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PubMed2003

Positive allosteric modulator of the human 5-HT2C receptor.

The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.

Allosteric Regulation↗
PubMed2002

A single residue contributes sensitivity to allosteric modulation of AMPA receptors by LY395153.

Previous studies have shown that a single point mutation (S(750)Q) in the splice variant region of rat Glu(1) subunits can eliminate positive allosteric modulation by cyclothiazide. The present study investigated the effects of mutating the equivalent residue (S(776)Q) in the human Glu(4) subunit on the activity and binding of a novel AMPA receptor potentiator, LY395153 (N-2-(4-benzamidophenylpropyl-2-propanesulfonamide)). The mutation markedly attenuated, but did not eliminate, potentiation by LY395153 and cyclothiazide. In addition, binding of [3H]LY395153 was significantly reduced by this mutation. These effects occurred in the absence of any change in the response to glutamate or the binding of a competitive AMPA receptor antagonist, [3H]Ro 48-8587 ([2,4,5-3H]9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]-triazolo[1,5-c]quinazoline-2,5-dione triethylammonium salt). Collectively, these results demonstrate that structurally diverse classes of potentiators are sensitive to mutations of this single Ser residue, suggesting that binding to this residue may be necessary for positive allosteric modulation of AMPA receptors.

Allosteric Regulation↗
PubMed2006

The 'allosteric modulator' SCH-202676 disrupts G protein-coupled receptor function via sulphydryl-sensitive mechanisms.

1. Previous studies suggest that the thiadiazole compound SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine) acts as an allosteric modulator of a variety of structurally distinct G protein-coupled receptors (GPCRs). It was postulated that SCH-202676 would directly bind a structural motif in the receptor molecule common to divergent members of the GPCR family. The molecular mechanisms of such a promiscuous action, however, remain obscure. 2. To clarify the mechanism of SCH-202676 action, we used the functional approach of [35S]GTPgammaS autoradiography with rat brain cryostat sections together with classical membrane [35S]GTPgammaS binding assays to evaluate how the thiadiazole affects G protein activity mediated by various receptors linked to the Gi-family of G proteins. 3. We found that in the absence of dithiotreitol (DTT), SCH-202676 (10(-7)-10(-5) M) elicits nonspecific effects in the [35S]GTPgammaS-based G protein activation assays, thereby severely compromising interpretations on the compounds ability to allosterically inhibit receptor-mediated G protein activity. Such a nonspecific behaviour was fully reversed upon addition of DTT (1 mM), revealing thiol-based mechanism of action. 4. In routine incubations containing DTT, SCH-202676 had no effect on receptor-driven G protein activity, as assessed for adenosine A1, alpha2-adrenergic, cannabinoid CB1, lysophosphatidic acid LPA1, muscarinic M2/M4, purinergic P2Y12 or sphingosine 1-phosphate receptors, suggesting that the thiadiazole does not act as an allosteric modulator of GPCR function. 5. 1H NMR analysis indicated that SCH-202676 underwent structural changes after incubation with the reducing agent DTT or with brain tissue. 6. We conclude that SCH-202676 modulates GPCRs via thiol modification rather than via true allosteric mechanisms.

Allosteric Regulation↗
PubMed1992

Complex allosteric modulation of cardiac muscarinic receptors by protamine: potential model for putative endogenous ligands.

A large number of diverse pharmacological agents bind to a secondary domain on the muscarinic receptor, to influence allosterically the interaction of ligands at the primary binding site. Based on common structural features of these antagonists, we examined the interaction of protamine, an endogenous polycationic peptide, and of polyamines with muscarinic receptors in rat heart. Our results provide several lines of qualitative evidence that protamine allosterically modulates the conformation of muscarinic receptors, in a marked negatively cooperative manner. It decelerated the dissociation of N-[3H]methylscopolamine ([3H] NMS) initiated by atropine, in a concentration-dependent fashion. Inhibition by protamine of [3H]NMS binding at equilibrium showed a distinct plateau, which increased in magnitude at higher ligand concentrations. Scatchard analysis of saturation isotherms of [3H]NMS binding in the absence and presence of protamine indicated that protamine did not alter Bmax in a statistically significant fashion, although there was a trend of a concentration-dependent increase in this parameter. On the other hand, it caused a marked concentration-dependent decrease in the affinity of [3H]NMS, and this effect reached a ceiling limit. However, there were marked quantitative deviations of the interaction of protamine from a simple ternary allosteric model. Some of these discrepancies could be explained by the tendency of protamine to increase Bmax. The allosteric actions of protamine demonstrated in kinetic and equilibrium experiments were selective for m1 and m2 muscarinic receptors, compared with m3, m4, and m5 receptors, as studied in Chinese hamster ovary cells transfected with the genes of the different muscarinic receptors. Arginine residues play an important role in the allosteric interaction of protamine, inasmuch as poly-L-arginine qualitatively mimicked the effects of protamine. In contrast, no effects of the polyamines spermine, spermidine, and putrescine were observed on [3H]NMS binding. This is the first report on the allosteric modulation of muscarinic receptors by an endogenous peptide.

