Using a radioalloster to test predictions of the cooperativity model for gallamine binding to the allosteric site of muscarinic acetylcholine M(2) receptors.

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.     

Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7

Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (NMS)-occupied hM1 receptor were measured directly by equilibrium and kinetic binding experiments. Identical values were obtained with the two types of assay methods, 14 pM and 0.9 nM in free or NMS-liganded receptor states, respectively, highlighting a strong negative cooperativity between this allosteric toxin and NMS. Identical results were obtained with indirect binding experiments with [3H]NMS using the ternary complex model, clearly demonstrating the reciprocal nature of this cooperativity. Furthermore, the effects of various orthosteric and allosteric agents on the dissociation kinetic of 125I-MT7ox were measured and show that, except for the MT1 toxin, all of the ligands studied [NMS, atropine, gallamine, brucine, tacrine, staurosporine, and (9S,10S,12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. Equilibrium binding experiments with 125I-MT7ox and [3H]NMS were conducted to reveal the effects of these ligands on the free receptor, and affinity constants (pKx values) were calculated using the allosteric ternary complex model. Our results suggest that MT7 toxin interacts with hM1 receptor at a specific allosteric site, which may partially overlap those identified previously for "classic" or "atypical" allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.     

Interaction studies of multiple binding sites on m4 muscarinic acetylcholine receptors

This study investigated the reciprocal cross-interactions between two distinct allosteric sites on the M(4) muscarinic acetylcholine receptor (mAChR) in the absence or presence of different orthosteric ligands. Initial studies revealed that two novel benzimidazole allosteric modulators, 17-beta-hydroxy-17-alpha-ethy nyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 62,577) and 17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 51,708), exhibited different degrees of positive, negative, or close-to-neutral cooperativity with the orthosteric site on M(1) or M(4) mAChRs, depending on the chemical nature of the orthosteric radioligand that was used [[(3)H]N-methylscopolamine ([(3)H]NMS) versus [(3)H]quinuclidinylbenzilate ([(3)H]QNB)]. The largest window for observing an effect (negative cooperativity) was noted for the combination of WIN 62,577 and [(3)H]QNB at the M(4) mAChR. Experiments involving the combination of these two ligands with unlabeled agonists [acetylcholine, 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), or xanomeline] revealed low degrees of negative cooperativity between WIN 62,577 and each agonist, whereas stronger negative cooperativity was observed against atropine. It is interesting that when these experiments were repeated using the prototypical modulators heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3-phth), alcuronium, or brucine (which act at a separate allosteric site), WIN 62,577 exhibited negative cooperativity with each modulator when the orthosteric site was unoccupied, but this switched to neutral cooperativity when the receptor was occupied by [(3)H]QNB. Dissociation kinetic experiments using [(3)H]NMS and combination of C(7)/3-phth with WIN 62,577 also provided evidence for neutral cooperativity between the two allosteric sites when the orthosteric site is occupied. Together, these results provide insight into the nature of the interaction between two distinct allosteric sites on the M(4) mAChR and how this interaction is perturbed upon occupancy of the orthosteric site.     

Atypical muscarinic allosteric modulation: cooperativity between modulators and their atypical binding topology in muscarinic M2 and M2/M5 chimeric receptors

The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are "atypical", having steep concentration-effect curves and not interacting competitively with "typical" allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg2+, and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M2/M5-receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the "common" allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M2-receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer.     

A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor

THRX-160209 is a potent antagonist at the M(2) muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M2 receptor subtype (apparent pK(I) = 9.51 +/- 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK(I) values < 6.0), 2) specificity of THRX-160209 for the M2 receptor subtype compared with the closely related M4 (apparent pK(I) = 8.78 +/- 0.24) and M1, M3, and M5 receptors (apparent pK(I) values 10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [3H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-[7-[4-[(4S)-4-methyl-1,3-oxazolidin-2-yl]phenoxy]heptyl]-1,2-oxazole (W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.     

