Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development

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Highlights

  • Blockade of airway M3 muscarinic receptors (M3Rs) triggers bronchodilation.

  • We solved the structure of the M3R–tiotropium (a clinically used drug) complex.

  • We reported the structure of the active M2 muscarinic receptor (M2R).

  • We elucidated the structures of the orthosteric and allosteric binding sites (M2R).

  • These new findings should facilitate the development of selective M3R antagonists.

Muscarinic acetylcholine receptor antagonists are widely used as bronchodilating drugs in pulmonary medicine. The therapeutic efficacy of these agents depends on the blockade of M3 muscarinic receptors expressed on airway smooth muscle cells. All muscarinic antagonists currently used as bronchodilating agents show high affinity for all five muscarinic receptor subtypes, thus increasing the likelihood of unwanted side effects. Recent X-ray crystallographic studies have provided detailed structural information about the nature of the orthosteric muscarinic binding site (the conventional acetylcholine binding site) and an ‘outer’ receptor cavity that can bind allosteric (non-orthosteric) drugs. These new findings should guide the development of selective M3 receptor blockers that have little or no effect on other muscarinic receptor subtypes.

Introduction

Many studies have shown that chronic obstructive pulmonary disease (COPD) and asthma are associated with increased pulmonary vagal tone [1, 2, 3]. As a result, muscarinic acetylcholine (ACh) receptor (mAChR) antagonists, including ipratropium and tiotropium, are of great clinical importance for the treatment of COPD and certain forms of asthma [1, 2, 3]. Various lines of evidence indicate that multiple mAChR subtypes are expressed in the airways of experimental animals and humans [4, 5].

In vitro and in vivo studies with mAChR knockout (KO) mice have provided convincing evidence that the bronchoconstricting effects of ACh are mediated predominantly by the M3 mAChR subtype (M3R) (reviewed in [5]). Interestingly, in M2R-deficient mice, vagal stimulation resulted in enhanced bronchoconstrictor responses [6]. This finding is in good agreement with the concept that M2Rs present on pulmonary parasympathetic nerve endings function as inhibitory autoreceptors to limit ACh release [2]. It has also been reported that airways express additional mAChRs including the M1R. For example, a study with M1R KO mice strongly suggests that activation of a subpopulation of pulmonary M1Rs inhibits M3R-mediated bronchoconstriction, perhaps by stimulating the secretion of a bronchorelaxing agent from airway epithelia or pulmonary nerves [7].

At present, muscarinic antagonists that can block M3Rs with a high degree of selectivity are not available. Since blockade of pulmonary M2Rs (and perhaps M1Rs) is predicted to lower the therapeutic efficacy of muscarinic antagonists, the development of M3R antagonists with greatly reduced affinity for other mAChR subtypes appears an attractive therapeutic goal. The clinical use of selective M3R antagonists should also reduce the incidence of unwanted side effects mediated by non-M3R mAChRs which are widely distributed both in the central nervous system and in peripheral tissues [5].

Recently, X-ray crystallographic studies have led to important novel insights into mAChR structure [8••, 9••, 10••]. These new studies provide detailed information about the structural features of mAChRs in their inactive (M2R [8••] and M3R [9••]) and their active (M2R) conformations [10••]. Importantly, Kruse et al. [10••] also reported the structure of the M2R in complex with an allosteric muscarinic modulator, providing the first direct structural information about how allosteric muscarinic agents interact with their target receptors. As discussed below, these recent structural studies offer new opportunities for the development of novel muscarinic drugs with increased affinity, efficacy, and/or mAChR subtype selectivity.

Section snippets

Structure of the M3R–tiotropium complex and implications for drug development

In 2012, X-ray crystallographic techniques yielded the first high-resolution mAChR structures, the structures of the inactive states of the human M2R [8••] and the rat M3R [9••]. The overall structures of the two receptors are similar to each other and to those of other biogenic amine G protein-coupled receptors (GPCRs) that have been crystallized during the past few years [8••, 9••] (Figure 1a). The M2R and M3R were crystallized in complex with a muscarinic antagonist/inverse agonist (M2R,

Structural features of the agonist-activated M2R

Recently, Kruse et al. [10••] also solved the structure of an agonist (iperoxo)-bound, active state of the human M2R stabilized by a G protein-mimetic antibody fragment (Figure 2a). Iperoxo is an orthosteric agonist that displays extraordinarily high potency at the M2R [20].

The key features of the active conformation of the M2R are a significant outward displacement of the cytoplasmic end of TM6, together with a smaller outward movement of the C-terminal portion of TM5 and a rearrangement of

Mode of binding of an allosteric modulator to the M2R

During the past decades, the M2R has served as a model system for studying the regulation of GPCR function by small allosteric modulators [15, 16, 17]. As discussed above, the inactive M2R and M3R feature a large extracellular vestibule, which is predicted to be involved in the binding of allosteric muscarinic ligands. Strikingly, agonist activation of the M2R triggers a pronounced contraction of this outer cavity, primarily due to the inward movement of the extracellular portion of TM6 [10••].

Concluding remarks

Recent X-ray crystallographic studies with two mAChR subtypes (M2R, M3R) have offered detailed insights into the structural configuration of the orthosteric and allosteric muscarinic binding sites. This new structural information should guide the development of novel muscarinic agents endowed with a high degree of selectivity for distinct mAChR subtypes. Such agents could include PAMs, NAMs, direct allosteric agonists, or bitopic muscarinic ligands. All muscarinic antagonists in current

References and recommended reading

Papers of particular interest, published within the period of the review, have been highlighted as:

•• of outstanding interest

Acknowledgements

The structural studies summarized in this review were supported by a National Science Foundation Graduate Research Fellowship to ACK and by National Science Foundation grant CHE-1223785 and National Institutes of Health grant U19GM106990 to BKK. The work of JH and JW was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH. We thank all of our coworkers and collaborators for their invaluable contributions to the work

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