Animals↗
PubMed2003

Positive allosteric modulation of the human metabotropic glutamate receptor 4 (hmGluR4) by SIB-1893 and MPEP.

We have identified 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) and 2-methyl-6-phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB-1893 and MPEP enhanced the potency and efficacy of L-2-amino-4-phophonobutyrate (L-AP4) in guanosine 5'-O-(3-[(35)S]thiotriphosphate ([(35)S]GTPgammaS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB-1893 and MPEP had no effects alone in GTPgammaS binding, effects were observed in the cell-based cAMP assay due to media-derived activation as indicated by CPPG inhibition. Positive modulation of the mGluR4 was a receptor-specific effect since SIB-1893 and MPEP had neither effects on mGluR2-expressing cells nor on the parent BHK cell line. In [(3)H]L-AP4 binding, a two-fold decrease in K(D) but not in B(max) was observed with 100 micro M SIB-1893, whereas MPEP affected neither parameter. Finally, SIB-1893 and MPEP failed to displace [(3)H]L-AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.

Allosteric Regulation↗
PubMed2006

Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates.

The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Effects of positive allosteric modulators of the GABAB receptor on cocaine self-administration in rats.

RATIONALE: Previous studies have strongly implicated a role for GABA(B) receptors in modulating the reinforcing effects of cocaine. OBJECTIVE: The purpose of the present study was to examine the efficacy of two novel positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, to decrease cocaine self-administration in rats responding under various schedules of reinforcement. METHODS: Rats were trained to self-administer cocaine under progressive ratio (PR), fixed ratio (FR) and discrete trials (DT) schedules of reinforcement, and the ability of CGP7930 and GS39783 to decrease cocaine-maintained responding was examined. RESULTS: On a PR schedule, CGP7930 markedly decreased break points maintained by 1.5 mg/kg per injection cocaine in a dose-dependent manner. GS39783 produced only modest decreases in cocaine-reinforced break points, with only the highest dose decreasing break points relative to baseline. On an FR1 schedule of reinforcement, both drugs decreased responding for a threshold dose of cocaine, but did not alter responding for higher doses of cocaine. In a DT procedure, 1.5 mg/kg per injection cocaine was made available during three 10-min trials each hour during 24-h sessions (DT3), engendering a circadian pattern of responding characterized by high numbers of infusions during the dark phase and low numbers of infusions during the light phase. Doses of 30 mg/kg CGP7930, 3.0 mg/kg GS39783 and 2.5 mg/kg baclofen significantly decreased cocaine-maintained responding when administered at the beginning of the dark phase of the cycle. Across all schedules, CGP7930 was more effective at decreasing cocaine self-administration than GS39783, a finding that may be due to differences in bioavailability between the two drugs. CONCLUSIONS: These findings suggest that positive allosteric modulators of the GABA(B) receptor may hold promise as potential pharmacotherapies for cocaine abuse and dependence.

Analysis of Variance↗
PubMed2004

Discovery of positive allosteric modulators for the metabotropic glutamate receptor subtype 5 from a series of N-(1,3-diphenyl-1H- pyrazol-5-yl)benzamides that potentiate receptor function in vivo.

This report describes the discovery of the first centrally active allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGluR5). Appropriately substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides (e.g., 8) have been identified as a novel class of potent positive allosteric modulators of mGluR5 that potentiate the response to glutamate. An iterative analogue library synthesis approach provided potentiators with excellent potency and selectivity for mGluR5 (vs mGluRs 1-4, 7, 8). Compound 8q demonstrated in vivo proof of concept in an animal behavior model where known antipsychotics are active, supporting the development of new antipsychotics based on the NMDA hypofunction model for schizophrenia.

Allosteric Regulation↗
PubMed1992

Novel amiloride analog allosterically modulates the alpha 2-adrenergic receptor but does not inhibit Na+/H+ exchange.

