mucosa and serosa were removed. Tissues were mounted in 5 or 10 ml organ baths containing Krebs solution, which was gassed with 95% O(2) and 5% CO(2). Resting tension of 1 g was obtained. When the contraction had stabilized, increasing concentrations of beta adrenoceptor agonists (non-selective, isoprenaline; beta(2)-selective, clenbuterol; beta(3)-selective, 138-355 and BRL37344) and propiverine (a non-selective anti-muscarinic antagonist) were added cumulatively and concentration-relaxation curves (CRCs) were obtained. CRCs to 138-355 were obtained in the absence and presence of SR59230A, a beta(3)-selective antagonist, and antagonist affinity values (pA(2)) were calculated from the Schild plot. Isoproterenol, clenbuterol, 138-355 and BRL37344 concentration-dependently relaxed isolated human urinary bladder strips with pEC(50) value being 6.76+/-0.17, 5.23+/-0.22, 5.80+/-0.26 and 5.90+/-0.28, respectively. Following antagonist assay, it was observed that concentration-relaxation curves to 138-355 was competitively antagonized by beta(3)- adrenoceptor antagonist, SR59230A, with a pA(2) value of 7.01+/-0.45 and with a Schild slope of 0.72+/-0.07. Involvement of the beta(3)-adrenoceptor appears to be greater than that of the beta(3)-adrenoceptor for relaxations of the human bladder. The relaxation response of 138-355 appears to be mediated via the beta(3)-adrenoceptor stimulation.

Synthesis of [3H]-labelled 4-[Ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]-2,3-[3H]-butan-1-ol: a high affinity radioligand for the corticotropin-releasing hormone type 1 receptor

[3H]-Labelled 4-[ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]-2,3-[3H]-butan-1-ol (3b) was prepared as a novel non-peptidic radiolabelled high affinity antagonist of the corticotropin-releasing hormone type 1 receptor (CRHR1) that could be useful as a more stable and receptor-selective alternative to the radiolabelled peptides now used to label the CRHR1 receptor for displacement studies in cell-based binding assays. The precursor (Z)-4-[ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]but-2-en-1-ol (2) was reduced with tritium gas using palladium as the catalyst. After HPLC purification 3b was obtained with a specific activity of 35 Ci/mmol in high radiochemical purity (>97%). Copyright © 2006 John Wiley & Sons, Ltd.

Identification of beta-adrenoceptor subtypes in lower urinary tract of the female pig.

We investigated the presence and functional role of beta-adrenoceptor subtypes in the bladder base and proximal urethra of the female pig. Saturation experiments were done with 7 concentrations (0.25 to 16 nM.) of [(3)H]-dihydroalprenolol (NEN Life Science Products, Boston, Massachusetts). Competition experiments with [(3)H]-dihydroalprenolol were performed with unlabeled antagonists (beta1 selective CGP20712A, beta2 selective ICI118551 and beta3 selective SR59230A). In functional studies concentration-relaxation curves to the beta3-agonist BRL37344 were obtained and antagonist affinities for SR59230A were determined. CGP20712A displaced [(3)H]-dihydroalprenolol with low affinity, suggesting that beta1-adrenoceptors were not present. Displacement with ICI118551 in the bladder base and urethra best fitted a 2-site model with 20% and 28% high affinity sites (beta2), respectively. Displacement experiments with SR59230A in the bladder base demonstrated that 59% of binding sites had high affinity (beta3). In the urethra displacement with SR59230A best fitted a 1-site model but with a pK(i) of 7.2 that was intermediate between that expected for beta2 and beta3-adrenoceptors. In functional studies BRL37344 induced relaxation with pEC50 values of 5.5 and 8, and a maximum relaxation response relative to 30 microM. isoprenaline of 79% and 90% in the bladder base and urethra, respectively. The affinity value of SR59230A for the response to BRL37344 was 7.87 and 7.71 in the bladder base and urethra, respectively, which were intermediate between those of beta2 and beta3-adrenoceptors. Apparently beta3-adrenoceptors are the predominant beta-adrenoceptor subtype present in the lower urinary tract of the pig.

Serotonin Receptor Antagonists Discriminate Between PKA- and PKC-Mediated Plasticity inAplysiaSensory Neurons

Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

Development of a Prostaglandin D2 Receptor Antagonist: Discovery of a New Chemical Lead.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Characterization of T Cell Receptors Engineered for High Affinity Against Toxic Shock Syndrome Toxin-1

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.

