Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists

Abstract

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

Keywords: G protein-coupled receptor; crystal structure; drug design; muscarinic receptor; subtype selectivity.

Fig. 1.

Comparison of the orthosteric binding sites of M2R and M3R. (A and B) Orthosteric binding pocket of M2R and M3R with conserved features of ligand recognition and binding affinities. The only nonconserved residue in the two binding pockets is located in the second extracellular loop (ECL2) (M2R: Phe181, M3R: Leu225). (C and D) Docking pose of compound 1c indicating that an enlarged upward-directed ring system can pass the nonconserved Phe181 of the M2 receptor.

Fig. 2.

Structure-based ligand design toward a selective M3R antagonist. (A) Spatial orientation of QNB ring B and the nonconserved Phe181 in the second extracellular loop (ECL2) of M2R. (B) Structural modifications 1, 2, and 3 confer an up-righting and rotation of ring B, as well as steric interactions with Phe181 for compound 6b (105-fold selectivity for M3 over M2).

Fig. 3.

In vivo selectivity of compound 6b. (A) Average (±SEM) heart rate (Top) and airway resistance (Bottom) response to methacholine with a 6b cumulative dose–response curve. Airway resistance was significantly decreased from the control response to methacholine, suggesting inhibition of the M3R at a 6b dose of 10⁹ mol/kg. Heart rate was significantly higher than that of the control, indicating inhibition of the M2R at a 6b dose of 10⁵ mol/kg. The asterisk indicates a significant difference from control: *P < 0.05, one-way repeated-measures ANOVA. (B) Average (±SEM) percent change from control heart rate (Top) and airway resistance (Bottom) response to methacholine dose–response with pretreatment of either saline (black) or 6b 10⁷ mol/kg (red). The airway resistance response to methacholine was significantly different in mice treated with 6b (*P < 0.05) compared with saline. There was no significant difference in the mean heart rate response between treatments. Cmpd, Compound.

Fig. 4.

Comparison of the orthosteric binding sites of M2R and M3R. (A and B) Crystal structure of M3R in complex with the selective antagonist 6o (BS46). (A) Overall structure of the M3R/mT4L/6o (BS46) complex. (B and C) Binding-pocket residues of M3R interacting with 6o (BS46). (D and E) Interaction of 6o (BS46) with a nonconserved position in the second extracellular loop (ECL2) of M2R and M3R. (D) Crystal structure shows an interaction of the fluorine group of 6o (BS46) with Leu225 in the ECL2 of M3R. (E) Superimposed structure of M2R on the M3R/6o (BS46) structure indicates a steric clash between Phe181 of M2R and the fluorine of 6o (BS46).