Two novel amiloride analogs have been synthesized during the course of efforts to develop a photoaffinity label for the amiloride allosteric domain on alpha 2-adrenergic receptors. One of these, 5-[N-2'-aminoethyl-N'-isopropyl]amiloride-N-[4"-azidosalicylamide] (A-EIA-AS), markedly accelerates the rate of dissociation of [3H]yohimbine from affinity-purified alpha 2-adrenergic receptors, an assay for allosteric modulation of receptor-adrenergic ligand interactions. In contrast, this agent does not appreciably inhibit Na+/H+ exchange, measured as 5-(N-ethyl-N-isopropyl)amiloride (EIA)-inhibitable 22Na+ uptake into cultured renal epithelial cells. A second analog, 5-[N-2'-(4"-azidosalicylamidino)ethyl-N'- isopropyl]amiloride (ASA-EIA), does not foster an accelerated rate of dissociation of [3H]yohimbine binding from the alpha 2 receptor but does block the ability of A-EIA-AS to do so, suggesting that ASA-EIA and A-EIA-AS interact at a common binding site. Interestingly, the ability of EIA to accelerate [3H]yohimbine dissociation is not blocked by ASA-EIA, a finding that may indicate that EIA and A-EIA-AS allosterically modulate alpha 2 receptor-ligand interactions via distinct or nonoverlapping binding sites.

Amiloride↗
PubMed2006

Cannabidiol is an allosteric modulator at mu- and delta-opioid receptors.

The mechanism of action of cannabidiol, one of the major constituents of cannabis, is not well understood but a noncompetitive interaction with mu opioid receptors has been suggested on the basis of saturation binding experiments. The aim of the present study was to examine whether cannabidiol is an allosteric modulator at this receptor, using kinetic binding studies, which are particularly sensitive for the measurement of allosteric interactions at G protein-coupled receptors. In addition, we studied whether such a mechanism also extends to the delta opioid receptor. For comparison, (-)-Delta9-tetrahydrocannabinol (THC; another major constituent of cannabis) and rimonabant (a cannabinoid CB1 receptor antagonist) were studied. In mu opioid receptor binding studies on rat cerebral cortex membrane homogenates, the agonist 3H-DAMGO bound to a homogeneous class of binding sites with a KD of 0.68+/-0.02 nM and a Bmax of 203+/-7 fmol/mg protein. The dissociation of 3H-DAMGO induced by naloxone 10 microM (half life time of 7+/-1 min) was accelerated by cannabidiol and THC (at 100 microM, each) by a factor of 12 and 2, respectively. The respective pEC50 values for a half-maximum elevation of the dissociation rate constant k(off) were 4.38 and 4.67; 3H-DAMGO dissociation was not affected by rimonabant 10 microM. In delta opioid receptor binding studies on rat cerebral cortex membrane homogenates, the antagonist 3H-naltrindole bound to a homogeneous class of binding sites with a KD of 0.24+/-0.02 nM and a Bmax of 352+/-22 fmol/mg protein. The dissociation of 3H-naltrindole induced by naltrindole 10 microM (half life time of 119+/-3 min) was accelerated by cannabidiol and THC (at 100 microM, each) by a factor of 2, each. The respective pEC50 values were 4.10 and 5.00; 3H-naltrindole dissociation was not affected by rimonabant 10 microM. The present study shows that cannabidiol is an allosteric modulator at mu and delta opioid receptors. This property is shared by THC but not by rimonabant.

Allosteric Regulation↗
PubMed1999

Positive allosteric modulators of the GABA(A) receptor: differential interaction of benzodiazepines and neuroactive steroids with ethanol.

Endogenous pregnane steroids, such as allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one; 3alpha, 5alpha-P) and pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one; 3alpha,5beta-P), allosterically modulate GABA(A) receptor function and exhibit behavioral effects similar to benzodiazepines, though acting at a distinct recognition site. Inasmuch as some positive allosteric modulators of GABA(A) receptor function exhibit profound interactions with ethanol, the effects of 3alpha,5alpha-P and 3alpha,5beta-P were compared to those of two benzodiazepines, triazolam and diazepam, on the motor function of mice and rats when administered either alone or in combination with ethanol. All four test compounds exhibited dose-related impairment of motor function in the horizontal wire task in mice and the rotorod task in rats. Ethanol caused a marked enhancement of triazolamand diazepam-induced motor impairment. In contrast, ethanol enhanced to a lesser extent the motor impairment induced by both neurosteroids in mice and not at all in rats. All four compounds increased ethanol-induced behavioral sleep time in mice, although the benzodiazepines did so at a much smaller fraction of their ataxic doses as compared to the neurosteroids. As one of the undesired side-effects of therapeutic use of benzodiazepines is their interaction with ethanol, development of neuroactive steroids as drugs may offer therapeutic advantages.

