Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.     

Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action.     

A Monod-Wyman-Changeux mechanism can explain G protein-coupled receptor (GPCR) allosteric modulation

The Monod-Wyman-Changeux (MWC) model was initially proposed to describe the allosteric properties of regulatory enzymes and subsequently extended to receptors. Yet despite GPCRs representing the largest family of receptors and drug targets, no study has systematically evaluated the MWC mechanism as it applies to GPCR allosteric ligands. We reveal how the recently described allosteric modulator, benzyl quinolone carboxylic acid (BQCA), behaves according to a strict, two-state MWC mechanism at the M1 muscarinic acetylcholine receptor (mAChR). Despite having a low affinity for the M1 mAChR, BQCA demonstrated state dependence, exhibiting high positive cooperativity with orthosteric agonists in a manner that correlated with efficacy but negative cooperativity with inverse agonists. The activity of BQCA was significantly increased at a constitutively active M1 mAChR but abolished at an inactive mutant. Interestingly, BQCA possessed intrinsic signaling efficacy, ranging from near-quiescence to full agonism depending on the coupling efficiency of the chosen intracellular pathway. This latter cellular property also determined the difference in magnitude of positive cooperativity between BQCA and the orthosteric agonist, carbachol, across pathways. The lack of additional, pathway-biased, allosteric modulation by BQCA was confirmed in genetically engineered yeast strains expressing different chimeras between the endogenous yeast G(pa1) protein and human Gα subunits. These findings define a chemical biological framework that can be applied to the study and classification of allosteric modulators across different GPCR families.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.     

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (E_max(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding E_max(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.     

Allosteric modulation of glycine receptors is more efficacious for partial rather than full agonists

Allosteric modulation of [3H]strychnine binding to glycine receptors (GlyRs) was examined in synaptosomal membranes of rat spinal cord. An allosteric model enabled us to determine the cooperativity factors of the allosteric agents with [3H]strychnine and glycine bindings (alpha and beta, respectively). We modified the allosteric model with a slope factor because the slope values of the displacement curves of partial agonists (beta-alanine, taurine and gamma-aminobutyric acid) were beyond unity. The slope factor was reduced only by 100 microM propofol. Further, propofol showed positive cooperativity (beta < 1) stronger with taurine than with glycine. The extent of the positive cooperativity of propofol was nearly independent from the potencies and structures of partial agonists. The steroidal alphaxalone and minaxolone also potentiated taurine better than glycine. Alphaxalone exerted weak negative cooperativity with [3H]strychnine binding. Displacement by taurine is attenuated by granisetron and m-chlorophenylbiguanide representing negative cooperativity (beta > 1) greater than with glycine. The results suggest a developmental role of elevated perinatal levels of taurine and neurosteroids as well as a better allosteric modulation of decreased agonist efficacies for impaired glycine receptor-ionophores.     

Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS*

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M₄ mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M₁/M₄ mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M₄ mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile⁹³ and Lys⁹⁵ as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe¹⁸⁶ in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp⁴³² in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr⁴³⁹ and Tyr⁴⁴³, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.     

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.     

Multiple allosteric sites on muscarinic receptors

Proteins and small molecules are capable of regulating the agonist binding and function of G-protein coupled receptors by multiple allosteric mechanisms. In the case of muscarinic receptors, there is the well-characterised allosteric site that binds, for example, gallamine and brucine. The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding. The binding of brucine and gallamine does not affect KT5720 binding nor its effects on the dissociation of [3H]-N-methylscopolamine from M1 receptors. Therefore it is possible to have a muscarinic receptor with three small ligands bound simultaneously. A model of the M1 receptor, based on the recently determined structure of rhodopsin, has the residues that have been shown to be important for gallamine binding clustered within and to one side of a cleft in the extracellular face of the receptor. This cleft may represent the access route of acetylcholine to its binding site.     

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.     

