Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Abstract

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 M 2 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]Nmethylscopolamine 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 M 2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M 1 and M 3 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.GPCR | allosteric modulators | ensemble docking | affinity | cooperativity

Gaussian Accelerated Molecular Dynamics: Theory, Implementation, and Applications

“…For systems of increasing size, the upper limit of the $\mathrm{normal\Delta }V$ standard deviation, ${\sigma }_0$ can be adjusted dynamically to ensure that the distribution width of the applied boost potential is narrow enough for accurate energetic reweighting using cumulant expansion to the second order. Therefore, GaMD serves as a promising tool for biomolecular conformation sampling, prediction of drug-receptor interactions and computer-aided drug design and discovery 108, 109 .…”

Computational chemical biology and drug design: Facilitating protein structure, function, and modulation studies

“…For example, methodology used to monitor receptor activity, detection of appropriate downstream signals, selection of orthosteric‐ligand probe, and assay time course must be considered when implementing high‐throughput screening (HTS) campaign for allosteric modulators . Moreover, for classical assays and analyses, such as radioligand binding and the assessment of compound affinity, the properties of allosteric modulators yield data that are less distinct than orthosteric ligand–receptor interactions . Consequently, some real interesting compounds will not be prioritized as screening hits by the conventional assay readouts.…”

“…In addition to protein–ligand interaction, protein flexibility also determines allostery and affects the recognition of binding partners involving protein–protein and protein–RNA/DNA interactions . There are some reports that ensemble receptor‐based protocols showed better performance on reproducing ligand‐binding poses, predicting binding affinity, and discriminating power between active and inactive molecular profiles compared to using a single crystal structure . Overall, protein ensemble has demonstrated its utility in various context, but it is still a rational and efficient methods to select and/or generate multiple protein conformations …”

“…99,100 Moreover, for classical assays and analyses, such as radioligand binding and the assessment of compound affinity, the properties of allosteric modulators yield data that are less distinct than orthosteric ligandreceptor interactions. 98,101 Consequently, some real interesting compounds will not be prioritized as screening hits by the conventional assay readouts. These factors and potential issues pose challenges to the identification of allosteric inhibitors.…”

Inhibiting Pneumococcal Surface Antigen A (PsaA) with Small Molecules Discovered through Virtual Screening: Steps toward Validating a Potential Target for Streptococcus pneumoniae

“…Such targeting has identified inhibitors in other diverse proteins such as Akt1, [53] Bcr-Abl, [54] and the muscarinic G proteincoupled receptor. [55] The examination of both open (3zk7) and closed (3ztt) conformations of the PsaA protein revealed a potential allosteric site that might also be druggable. This site is formed in part by two loops (see Figure 4F) that show significant translation between the open and closed conformations.…”