Allosteric Regulation↗
PubMed2002

Elevation of ligand binding to muscarinic M(2) acetylcholine receptors by bis(ammonio)alkane-type allosteric modulators.

Bis(ammonio)alkane-type compounds are archetypal muscarinic allosteric modulators. Phthalimido-substituted hexane-bis-ammonium agents were methylated in the phthalimide moieties and the lateral propyl side chains. All compounds retarded allosterically the dissociation of the orthosteric ligand [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine heart M(2) receptors. [(3)H]NMS equilibrium binding was reduced, left unaltered, or elevated, depending on the degree and position of methylation. This is the first time that an allosteric elevation of ligand binding is demonstrated for bis(ammonio)alkane-type compounds.

Allosteric Regulation↗
PubMed2001

Muscarinic allosteric modulation: M2/M3 subtype selectivity of gallamine is independent of G-protein coupling specificity.

Among the five subtypes of muscarinic acetylcholine receptors, the sensitivity towards allosteric modulation is generally higher in M2 and M4 receptors that preferentially couple to inhibitory G-proteins of the Gi/o type than in M1, M3, and M5 that preferentially couple to stimulatory G-proteins such as Gq/11. We aimed to check whether the high allosteric sensitivity of the M2 receptor compared to M3 is related to the differential G-protein coupling preference. As the third intracellular loop (i3) is known to be the major determinant in receptor G-protein coupling specificity, we used wild-type M2 and M3 receptors and the related chimeric constructs with exchanged i3-loops, i.e., M2 containing M3-i3 (M2/M3-i3) and M3 containing M2-i3 (M3/M2-i3). The allosteric effect of the archetypal modulator gallamine on the dissociation and the equilibrium binding of [3H]N-methylscopolamine ([3H]NMS) was measured in membranes of mouse A9L cells stably expressing the wild-type and the chimeric receptors (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C). The dissociation of [3H]NMS was monophasic under all conditions studied. Control values of t 1/2 were (means +/- SEM, n = 4-7): M2: 3.8 +/- 0.2 min, M2/M3-i3: 4.8 +/- 0.3 min, M3:43.3 +/- 4.2 min, M3/M2-i3: 41.1 +/- 3.6 min. At M2 receptors, 0.2 microM gallamine allosterically reduced the apparent rate constant of dissociation k-1 to 51 +/- 5% of the control value (n = 5). At M2/M3-i3 the allosteric potency of gallamine was not significantly changed (0.2 microM gallamine --> k-1 = 61 +/- 4%, n = 7). At M3, a 20-fold higher concentration was required for an equieffective allosteric action (10 microM gallamine --> k-1 = 51 +/- 5%, n = 5). The potency of gallamine at M3/M2-i3 was not increased compared with M3 receptors (10 microM gallamine --> k-1 = 73 +/- 2%, n = 4) but even significantly diminished. [3H]NMS equilibrium binding experiments revealed that neither the binding constants of gallamine at free receptor subtypes (pKA,M2: 7.57 +/- 0.04, n = 4; pKA,M3: 5.56 +/- 0.13, n = 3) nor the factors of negative cooperativity with [3H]NMS (alphaM2 = 31 +/- 1, alphaM3 = 3 +/- 0.4) were affected by the exchanged i3-loops (pKA,M2/M3-i3: 7.65 +/- 0.03, pKA,M3/M2-i2: 5.35 +/- 0.24, alphaM2/M3-i3= 30 +/- 2, alphaM3/M2-i2 = 3 +/- 0.7). In conclusion, the different sensitivities of M2 and M3 receptors towards allosteric modulation by gallamine are not related to the G-protein coupling specificity of the receptors.

Allosteric Regulation↗
PubMed2004

Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi alpha-subunits.

We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated G(i1) alpha-subunits (A1-Gialpha) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1-Gialpha fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPgammaS binding experiments to study receptor and G protein activation of selected A1-Gialpha fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1-Gialpha fusion proteins (351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A1-Gialpha fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1-Gialpha fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [35S]GTPgammaS binding experiments, we learned that all the A1-Gialpha fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPgammaS binding was increased for the five A1-Gialpha fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1-Gialpha fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi alpha-subunit. In conclusion, A1-Gialpha fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [35S]GTPgammaS binding experiments.

Adenosine↗
PubMed2000

Allosteric modulators affect the efficacy of partial agonists for recombinant GABA(A) receptors.

