Accelerated structure based design of chemically diverse allosteric modulators of a muscarinic G protein coupled receptor
Significance Chemical diversity has recently risen as key structural feature for the discovery of novel selective drugs of G protein coupled receptors (GPCRs) However, the traditional drug discovery technique of combinatorial chemistry coupled to high throughput screening has become less attractive because of its immense financial impact. To address this problem, we implemented a computer aided drug design approach, using the M2 muscarinic acetylcholine receptor (mAChR) as a GPCR model, and performed computational enhanced sampling simulations to account for the receptor flexibility. Through iterative molecular docking and experimental testing, half of the 38 computationally selected National Cancer Institute compounds were validated as allosteric modulators of the M2 mAChR. Our method successfully identified positive and negative allosteric modulators of M2 mAChR with unprecedented chemical diversity. 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 [3H]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 mM. 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.
Proceedings of the National Academy of Sciences