Proton Activation Does Not Alter Antagonist Interaction with the Capsaicin-Binding Pocket of TRPV1

Vanilloid receptor 1 (TRPV1) is activated by chemical ligands (e.g., capsaicin and protons) and heat. In this study, we show that (2E)-3-[2-piperidin-1-yl-6-(trifluoromethyl)pyridin-3-yl]-N-quinolin-7-ylacrylamide (AMG6880), 5-chloro-6-[(3R)-3-methyl-4-[6-(trifluoromethyl)-4-(3,4,5-trifluorophenyl)-1H-benzimidazol-2-yl]piperazin-1-yl]pyridin-3-yl)methanol (AMG7472), and N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) are potent antagonists of rat TRPV1 activation by either capsaicin or protons (pH 5) (defined here as group A antagonists), whereas (2E)-3-(6-tert-butyl-2-methylpyridin-3-yl)-N-(1H-indol-6-yl)acrylamide (AMG0610), capsazepine, and (2E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)acrylamide (SB-366791) are antagonists of capsaicin, but not proton, activation (defined here as group B antagonists). By using capsaicin-sensitive and insensitive rabbit TRPV1 channels, we show that antagonists require the same critical molecular determinants located in the transmembrane domain 3/4 region to block both capsaicin and proton activation, suggesting the presence of a single binding pocket. To determine whether the differential pharmacology is a result of proton activation-induced conformational changes in the capsaicin-binding pocket that alter group B antagonist affinities, we have developed a functional antagonist competition assay. We hypothesized that if group B antagonists bind at the same or an overlapping binding pocket of TRPV1 as group A antagonists, and proton activation does not alter the binding pocket, then group B antagonists should compete with and prevent group A antagonism of TRPV1 activation by protons. Indeed, we found that each of the group B antagonists competed with and prevented BCTC, AMG6880 or AMG7472 antagonism of rat TRPV1 activation by protons with pA2 values similar to those for blocking capsaicin, indicating that proton activation does not alter the conformation of the TRPV1 capsaicin-binding pocket. In conclusion, group A antagonists seem to lock the channel conformation in the closed state, blocking both capsaicin and proton activation.

Evidence for a Secondary State of the Human β3-Adrenoceptor

There are three members of the beta-adrenoceptor family, all of which are primarily coupled to G(s) proteins. Recent studies using the huge range of beta-ligands now available have given remarkable new insights into their pharmacology. beta1-adrenoceptors exist in at least two active conformations, whereas beta2-adrenoceptors are able to induce signaling via different agonist-induced receptor conformational states, and their affinity for antagonists can be altered by highly efficacious agonists. This study therefore examined the pharmacology of the human beta3-adrenoceptor stably expressed in Chinese hamster ovary cells. Several compounds described previously as beta-antagonists have agonist properties at the beta3-adrenoceptor. Antagonist affinity measurements varied at the beta3-adrenoceptor in a manner similar to those observed at human beta1-adrenoceptors and unlike those seen at beta2-adrenoceptors. Some ligands (e.g., fenoterol and cimaterol) were more readily inhibited by all antagonists, whereas other ligands [e.g., alprenolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride [SR 59230A]) stimulated responses that were more resistant to antagonism. Alprenolol inhibited fenoterol-induced beta3-adrenoceptor responses while acting as an agonist at higher concentrations. This is highly suggestive of two active conformational states of the beta3-adrenoceptor. (S)-4-[2-Hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide (ZD 7114) stimulated a two-component response, of which the first component was more readily antagonized than the second. Taken together, these experiments suggest that the human beta3-adrenoceptor exists in at least two different agonist conformations with a similar high- and low-affinity pharmacology analogous to, if not as pronounced as, the beta1-adrenoceptor. Both conformations are present in living cells and can be distinguished by their pharmacological characteristics. In this respect, the human beta3-adrenoceptor seems similar to the human beta1-adrenoceptor.

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

Activation of nicotinic acetylcholine receptors is initiated by binding of agonists, and as a consequence, specific domains transmit the chemical signal to the channel gate through a sequence of conformational changes. Recent high-resolution structural data from a snail acetylcholine binding protein have shown that the side chain of a lysine residue, located in the beta-strand beta7 and strictly conserved in alpha subunits of nicotinic receptors, systematically moves upon agonist binding, suggesting that it might be involved in both binding and gating. To test this hypothesis in neuronal nicotinic receptors, Lys145 was substituted by other amino acids in the alpha7 nicotinic receptor, and expression levels and electrophysiological responses for several nicotinic agonists and antagonists were determined. Substitutions of Lys145 showed a variety of functional effects: 1) strong reductions in the functional responses to acetylcholine, nicotine, and dimethylphenylpiperazinium, the latter becoming an antagonist; 2) increases in the agonist EC50 values (up to 80-fold with acetylcholine); 3) heterogeneous behavior of the different agonists, with epibatidine and cytisine being less affected by the substitutions; 4) decreases of agonist affinities for the desensitized receptors; and 5) small changes in the affinity of nicotinic antagonists. It is concluded that the presence of a polar or positively charged side chain at this position improves the gating function with acetylcholine and nicotine, although the lysine side chain seems to be necessary for retaining the binding properties of acetylcholine. The results are compatible with the involvement of Lys145 in the early steps of channel activation by acetylcholine.

