Allosteric modulation of muscarinic acetylcholine receptors

Abstract

Muscarinic acetylcholine receptors (mAChRs) are prototypical Family A G protein coupled-receptors. The five mAChR subtypes are widespread throughout the periphery and the central nervous system and, accordingly, are widely involved in a variety of both physiological and pathophysiological processes. There currently remains an unmet need for better therapeutic agents that can selectively target a given mAChR subtype to the relative exclusion of others. The main reason for the lack of such selective mAChR ligands is the high sequence homology within the acetylcholine-binding site (orthosteric site) across all mAChRs. However, the mAChRs possess at least one, and likely two, extracellular allosteric binding sites that can recognize small molecule allosteric modulators to regulate the binding and function of orthosteric ligands. Extensive studies of prototypical mAChR modulators, such as gallamine and alcuronium, have provided strong pharmacological evidence, and associated structure-activity relationships (SAR), for a "common" allosteric site on all five mAChRs. These studies are also supported by mutagenesis experiments implicating the second extracellular loop and the interface between the third extracellular loop and the top of transmembrane domain 7 as contributing to the common allosteric site. Other studies are also delineating the pharmacology of a second allosteric site, recognized by compounds such as staurosporine. In addition, allosteric agonists, such as McN-A-343, AC-42 and N-desmethylclozapine, have also been identified. Current challenges to the field include the ability to effectively detect and validate allosteric mechanisms, and to quantify allosteric effects on binding affinity and signaling efficacy to inform allosteric modulator SAR.

Keywords: Acetylcholine; G protein-coupled receptor; allosteric interaction; molecular modeling; muscarinic acetylcholine receptor; mutagenesis; radioligand binding; structure-activity studies; ternary complex model..

Fig. (1)

Allosteric GPCR models. A) The simple allosteric ternary complex model (ATCM), which describes the interaction between an orthosteric ligand, A, and allosteric modulator, B, in terms of their equilibrium dissociation constants (K_A, K_B) and the cooperativity factor, α, which describes the magnitude and direction of the allosteric effect on ligand binding affinity. B) The allosteric two state model (ATSM), which describes allosteric modulator effects on affinity, efficacy and the distribution of the receptor between active (R*) and inactive (R) states, in terms of distinct conformations selected by ligands according to their cooperativity factors for the different states.

Fig. (2)

Interaction between the allosteric modulators gallamine or alcuronium with the orthosteric radioligand, [³H]N-methylscopolamine ([³H]NMS) in membranes from CHO cell stably expressing the human M₃ mAChR. Curves superimposed on the data represent the best fit of the simple ATCM. The dashed line denotes the residual level of specific [³H]NMS binding in the presence of saturating gallamine concentrations.

Fig. (3)

A) Interaction between acetylcholine and gallamine at native M₂ mAChRs in the guinea pig electrically-driven left atrium. Data taken from [ 16]. B) Concentration-ratios (CR) were derived from the data in panel A and plotted in the form of a Schild regression. Solid curve denotes the fit of the ATCM to the data. Dashed line denotes the expected Schild regression for a simple competitive interaction.

Fig. (4)

Allosteric modulation of orthosteric agonist efficacy. Interaction between alcuronium and pilocarpine at human M₂ mAChRs stably expressed in CHO cells. Receptor activation was quantified as a change in the extracellular whole cell acidification rate with a Cytosensor microphysiometer.

Fig. (5)

Prototypical “common-allosteric site” mAChR modulators.

Fig. (6)

Representative “second-site” and “atypical” mAChR modulators.

Fig. (7)

Putative allosteric mAChR agonists.

Fig. (8)

Schematic representation of the relationship between residues comprising the orthosteric and “common” allosteric site on the M2 mAChR, using a homology model based on the crystal structure of inactive state bovine rhodopsin. Regions highlighted in blue incorporate the following orthosteric-site residues: W⁹⁹, D¹⁰³, S¹⁰⁷, Y¹¹⁰, W¹⁵⁵, T¹⁸⁷, T¹⁹⁰, W⁴⁰⁰, Y⁴⁰³, N⁴⁰⁴, Y⁴²⁶, Y⁴³⁰. Regions highlighted in purple incorporate the following allosteric site residues: ¹⁷²EDGE¹⁷⁵, Y¹⁷⁷, N⁴¹⁹, N⁴²², N⁴²³. The residues in yellow represent a cysteine pair, and corresponding disulphide bond between the second extracellular loop and top of TM3, that are highly conserved in over 90% of GPCRs.