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

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

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells

The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25–81) and a C-terminal peptide fragment GPR15L(71–81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3′,5′-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs, Gq/11 and G12/13 is unclear. Here, GPR15L(25–81) and GPR15L(71–81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1, Gi2, Gi3, GoA, GoB and Gz). The Emax and activation rates of Gi1, Gi2, Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin

Rules and mechanisms governing G protein coupling selectivity of GPCRs

Summary: G protein-coupled receptors (GPCRs) convert extracellular stimuli into intracellular signaling by coupling to heterotrimeric G proteins of four classes: Gi/o, Gq, Gs, and G12/13. However, our understanding of the G protein selectivity of GPCRs is incomplete. Here, we quantitatively measure the enzymatic activity of GPCRs in living cells and reveal the G protein selectivity of 124 GPCRs with the exact rank order of their G protein preference. Using this information, we establish a classification of GPCRs by functional selectivity, discover the existence of a G12/13-coupled receptor, G15-coupled receptors, and a variety of subclasses for Gi/o-, Gq-, and Gs-coupled receptors, culminating in development of the predictive algorithm of G protein selectivity. We further identify the structural determinants of G protein selectivity, allowing us to synthesize non-existent GPCRs with de novo G protein selectivity and efficiently identify putative pathogenic variants

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 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]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 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

Eigenstate Thermalization, Random Matrix Theory and Behemoths

The eigenstate thermalization hypothesis (ETH) is one of the cornerstones in our understanding of quantum statistical mechanics. The extent to which ETH holds for nonlocal operators is an open question that we partially address in this paper. We report on the construction of highly nonlocal operators, Behemoths, that are building blocks for various kinds of local and non-local operators. The Behemoths have a singular distribution and width w∼D−1 (D being the Hilbert space dimension). From them, one may construct local operators with the ordinary Gaussian distribution and w∼D−1/2 in agreement with ETH. Extrapolation to even larger widths predicts sub-ETH behavior of typical nonlocal operators with w∼D−δ, 0<δ<1/2. This operator construction is based on a deep analogy with random matrix theory and shows striking agreement with numerical simulations of non-integrable many-body systems