S-nitrosoglutathione inhibits α1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery

Endogenous nitric oxide donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-hydroxytryptamine (5-HT)-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G protein-coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to G(q), we hypothesized that S-nitrosothiols modify the alpha1-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g., phenylephrine (PE). Rat pulmonary artery rings were pretreated for 30 min with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE (10(-8)-10(-6) M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or LY-83583} or G kinase inhibitor (KT-5823), and a thiol reductant [dithiothreitol (DTT)] was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose response. The GSNO effect was not prevented by LY-83583, ODQ, or KT-5823, indicating cGMP independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the alpha1-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of alpha1-adrenergic receptor subtype expression and receptor density by saturation binding with 125I-HEAT showed that GSNO decreased alpha1-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible alpha1-adrenergic receptor inhibition by S-nitrosothiols.

Increased αCGRP potency and CGRP‐receptor antagonist affinity in isolated hypoxic porcine intramyocardial arteries

1. This study describes the effects of hypoxia on relaxing responses and cAMP production induced by the known vasodilator peptides: alphaCGRP, amylin (AMY) and adrenomedullin (AM) on isolated pig coronary arteries in vitro. 2. Hypoxic incubation increased the vasorelaxant effect of alphaCGRP (four-fold; P<0.05), AMY (3.2-fold; P<0.05), but not significantly for AM (two-fold; NS). 3. Whereas hypoxia had no effect on arterial cAMP levels, it significantly potentiated the production of cAMP stimulated of alphaCGRP and AMY, but not of AM. 4. The antagonist alphaCGRP(8-37) also exerted an increased effect in hypoxia. The Schild plot-derived pK(B) values revealed an increase in the apparent affinity of the antagonist for the CGRP(1) receptor from 7.0 to 7.2 under control conditions versus 8.0 in hypoxia. 5. Removal of endothelium, peptidase inhibitors, preincubation with the adenosine A(2A) receptor antagonist CSC (10(-3) M), the ATP-sensitive K-channel inhibitor glibenclamide (10(-5) M), the cyclooxygenase inhibitor indomethacin (10(-3) M) or NG-monomethyl-L-arginine (10(-4) M) had no effect on the alphaCGRP-induced vasorelaxation in hypoxia; neither did hypoxia influence the levels of CGRP and AM receptor mRNA. 6. We conclude that hypoxic incubation increases the relaxation and cAMP production induced by alphaCGRP and AMY in rings of porcine coronary arteries in vitro. A concomitant release of adenosine, a cyclooxygenase product, an endothelium-derived substance, activation of vascular ATP-sensitive K-channels, peptidase inhibitors or changes in CGRP and AM receptor mRNA cannot account for the changes observed in hypoxia. Moreover, alphaCGRP(8-37) showed increased affinity at the CGRP(1) receptor during hypoxia, possibly due to a conformational change at the CGRP(1) receptor site.

GPCR Dimers Are For Real

Waldhoer et al. report new evidence to support the notion that heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors can signal as heterodimers in vivo. The authors noted that the opioid receptor agonist 6′-guanidinonaltrindole (6′-GNTI) was a more potent inhibitor of signaling in cells expressing both kappa opioid peptide (KOP-Rs) and delta opioid peptide receptors (DOP-Rs). Selective agonists for either the KOP-Rs or DOP-Rs could block 6′-GNTI-mediated signaling in cells expressing both receptors, which indicates that 6′-GNTI was acting on heterodimers. Binding affinities for the specific antagonists were different from those measured in cells expressing only one receptor type, which may indicate that it will be possible to selectively modulate heterodimer function with therapeutic agents. Indeed, in mice, 6′-GNTI was a more potent and effective analgesic when applied to the spinal cord than when applied to the brain. Such a tissue-selective agonist could be very useful, because side effects of opiates like morphine are reduced when the drug is applied directly to the spinal cord. M. Waldhoer, J. Fong, R. M. Jones, M. M. Lunzer, S. K. Sharma, E. Kostenis, P. S. Portoghese, J. L. Whistler, A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc. Natl. Acad. Sci. U.S.A. 102 , 9050-9055 (2005). [Abstract] [Full Text]

Synthesis and Biological Evaluation of Benzodioxanylpiperazine Derivatives as Potent Serotonin 5-HT1A Antagonists: The Discovery of Lecozotan

A series of benzodioxanylpiperazine derivatives possessing a 4-aryl amide substituent was prepared and evaluated for 5-HT(1A) affinity and functional antagonist activity in vitro and in vivo. All of the compounds in this series possessed high affinity for the human 5-HT(1A) receptor and many displayed potent antagonist activity in vitro and varying degrees of intrinsic activity in vivo. Compound 11c (Lecozotan) was selected for further development and is currently in clinical trials.