Analogs of WIN 62,577 define a second allosteric site on muscarinic receptors

WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) and WIN 62,577 (17-beta-hydroxy- 17-alpha-ethynyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) are potent and centrally active antagonists at rat, but not human, NK(1) receptors. The interactions of these compounds and some analogs with [(3)H]N-methyl scopolamine ([(3)H]NMS) and unlabeled acetylcholine (ACh) at M(1)-M(4) muscarinic receptors have been studied using equilibrium and nonequilibrium radioligand binding methods. The results are consistent with the predictions of the allosteric ternary complex model. The WIN compounds have log affinities for the unliganded receptor in the range 5 to 6.7, and exhibit positive, negative, or neutral cooperativity with [(3)H]NMS and ACh, depending on the receptor subtype and nature of the interacting ligands. WIN 62,577 is an allosteric enhancer of ACh affinity at M(3) receptors. Although interacting allosterically, WIN 62,577 and WIN 51,708 do not affect [(3)H]NMS dissociation from M(3) receptors. Certain analogs have higher affinities than WIN 62,577, and truncated forms of WIN 62,577, including steroids, also act allosterically. One analog, 17-beta-hydroxy-17-alpha-Delta(4)-androstano[3,2-b]pyrido[2,3-b]indole (PG987), has the unique effect of speeding [(3)H]NMS dissociation; its largest effect, 2.5-fold, is at M(3) receptors. The interaction between PG987 and other allosteric agents on [(3)H]NMS dissociation from M(3) receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M(3) receptors as KT5720, staurosporine, and WIN 51,708. Therefore, in addition to the allosteric site that binds strychnine (and probably chloromethyl brucine, another allosteric enhancer) there is a second, nonoverlapping, pharmacologically distinct allosteric site on M(3) receptors that also supports positive cooperativity with ACh.     

Allosteric site in M2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist

Muscarinic acetylcholine receptors contain two distinct ligand binding sites, i.e. the orthosteric site for acetylcholine and other conventional ligands, and an allosteric site located at the entrance of the ligand binding pocket. We used a set of allosteric agents to probe whether muscarinic M₂ receptors whose orthosteric site is occupied by an agonist still reveal the common allosteric site that has been identified in M₂ receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [³H]oxotremorine M ([³H]OxoM) or the antagonist [³H]NMS. The affinities of the allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M₂ receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [³H]OxoM-liganded receptors compared with [³H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [³H]OxoM- and [³H]NMS-occupied receptors. The allosteric antagonistic tool obidoxime inhibited WDuo3 action in [³H]OxoM-liganded receptors with the same potency as in [³H]NMS-liganded receptors. We conclude that the common allosteric site is still present in OxoM-liganded M₂ receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.     

The allosteric binding profile of himbacine: a comparison with other cardioselective muscarinic antagonists

The possibility of an allosteric interaction by himbacine, a cardioselective antagonist, with rat cardiac muscarinic receptors was studied. Himbacine allosterically decelerated the dissociation of bound [3H]N-methylscopolamine [( 3H]NMS) in a concentration-dependent manner with an IC50 value of 103.7 microM. When compared to the IC50 values of other cardioselective antagonists, the rank order of potencies was: methoctramine greater than gallamine greater than himbacine greater than AF-DX 116. In contrast, the potencies of these compounds to displace [3H]NMS binding were: himbacine greater than methoctramine greater than AF-DX 116 greater than gallamine. The allosteric potencies were found not to be correlated with binding potencies (correlation coefficient = -0.15). A striking common feature of the cardioselective antagonists is their ability to bind to an allosteric site on cardiac muscarinic receptors.     

Modes of allosteric interactions with free and [3H]N-methylscopolamine-occupied muscarinic M2 receptors as deduced from buffer-dependent potency shifts

