Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M₁ Muscarinic Acetylcholine Receptors

Abstract

Point mutations and molecular modeling have been used to study the activation of the M 1 muscarinic acetylcholine receptor (mAChR) by the functionally selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them with N-desmethylclozapine (NDMC) and acetylcholine (ACh). Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although they were reduced by mutations of Tyr408. Signaling by AC-42, 77-LH-28-1, and NDMC was reduced by L102A and abolished by D105E, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105 to activate the M 1 mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by W101A, and their signaling activities were abolished by Y82A. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M 1 mAChR. We suggest the hypothesis that the side chain of Trp101 undergoes conformational isomerization, opening a novel binding site for the aromatic side chain of the AC-42 analogs. This may allow the positively charged piperidine nitrogen of the ligands to access the neighboring Asp105 carboxylate to activate signaling following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not seem to use this mechanism. Subtype-specific differences in the free energy of rotation of the side chain and indole ring of Trp101 might underlie the M 1 selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design.The M 1 muscarinic receptor (mAChR) is an attractive drug target to treat Alzheimer's disease and schizophrenia (Langmead et al., 2008b). The amino acid side chains that contact acetylcholine are modeled on the inward-facing segments of transmembrane (TM) helices 3, 4, 5, 6, and 7 within the outer leaflet of the lipid bilayer (Hulme et al., 2003). Their conservation between the five receptor subtypes has restricted the discovery of selective agonists.AC-42 is a novel ligand that activates M 1 mAChRs selectively (Spalding et al., 2002). Activation is little affected by alanine substitution of the orthosteric binding pocket residues Tyr381 (6.51; Ballesteros and Weinstein, 1995) and Asn382 (6.52) but requires sequences in the N terminus and TM1, extracellular loop 3, and the outer part of TM7. Thus AC-42 may activate M 1 mAChRs from a distinct "ectopic" site (Spalding et al., 2002). Supporting this, AC-42 can display allosteric interactions with N-methyl scopolamine (NMS) and atropine (Langmead et al., 2006). A study of TM3 showed that activation of M 1 mAChRs by AC-42 analogs was unaffected by alanine substitution of Tyr106 (3.33) neighboring the ACh counter-ion Asp105 (3.32) or Ser109 (3.36) one helical turn below but was inhibited by L102A (3.29) and paradoxically very ...

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

“…Consistent with previous reports (15,22,23), improved binding of TBPB and 77-LH-28-1 was detected at the W101 3.28 A receptor. This was also observed for the agonist fragment derivative of TBPB (VCP794), and all three ligands exhibited improved binding at the Y381 6.51 A receptor (Fig.…”

“…In comparison, the efficacy of 77-LH-28-1-activated pERK1/2 was reduced at the F77 2.56 I mutant (Fig. 10C), consistent with previous reports (15,22,23). However, its efficacy in [Ca 2ϩ ] i mobilization experiments was unchanged (Fig.…”

“…Intriguingly, although 77-LH-28-1 exhibited a small but significant reduction in affinity at the F77 2.56 I mutation, consistent with previous findings (15), it also exhibited reduced affinity (ϳ3-fold) at the Y82 2.61 A receptor (Fig. 3D), in contrast to an earlier report (22). Furthermore, unlike the structurally similar TBPB, VCP794 displayed reduced affinity for the F77 2.56 I mutant (Fig.…”

“…13C) and VCP794 and is concordant with the effects of alanine substitution of this residue. However, although a gauche ϩ conformation of Trp-101 3.28 has been suggested as permitting 77-LH-28-1 binding to the M 1 mAChR (10,15,22), our simulations did not predict such a pronounced rotamerization of the residue. In agreement with the enhanced bitopic ligand binding affinities at the Y381 6.51 A mutant, the final ligand poses reveal only Tyr-381 6.51 positioned away from the orthosteric pocket (Fig.…”

“…3.28 A and F77 2.56 I because of their unique discriminatory effects on the function of TBPB and 77-LH-28-1 relative to other prototypical ligands (15,22,23).…”