Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor.
Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [(3)H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.
Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093; Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093; yimiao@ucsd.edu jmccammon@ucsd.edu celine.valant@monash.edu.
Yinglong
Y
Miao
Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093;
Dahlia Anne
DA
Goldfeld
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia;
Ee Von
EV
Moo
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia;
Patrick M
PM
Sexton
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia;
Arthur
A
Christopoulos
Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093; Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093 yimiao@ucsd.edu jmccammon@ucsd.edu celine.valant@monash.edu.
J Andrew
JA
McCammon
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville VIC 3052, Australia; yimiao@ucsd.edu jmccammon@ucsd.edu celine.valant@monash.edu.
Celine
C
Valant
D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D007854:Lead / Q000737:chemistry* / Q000494:pharmacology; D008024:Ligands; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation / Q000187:drug effects; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry; D043585:Receptor, Muscarinic M2 / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry; D013329:Structure-Activity Relationship
D008024:Ligands; D043643:Receptor, Muscarinic M1; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D007854:Lead
GPCR; affinity; allosteric modulators; cooperativity; ensemble docking