Muscarinic M2 acetylcholine receptors contain an allosteric site that is probably located at the entrance of the ligand binding pocket above the orthosteric binding site. With the orthosteric area not occupied, allosteric agents might gain access to this site. The interaction of allosteric agents with orthoster-occupied receptors is known to depend on the buffer conditions in an alloster-specific fashion. Utilizing the buffer-dependent potency shift as an indicator, we aimed to find out for two rod-like shaped and flexible allosteric agents whether or not there is evidence for a switch in the site of attachment in free compared with [3H]N-methylscopolamine ([3H]NMS)-occupied porcine heart M2 receptors. These agents are the bispyridinium compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl] propane dibromide) and Duo3 (4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype allosteric agents gallamine and alcuronium were included. Inhibition of [3H]NMS association was taken to reflect alloster interaction with free receptors, inhibition of [3H]NMS dissociation indicated binding to [3H]NMS-occupied receptors. In Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degrees C) compared with Mg,Tris,Cl,Pi buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degrees C) WDuo3 underwent the same loss of potency for the interaction with either free or [3H]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentrations of the modulator leading to a half-maximal delay of [3H]NMS association or dissociation, respectively, in Mg,Tris,Cl,Pi compared with Na,K,Pi. For WDuo3 the ratios were PRass=27 and PRdiss=22, respectively. For Duo3, the interaction with free and [3H]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast to the other agents, the concentration-effect curves of which had slope factors nH not different from unity, the curves of Duo3 were steep (nH about -1.6). For alcuronium the shift factors amounted to PRass=29 and PRdiss=25, for gallamine to PRass=216 and PRdiss=159. In conclusion, there was a wide variation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction with either free or [3H]NMS-occupied receptors was always characterized by the same buffer-dependent shift. Thus, even the applied rod-shaped allosteric agents do not appear to switch to the orthosteric site in free compared with orthoster-occupied M2 receptors.     

Systematic development of high affinity bis(ammonio)alkane-type allosteric enhancers of muscarinic ligand binding

Bis(ammonio)alkane compounds carrying lateral phthalimidopropyl substituents on the nitrogen atoms belong to the archetypal muscarinic allosteric agents. Herein, a series of symmetrical and nonsymmetrical compounds was synthesized in which the phthalimide residues were replaced by differently substituted imide moieties. The allosteric action was measured in porcine heart muscarinic M(2) receptors using [(3)H]N-methylscopolamine (NMS) as a ligand for the orthosteric receptor site in equilibrium binding and dissociation experiments. 1,8-Naphthalimido residues conferred an up to 100-fold gain in affinity leading into the low nanomolar range, while the inhibition of NMS binding was maintained. Additional propyl chain methylation was accompanied by an allosteric elevation of orthosteric ligand binding. In general, the gain in allosteric activity achieved by ring variation plus propyl chain methylation on one side of the molecule could not be augmented by symmetrical variations. The elevation of the ligand binding can be explained by different quantitative structure-activity relationships for the affinities to the free and the orthoster-liganded receptor.     

Testing the specificity of allosteric modulators of muscarinic receptors in phylogenetically closely related histamine H1-receptors

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.     

Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M(3) receptors

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M(3) receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M(2) receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) and the negative effect of gallamine on the affinity for classical antagonist N-[(3)H]methylscopolamine ([(3)H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M(3) receptors was made identical with the o3 loop of M(2) receptors, and alcuronium acquired positive influence on the affinity for [(3)H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M(2) to M(3) receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [(3)H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M(3) receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [(3)H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.     

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.     

Allosteric interactions of staurosporine and other indolocarbazoles with N-[methyl-(3)H]scopolamine and acetylcholine at muscarinic receptor subtypes: identification of a second allosteric site

We have studied the interactions of five indolocarbazoles with N-[methyl-(3)H]scopolamine (NMS) and unlabeled acetylcholine at M(1)-M(4) muscarinic receptors, using equilibrium and nonequilibrium radioligand binding studies. The results are consistent with an allosteric model in which the primary and allosteric ligands bind simultaneously to the receptor and modify each other's affinities. The compounds were generally most active at M(1) receptors. [(3)H]NMS binding was enhanced by staurosporine, KT5720, and KT5823 at M(1) and M(2) receptors, and by K-252a at M(1) receptors. G? 7874 reduced [(3)H]NMS affinity by up to threefold for all subtypes. A range of cooperative effects with acetylcholine was seen, and, at the M(1) receptor, KT5720 had a log affinity of 6.4 and enhanced acetylcholine affinity by 40%. The compounds inhibited the dissociation of [(3)H]NMS to different extents across the receptor subtypes, with the largest effects at M(1) receptors. In equilibrium binding studies the inhibitory potency of gallamine at M(1) receptors was not affected by KT5720, indicating that these agents bind to two distinct allosteric sites and have neutral cooperativity with each other. In contrast, gallamine and staurosporine had a negatively cooperative or competitive interaction at M(1) receptors. Similarly, the potency and relative effectiveness of KT5720 for inhibiting [(3)H]NMS dissociation from M(1) receptors were not affected by gallamine or brucine, but were affected in a complex manner by staurosporine. These results demonstrate that there are at least two distinct allosteric sites on the M(1) receptor, both of which can support positive cooperativity with acetylcholine.     

