We expanded on a series of pyrido[2,1-f]purine-2,4-dione derivatives as human adenosine A3 receptor (hA3R) antagonists to determine their kinetic profiles and affinities. Many compounds showed high affinities and a diverse range of kinetic profiles. We found hA3R antagonists with very short residence time (RT) at the receptor (2.2 min for 5) and much longer RTs (e.g., 376 min for 27 or 391 min for 31). Two representative antagonists (5 and 27) were tested in [35S]GTPγS binding assays, and their RTs appeared correlated to their (in)surmountable antagonism. From a kon–koff–KD kinetic map, we divided the antagonists into three subgroups, providing a possible direction for the further development of hA3R antagonists. Additionally, we performed a computational modeling study that sheds light on the crucial receptor interactions, dictating the compounds’ binding kinetics. Knowledge of target binding kinetics appears useful for developing and triaging new hA3R antagonists in the early phase of drug discovery.

The human M5muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2and M5mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

Recent breakthroughs and developments in structural biology have led to a spate of crystal structures for G protein-coupled receptors (GPCRs). This is the case for the muscarinic acetylcholine receptors (mAChRs) where inactive-state structures for four of the five subtypes and two active-state structures for one subtype are available. These mAChR crystal structures have provided new insights into receptor mechanisms, dynamics, and allosteric modulation. This is highly relevant to the mAChRs given that these receptors are an exemplar model system for the study of GPCR allostery. Allosteric mechanisms of the mAChRs are predominantly consistent with a two-state model, albeit with some notable recent exceptions. Herein, we discuss the mechanisms for positive and negative allosteric modulation at the mAChRs and compare and contrast these to evidence offered by pharmacological, biochemical, and computational approaches. This analysis provides insight into the fundamental pharmacological properties exhibited by GPCR allosteric modulators, such as enhanced subtype selectivity, probe dependence, and biased modulation while highlighting the current challenges that remain. Though complex, enhanced molecular understanding of allosteric mechanisms will have considerable influence on our understanding of GPCR activation and signaling and development of therapeutic interventions.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.