Xanomeline displays concomitant orthosteric and allosteric binding modes at the M4 mAChR.

The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M4 mAChR in complex with the heterotrimeric Gi1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M4 mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M4 mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.

Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors.

Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

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.

Recessive CHRM5 variant as a potential cause of neurogenic bladder.

Neurogenic bladder is caused by disruption of neuronal pathways regulating bladder relaxation and contraction. In severe cases, neurogenic bladder can lead to vesicoureteral reflux, hydroureter, and chronic kidney disease. These complications overlap with manifestations of congenital anomalies of the kidney and urinary tract (CAKUT). To identify novel monogenic causes of neurogenic bladder, we applied exome sequencing (ES) to our cohort of families with CAKUT. By ES, we have identified a homozygous missense variant (p.Gln184Arg) in CHRM5 (cholinergic receptor, muscarinic, 5) in a patient with neurogenic bladder and secondary complications of CAKUT. CHRM5 codes for a seven transmembrane-spanning G-protein-coupled muscarinic acetylcholine receptor. CHRM5 is shown to be expressed in murine and human bladder walls and is reported to cause bladder overactivity in Chrm5 knockout mice. We investigated CHRM5 as a potential novel candidate gene for neurogenic bladder with secondary complications of CAKUT. CHRM5 is similar to the cholinergic bladder neuron receptor CHRNA3, which Mann et al. published as the first monogenic cause of neurogenic bladder. However, functional in vitro studies did not reveal evidence to strengthen the status as a candidate gene. Discovering additional families with CHRM5 variants could help to further assess the genes' candidate status.

Structural basis of efficacy-driven ligand selectivity at GPCRs.

A drug's selectivity for target receptors is essential to its therapeutic utility, but achieving selectivity between similar receptors is challenging. The serendipitous discovery of ligands that stimulate target receptors more strongly than closely related receptors, despite binding with similar affinities, suggests a solution. The molecular mechanism of such 'efficacy-driven selectivity' has remained unclear, however, hindering design of such ligands. Here, using atomic-level simulations, we reveal the structural basis for the efficacy-driven selectivity of a long-studied clinical drug candidate, xanomeline, between closely related muscarinic acetylcholine receptors (mAChRs). Xanomeline's binding mode is similar across mAChRs in their inactive states but differs between mAChRs in their active states, with divergent effects on active-state stability. We validate this mechanism experimentally and use it to design ligands with altered efficacy-driven selectivity. Our results suggest strategies for the rational design of ligands that achieve efficacy-driven selectivity for many pharmaceutically important G-protein-coupled receptors.

Importance of receptor expression in the classification of novel ligands at the M2 muscarinic acetylcholine receptor.

BACKGROUND AND PURPOSE: Affinity-based, selective orthosteric ligands for the muscarinic acetylcholine receptors (mAChRs) are difficult to develop due to high sequence homology across the five subtypes. Selectivity can also be achieved via the selective activation of a particular subtype or signalling pathway. Promisingly, a prior study identified compounds 6A and 7A as functionally selective and Gi biased compounds at the M2 mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M2 mAChR. EXPERIMENTAL APPROACH: G protein activation was measured with the TRUPATH assay in M2 mAChR FlpIn CHO cells. Activity in downstream signalling pathways was determined using the cAMP CAMYEL BRET sensor and assay of ERK 1/2 phosphorylation. KEY RESULTS: M2 mAChRs coupled to Gɑi1 , GɑoA and Gɑs , but not Gɑq , in response to canonical orthosteric agonists. Compounds 6A and 7A did not elicit any G protein activation, cAMP inhibition or stimulation, or ERK 1/2 phosphorylation. Instead, a Schild analysis indicates a competitive, antagonistic interaction of compounds 6A and 7A with ACh in the Gɑi1 activation assay. Overexpression of the M2 mAChR may suggest an expression-dependent activation profile of compounds 6A and 7A. CONCLUSIONS AND IMPLICATIONS: These data confirm that the M2 mAChR preferentially couples to Gɑi/o and to a lesser extent to Gɑs in response to canonical orthosteric ligands. However, this study was not able to detect Gɑi bias of compounds 6A and 7A, highlighting the importance of cellular background when classifying new ligands.

Identification of a Novel Allosteric Site at the M5 Muscarinic Acetylcholine Receptor.

The M5 muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M5 mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M5 mAChR selective modulators. To determine the possible location of the ML375 binding site, we used radioligand binding and functional assays to show that ML375 does not interact with the well-characterized "common" mAChR allosteric site located in the receptor's extracellular vestibule, nor a previously proposed second allosteric site recognized by the modulator, amiodarone. Molecular docking was used to predict potential allosteric sites within the transmembrane (TM) domain of the M5 mAChR. These predicted sites were assessed using M5-M2 mAChR receptor chimeras and further targeted with site-directed mutagenesis, which enabled the identification of a putative binding site for ML375 at the interface of TMs 2-4. Collectively, these results identify a third allosteric site at the M5 mAChR and highlight the ability of allosteric modulators to selectively target highly conserved proteins.

Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic 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 M5 mAChR 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 M2 and M5 mAChRs, 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.

6-Phenylpyrimidin-4-ones as Positive Allosteric Modulators at the M1 mAChR: The Determinants of Allosteric Activity.

Targeting allosteric sites of the M1 muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer's disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M1 mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M1 mAChR PAMs.

Toward an understanding of the structural basis of allostery in muscarinic acetylcholine receptors.

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.

Structure-Affinity Relationships and Structure-Kinetics Relationships of Pyrido[2,1-f]purine-2,4-dione Derivatives as Human Adenosine A3 Receptor Antagonists.

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.