Allosteric Inhibition of Serotonin 5-HT<Subscript>7</Subscript> Receptors by Zinc Ions

The allosteric regulation of G protein-coupled receptors (GPCRs) is a well-known phenomenon, but there are only a few examples of allosteric modulation within the metabotropic serotonergic receptor family. Recently, we described zinc non-competitive interactions toward agonist binding at serotonin 5-HT_1A receptors, in which biphasic effects, involving potentiation at sub-micromolar concentrations (10 μM) and inhibition at sub-millimolar concentrations (500 μM) of Zn²⁺ in radioligand binding assays, were consistent with both the agonist and antagonist-like effects of zinc ions observed in in vivo studies. Here, we showed new data demonstrating zinc allosteric inhibition of both agonist and antagonist binding at human recombinant 5-HT₇ receptors stably expressed in HEK293 cells as observed by radioligand binding studies as well as zinc neutral antagonism displayed by the concentration of 10 μM in the functional LANCE assay. The allosteric nature of the effect of Zn on 5-HT₇ receptors was confirmed (1) in saturation studies in which zinc inhibited the binding of potent orthosteric 5-HT₇ receptor radioligands, the agonist [³H]5-CT, and the two antagonists [³H]SB-269970 and [³H]mesulergine, showing ceiling effect and differences in the magnitude of negative cooperativity (α = 0.15, 0.06, and 0.25, respectively); (2) in competition experiments in which 500 μM of zinc inhibited all radioligand displacements by non-labeled orthosteric ligands (5-CT, SB-269970, and clozapine), and the most significant reduction in affinity was observed for the 5-CT agonist (4.9–16.7-fold) compared with both antagonists (1.4–3.9-fold); and (3) in kinetic experiments in which 500 μM zinc increased the dissociation rate constants for [³H]5-CT and [³H]mesulergine but not for [³H]SB-269970. Additionally, in the functional LANCE test using the constitutively active HEK293 cell line expressing the 5-HT₇ receptor, 10 μM zinc had features of neutral antagonism and increased the EC₅₀ value of the 5-CT agonist by a factor of 3.2. Overall, these results showed that zinc can act as a negative allosteric inhibitor of 5-HT₇ receptors. Given that the inhibiting effects of low concentrations of zinc in the functional assay represent the most likely direction of zinc activity under physiological conditions, among numerous zinc-regulated proteins, the 5-HT₇ receptor can be considered a serotonergic target for zinc modulation in the CNS.     

Designing human m1 muscarinic receptor-targeted hydrophobic eigenmode matched peptides as functional modulators

A new proprietary de novo peptide design technique generated ten 15-residue peptides targeting and containing the leading nontransmembrane hydrophobic autocorrelation wavelengths, “modes”, of the human m₁ muscarinic cholinergic receptor, m₁AChR. These modes were also shared by the m₄AChR subtype (but not the m₂, m₃, or m₅ subtypes) and the three-finger snake toxins that pseudoirreversibly bind m₁AChR. The linear decomposition of the hydrophobically transformed m₁AChR amino acid sequence yielded ordered eigenvectors of orthogonal hydrophobic variational patterns. The weighted sum of two eigenvectors formed the peptide design template. Amino acids were iteratively assigned to template positions randomly, within hydrophobic groups. One peptide demonstrated significant functional indirect agonist activity, and five produced significant positive allosteric modulation of atropine-reversible, direct-agonist-induced cellular activation in stably m₁AChR-transfected Chinese hamster ovary cells, reflected in integrated extracellular acidification responses. The peptide positive allosteric ligands produced left-shifts and peptide concentration-response augmentation in integrated extracellular acidification response asymptotic sigmoidal functions and concentration-response behavior in Hill number indices of positive cooperativity. Peptide mode specificity was suggested by negative crossover experiments with human m₂ACh and D₂ dopamine receptors. Morlet wavelet transformation of the leading eigenvector-derived, m₁AChR eigenfunctions locates seven hydrophobic transmembrane segments and suggests possible extracellular loop locations for the peptide-receptor mode-matched, modulatory hydrophobic aggregation sites.     