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.     

Interaction of Mg2+ with the allosteric site of muscarinic M2 receptors

Mg²⁺-ions have been suspected to attenuate the inhibitory effect of allosteric modulators on the dissociation of orthosteric ligands from muscarinic M₂ receptors. It was aimed to gain more insight into the molecular events underlying the effect of Mg²⁺. The interaction of Mg²⁺ with the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3’-phthalimidopropylammonium bromide]) and Chin3/6 (hexane-1,6-bis[dimethyl-3’-{4-oxo-2-phenyl-3,4-dihydro-2H-quinazolin-1-yl}propylammonium bromide]) was studied in porcine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [³H]N-methylscopolamine ([³H]NMS). The incubation buffer was composed of 4 mM Na₂HPO₄ and 1 mM KH₂PO₄ (pH 7.4, 23°C). The retardation of [³H]NMS dissociation (control t_1/2 = 5.6 min) induced by the allosteric test compounds was diminished by 3 mM Mg²⁺ to a greater extent than to be expected with regard to its contribution to the ionic strength of the buffer solution. Concentration-effect curves for the allosteric retardation of [³H]NMS dissociation by W84 (half maximal effective concentration EC_0.5 = 24 nM in the absence of Mg²⁺) and by Chin3/6 (EC_0.5 = 28 nM) were shifted by Mg²⁺ to the right in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg²⁺ and the modulators. The pK _b-values as a measure of the antagonistic potency of Mg²⁺, however, differed depending on the modulator, i.e. pK _b = 3.4 with W84 and pK _b = 2.8 with Chin3/6. Mg²⁺ itself was capable of slowing the dissociation of [³H]NMS; the maximal retardation of [³H]NMS dissociation was about 3fold, the concentration-effect relationship was compatible with a two-site model using the above-mentioned pK _b-values as affinity constants. Since the equilibrium-binding of [³H]NMS remained unchanged up to a Mg²⁺-concentration of 3 mM, the cation appears to inhibit the association and dissociation of [³H]NMS to the same extent in this concentration range. Taken together, the findings indicate that Mg²⁺ may bind to the allosteric region of muscarinic M₂ receptors and that more than one site is involved in this interaction. The sites of action may represent divalent cation binding sites. Received: 1 October 1997 / Accepted: 20 January 1998     

Probing the molecular mechanism of interaction between 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the muscarinic M(1) receptor: direct pharmacological evidence that AC-42 is an allosteric agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M(1) receptor previously suggested to interact with an "ectopic" site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M(1) receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [(3)H]N-methylscopolamine ([(3)H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [(3)H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [(3)H]NMS dissociation from CHO-hM(1) cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M(1) receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M(1) receptors in the absence of the orthosteric agonist.     

Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors

Two fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore (in ortho and para positions) to AC42-NH(2). This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore (alone or linked to an alkyl chain). All these compounds are antagonists, but examination of their ability to inhibit or modulate orthosteric [(3)H]NMS binding revealed that para-LRB-AC42 shared several properties with AC-42. Carefully designed experiments allowed para-LRB-AC42 to be used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium binding conditions, orthosteric ligands, AC-42, and the allosteric modulator gallamine behaved as competitors of para-LRB-AC42 binding whereas other allosteric compounds such as WIN 51,708 and N-desmethylclozapine were noncompetitive inhibitors. Finally, molecular modeling studies focused on putative orthosteric/allosteric bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human M1 receptor.     

Allosteric binding sites on muscarinic acetylcholine receptors

In this issue of Molecular Pharmacology, Tr?nkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tr?nkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tr?nkle et al. should be of considerable general interest.     

New insights into the function of M4 muscarinic acetylcholine receptors gained using a novel allosteric modulator and a DREADD (designer receptor exclusively activated by a designer drug)

The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.