Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

Further optimization of the M5 NAM MLPCN probe ML375: tactics and challenges.

This Letter describes the continued optimization of the MLPCN probe ML375, a highly selective M5 negative allosteric modulator (NAM), through a combination of matrix libraries and iterative parallel synthesis. True to certain allosteric ligands, SAR was shallow, and the matrix library approach highlighted the challenges with M5 NAM SAR within in this chemotype. Once again, enantiospecific activity was noted, and potency at rat and human M5 were improved over ML375, along with slight enhancement in physiochemical properties, certain in vitro DMPK parameters and CNS distribution. Attempts to further enhance pharmacokinetics with deuterium incorporation afforded mixed results, but pretreatment with a pan-P450 inhibitor (1-aminobenzotriazole; ABT) provided increased plasma exposure.

Development of a highly potent, novel M5 positive allosteric modulator (PAM) demonstrating CNS exposure: 1-((1H-indazol-5-yl)sulfoneyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380).

A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).

Discovery of the first M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetylcholine receptor: (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one (ML375).

A functional high throughput screen and subsequent multidimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with submicromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1-M4 IC50 > 30 µM), excellent multispecies PK, high CNS penetration, and enantiospecific inhibition.

Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM(5) ).

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with some nerve agents. Promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128 which was partly attributed to its interaction with nicotinic acetylcholine receptors. Previous studies indicate that bispyridinium compounds interact with muscarinic acetylcholine receptors as well. The muscarinic M(5) receptor is not well investigated compared to other subtypes, but could be important in the search for new drugs for treating nerve agent poisoning. A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M(5) muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.

Molecular conversion of muscarinic acetylcholine receptor M(5) to muscarinic toxin 7 (MT7)-binding protein.

Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M(1) muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M(1) receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M(5) subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M(1) receptor. An M(5) receptor construct (M(5)-E(175)Y(184)E(474)), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent K(i) = 24 nM; apparent K(i) for M(1) = 0.5 nM). After screening for different domains and residues, we found a specific residue (P(179) to L in M(5)) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M(5)-EL(179)YE, apparent K(i) = 0.5 nM). Mutation of P(179) to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors.

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.

The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization.

Besides some pharmacological, biochemical and biophysical evidences support the contention that muscarinic acetylcholine receptors can form homo- and heterodimers, the existence of specific M(3) and M(5) muscarinic receptors oligomers in living cells is a new concept. Interestingly, this phenomenon might have relevance in lymphocytic cholinergic function since both T- and B-cells naturally express high levels of these two receptor subtypes. Here, by means of co-immunoprecipitation and bioluminescence resonance energy transfer methods we demonstrated that M(3) and M(5) muscarinic receptors could form constitutive homo- and heterodimers in transiently transfected HEK-293T cells. Interestingly, this receptor-receptor interaction was unaltered by carbachol treatment but it was affected by the expression of a peptide corresponding to a portion of the third intracellular loop of the M(5) muscarinic receptor. In addition, the same peptide was able to abrogate the carbachol-induced mitogen-activated protein kinase phosphorylation and the carbachol-enhanced PHA-induced IL-2 production in derived lymphocytic T cells. Overall, these results suggest that the third intracellular loop of the M(5) muscarinic receptor might play a regulatory role in receptor function and heteromerization, thus providing the molecular framework for a potential cholinergic-based therapeutic intervention of the immune system.

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.