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

Abstract

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.

Figure 1

Comparison of the M2R and M3R structures in their inactive states. (A) The overall structure of the M3R bound to tiotropium (orange) is similar to that of the M2R subtype both in overall fold and in the specific binding site contacts, shown in (B). Extracellular view of the orthosteric ligand binding site. Polar contacts are indicated with dotted lines. Residues are numbered according to the rat M3R sequence. (C) The chemical structures of tiotropium and QNB, the two inverse agonists used to stabilize the M3R and M2R, respectively, for crystallographic studies. (D) A cross-section through the M3R shows the buried orthosteric ligand binding pocket and the large extracellular vestibule located superficial to it. This latter site is the target of a wide variety of allosteric modulators.

Figure 2

Activation of the M2R. (A) The overall structure of the active M2R is shown in orange. The active state-stabilizing nanobody (Nb9-8) is highlighted in magenta. The bound orthosteric agonist iperoxo is represented by yellow spheres. (B) A cross-sectional view through the active M2R shows that the ligand binding pocket has closed completely over the orthosteric agonist. (C) The binding pocket conformation for the M2R is shown in both active (orange) and inactive (blue) conformations. Polar contacts are indicated with dotted lines, and major conformational changes associated with M2R activation are highlighted with red arrows. (D) M2R activation involves closure of the tyrosine lid over the orthosteric agonist, forming a hydrogen bond network that occludes the agonist from solvent.

Figure 3

Allosteric modulation of the M2R. (A) The structure of the active M2R bound to both the orthosteric agonist iperoxo and the allosteric modulator LY2119620 is shown with transmembrane domains 6 and 7 removed for clarity. The chemical structures of the two compounds are shown to the right. (B) Contacts between the allosteric modulator and the M2R are shown, with polar contacts indicated by dotted lines.