Different alpha subunits of human gamma-aminobutyric acid type A (GABA(A)) receptors were transiently expressed together with beta(3) and gamma(2) subunits in Xenopus oocytes to examine the interactions of various GABA(A) agonists and representative allosteric modulators. Chloride currents elicited by agonists were measured using two electrode voltage clamp electrophysiology. Where compounds behaved as full agonists, i.e. GABA on all subtypes and 4,5,6, 7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) on alpha2beta(3)gamma(2) GABA(A) receptors, agonist concentration-response curves were shifted to the left by the benzodiazepine full agonist chlordiazepoxide and the anticonvulsant loreclezole, or to the right by the inverse agonist 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (DMCM), with no effect on the maximal currents (I(max)). In contrast, maximal responses for different partial GABA(A) agonists on all benzodiazepine-sensitive alpha(x)beta(3)gamma(2) GABA(A) receptors were enhanced by chlordiazepoxide. I(max) values for piperidine-4-sulphonic acid (P4S) on alpha(1)beta(3)gamma(2), THIP on alpha(3)beta(3)gamma(2), and 5-(4-piperidyl)isothiazol-3-ol (thio-4-PIOL) on alpha(2)beta(3)gamma(2) and alpha(5)beta(3)gamma(2) GABA(A) receptors were increased by chlordiazepoxide, while that for P4S on alpha(1)beta(3)gamma(2) receptors was decreased by DMCM. The I(max) values for partial agonists were also enhanced by pentobarbitone, the neurosteroid allopregnanolone and loreclezole irrespective of receptor subtype or the nature of the partial agonist. In the light of models of ligand-gated ion channel receptor activation we suggest two possible mechanisms of action for the effects of allosteric modulators on partial agonist receptor activation: either selective modulation of agonist affinity for the open/closed state, or direct modulation of the gating process itself.

Allosteric Regulation↗
PubMed1995

Allosteric modulation of [3H]flunitrazepam binding to recombinant GABAA receptors.

The allosteric modulation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), pentobarbital, (+)-etomidate, etazolate, alphaxalone, propofol and chlormethiazole was investigated in cerebellar membranes and membranes from human embryonic kidney (HEK) 193 cells transfected with alpha 1 beta 3 gamma 2 or alpha 1 gamma 2 subunits. Results obtained indicate that [3H]flunitrazepam binding to recombinant GABAA receptors consisting of alpha 1 beta 3 gamma 2 subunits could be modulated by these compounds in a way and with a potency similar to that observed in cerebellar membranes. In addition, it was demonstrated that not only receptors consisting of alpha 1 beta 3 gamma 3, but also those consisting of alpha 1 gamma 2 subunits exhibited [3H]flunitrazepam binding which could be stimulated by GABA. In contrast to alpha 1 beta 3 gamma 2 receptors, however, [3H]flunitrazepam binding to recombinant alpha 1 gamma 2 receptors was inhibited by pentobarbital, (+)-etomidate, etazolate, alphaxalone, propofol and chlormethiazole. This seems to indicate that binding sites for these compounds are present on alpha 1 gamma 2 receptors, but that their allosteric interaction with [3H]flunitrazepam binding sites is different from that of alpha 1 beta 3 gamma 2 receptors.

Allosteric Regulation↗
PubMed2006

Allosteric modulation, thermodynamics and binding to wild-type and mutant (T277A) adenosine A1 receptors of LUF5831, a novel nonadenosine-like agonist.

The interaction of a new nonribose ligand (LUF5831) with the human adenosine A1 receptor was investigated in the present study. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A1 receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N6-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A1 receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A1 receptor (Ki=122+/-22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively. The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A1 receptor showed that LUF5831 had a submaximal effect (37+/-1%) in comparison to CPA (66+/-5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A1 receptor.

Adenosine↗
PubMed1992

Two allosteric modulators interact at a common site on cardiac muscarinic receptors.

The abilities of gallamine, obidoxime, tetrahydroaminoacridine (THA), and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) to alter the rate of dissociation of N-[3H]methylscopolamine from rat cardiac muscarinic receptors were investigated. All four ligands monotonically slowed the dissociation, with the order of potency gallamine > TMB-8 > THA > obidoxime. There was a dramatic difference in the efficacy of these allosteric modulators. Gallamine, TMB-8, and THA slowed the dissociation of N-methylscopolamine by > 90% at maximally effective concentrations, whereas obidoxime was capable of slowing it by only about 50%. In a manner analogous to the action of a partial agonist, obidoxime was able to partially reverse the effects of the other three modulators. Furthermore, the concentration-dependent effects of combinations of obidoxime and gallamine were in good agreement with the model of competitive interaction between these two ligands. These results provide the first evidence that two muscarinic allosteric modulators interact competitively at a well defined site.

Allosteric Regulation↗