Positive and negative inotropic effects of muscarinic receptor stimulation in mouse left atria

In isolated mouse left atria, acetylcholine (ACh) produced a biphasic inotropic response; a transient decrease in developed tension was followed by an increase. Both negative and positive responses were concentration dependent and were inhibited by atropine. The negative and positive inotropic responses were also observed with a nonselective muscarinic stimulant, oxotremorine-M, but not with an M1-receptor selective stimulant, McN-A343. Pirenzepine, an M1-receptor antagonist, inhibited both negative and positive inotropic responses at high concentrations. Gallamine, an M2-receptor antagonist, inhibited the negative response. Hexahydro-siladifenidol hydrochloride, p-fluoro analog (p-F-HHSiD), an M3-receptor antagonist, inhibited the positive response with no effect on the negative phase. In pertussis toxin (PTX) treated preparations, negative inotropic response to ACh was not observed. These results suggest that the negative and positive inotropic responses to acetylcholine in mouse atria are mediated by M2 and M3 receptors, respectively. The negative phase, but not the positive phase, was mediated by a PTX-sensitive G protein.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

Effect of organophosphates on dopamine and muscarinic receptor binding in rat brain

The effect of Soman, Sarin and Vx, known potent cholinesterase inhibitors, on the binding of several neurotransmitter receptors in various regions of brain was studied. Vx, exhibited considerable inhibition of binding of 3H-N-methylscopolamine (3H-NMS) to muscarinic receptors and of 3H-spiperone to dopamine D2 receptors in the striatum. 3H-NMS binding was 50% inhibited at 10(-6)M and 90% at 10(-3)M Vx. Inhibition of 3H-spiperone binding by Vx in striatum had an ID50 of 10(-5)M. KD of the treatment was affected more than Bmax. Binding inhibition of both 3H-NMS and 3H-spiperone in post-mortem brain of rats pre-treated with Vx confirmed the specificity of the organophosphates effect, since other organophosphates and ligands failed to show any activity.     

The allosteric site regulates the voltage sensitivity of muscarinic receptors

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein–coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M₁-Rs and M₃-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G_q protein cycle. In the presence of ACh, M₁-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M₃-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed “allosteric site” M₃/M₁-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M₃-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.     

Synthesis of (nor)tropeine (di)esters and allosteric modulation of glycine receptor binding

(Hetero)aromatic mono- and diesters of tropine and nortropine were prepared. Modulation of [3H]strychnine binding to glycine receptors of rat spinal cord was examined with a ternary allosteric model. The esters displaced [3H]strychnine binding with nano- or micromolar potencies and strong negative cooperativity. Coplanarity and distance of the ester moieties of diesters affected the binding affinity being nanomolar for isophthaloyl-bistropane and nortropeines. Nortropisetron had the highest affinity (K(A) approximately 10 nM). Two esters displayed negative cooperativity with glycine in displacement, while three esters of low-affinity and nortropisetron exerted positive cooperativity with glycine.     

A missense mutation in the seven-transmembrane domain of the human Ca2+ receptor converts a negative allosteric modulator into a positive allosteric modulator

G protein-coupled receptors (GPCRs) are the most common targets of drug action. Allosteric modulators bind to the seven-transmembrane domain of family 3 GPCRs and offer enhanced selectivity over orthosteric ligands that bind to the large extracellular N terminus. We characterize a novel negative allosteric modulator of the human Ca(2+) receptor, Compound 1, that retains activity against the E837A mutant that lacks a response to previously described positive and negative modulators. A related compound, JKJ05, acts as a negative allosteric modulator on the wild type receptor but as a positive modulator on the E837A mutant receptor. This positive modulation critically depends on the primary amine in JKJ05, which appears to interact with acidic residue Glu(767) in our model of the seven-transmembrane domain of the receptor. Our results suggest the need for identification of possible genetic variation in the allosteric site of therapeutically targeted GPCRs.