Mechanisms of ligand recognition and activation of melanin-concentrating hormone receptors.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that regulates food intake, energy balance, and other physiological functions by stimulating MCHR1 and MCHR2 receptors, both of which are class A G protein-coupled receptors. MCHR1 predominately couples to inhibitory G protein, Gi/o, and MCHR2 can only couple to Gq/11. Here we present cryo-electron microscopy structures of MCH-activated MCHR1 with Gi and MCH-activated MCHR2 with Gq at the global resolutions of 3.01 Å and 2.40 Å, respectively. These structures reveal that MCH adopts a consistent cysteine-mediated hairpin loop configuration when bound to both receptors. A central arginine from the LGRVY core motif between the two cysteines of MCH penetrates deeply into the transmembrane pocket, triggering receptor activation. Integrated with mutational and functional insights, our findings elucidate the molecular underpinnings of ligand recognition and MCH receptor activation and offer a structural foundation for targeted drug design.

Ligand recognition and G protein coupling of the human itch receptor MRGPRX1.

MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception and targeting MRGPRX1 may have potential to treat both chronic itch and pain. Here we report cryo-EM structures of the MRGPRX1-Gi1 and MRGPRX1-Gq trimers in complex with two peptide ligands, BAM8-22 and CNF-Tx2. These structures reveal a shallow orthosteric pocket and its conformational plasticity for sensing multiple different peptidic itch allergens. Distinct from MRGPRX2, MRGPRX1 contains a unique pocket feature at the extracellular ends of TM3 and TM4 to accommodate the peptide C-terminal "RF/RY" motif, which could serve as key mechanisms for peptidic allergen recognition. Below the ligand binding pocket, the G6.48XP6.50F6.51G6.52X(2)F/W6.55 motif is essential for the inward tilting of the upper end of TM6 to induce receptor activation. Moreover, structural features inside the ligand pocket and on the cytoplasmic side of MRGPRX1 are identified as key elements for both Gi and Gq signaling. Collectively, our studies provide structural insights into understanding itch sensation, MRGPRX1 activation, and downstream G protein signaling.

Structural genomics of the human dopamine receptor system.

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson's Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

Structural basis for motilin and erythromycin recognition by motilin receptor.

Motilin is an endogenous peptide hormone almost exclusively expressed in the human gastrointestinal (GI) tract. It activates the motilin receptor (MTLR), a class A G protein-coupled receptor (GPCR), and stimulates GI motility. To our knowledge, MTLR is the first GPCR reported to be activated by macrolide antibiotics, such as erythromycin. It has attracted extensive attention as a potential drug target for GI disorders. We report two structures of Gq-coupled human MTLR bound to motilin and erythromycin. Our structures reveal the recognition mechanism of both ligands and explain the specificity of motilin and ghrelin, a related gut peptide hormone, for their respective receptors. These structures also provide the basis for understanding the different recognition modes of erythromycin by MTLR and ribosome. These findings provide a framework for understanding the physiological regulation of MTLR and guiding drug design targeting MTLR for the treatment of GI motility disorders.

GPCRs steer Gi and Gs selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.

Structural insights into the peptide selectivity and activation of human neuromedin U receptors.

Neuromedin U receptors (NMURs), including NMUR1 and NMUR2, are a group of Gq/11-coupled G protein-coupled receptors (GPCRs). NMUR1 and NMUR2 play distinct, pleiotropic physiological functions in peripheral tissues and in the central nervous system (CNS), respectively, according to their distinct tissue distributions. These receptors are stimulated by two endogenous neuropeptides, neuromedin U and S (NMU and NMS) with similar binding affinities. NMURs have gathered attention as potential drug targets for obesity and inflammatory disorders. Specifically, selective agonists for NMUR2 in peripheral tissue show promising long-term anti-obesity effects with fewer CNS-related side effects. However, the mechanisms of peptide binding specificity and receptor activation remain elusive. Here, we report four cryo-electron microscopy structures of Gq chimera-coupled NMUR1 and NMUR2 in complexes with NMU and NMS. These structures reveal the conserved overall peptide-binding mode and the mechanism of peptide selectivity for specific NMURs, as well as the common activation mechanism of the NMUR subfamily. Together, these findings provide insights into the molecular basis of the peptide recognition and offer an opportunity for the design of the selective drugs targeting NMURs.

Pharmacokinetics of S-epacadostat, an indoleamine 2,3-dioxygenase 1 inhibitor, in dog plasma and identification of its metabolites in vivo and in vitro.

S-epacadostat (S-EPA) is an efficient and selective small-molecule inhibitor of indoleamine 2,3-dioxygenase 1. It is an EPA analog with a sulfur atom instead of a nitrogen atom at the furazan C3 position. This study documents the pharmacokinetics of S-EPA in dogs and its metabolic pathway. After an oral administration of 15 mg/kg of S-EPA in dogs, the time to peak concentration was 0.80 h, the mean elimination half-life was 7.3 h, and the absolute bioavailability was 55.8%. Furthermore, we identified S-EPA metabolites in dog plasma and dog liver microsomes by UPLC-Q Exactive Orbitrap HRMS. In dog plasma, we found five metabolites, which came from glucuronidation (M1 and M2), deoxygenation (the amidine M4), glucuronidation of M4 (M3), and desulfonamidation and oxidation of M4 (the carboxylic acid M5). In dog liver microsomes, we identified three major metabolites, namely, the glucuronide conjugate (M6), a mono-oxidation product (M7), and a desulfonamidation and oxidation product (M8). Gut microbiota may cause the differences between in vivo and in vitro oxidation metabolisms. Contrary to EPA, S-EPA did not undergo dealkylation, suggesting that substituting the nitrogen with sulfur affects the metabolism of the adjacent alkyl side chain.

Clozapine affects the pharmacokinetics of risperidone and inhibits its metabolism and P-glycoprotein-mediated transport in vivo and in vitro: A safety attention to antipsychotic polypharmacy with clozapine and risperidone.

Antipsychotic polypharmacy (APP), as one maintenance treatment strategy in patients with schizophrenia, has gained popularity in real-world clinical settings. Risperidone (RIS) and clozapine (CLZ) are the most commonly prescribed second-generation antipsychotics, and they are often used in combination as APP. In this study, the pharmacokinetics of RIS and CLZ in rats were examined after co-administration to explore the reliability and rationality of co-medication with RIS and CLZ. In addition, the effects of CLZ on RIS metabolism and transport in vitro were investigated. The results illustrated that in the 7-day continuous administration test in rats, when co-administered with CLZ, the area under curve and peak concentrations of RIS were increased by 2.2- and 3.1-fold at the first dose, respectively, increased by 3.4- and 6.2-fold at the last dose, respectively. The metabolite-to-parent ratio of RIS was approximately 22% and 33% lower than those of RIS alone group at the first and last doses, respectively. Moreover, CLZ significantly increased RIS concentrations in the brain (3.0-4.8 folds) and cerebrospinal fluid (2.1-3.5 folds) in rats, which was slightly lower than the impact of verapamil on RIS after co-medication. Experiments in vitro indicated that CLZ competitively inhibited the conversion of RIS to 9-hydroxy-RIS with the inhibition constants of 1.36 and 3.0 µM in rat and human liver microsomes, respectively. Furthermore, the efflux ratio of RIS in Caco-2 monolayers was significantly reduced by CLZ at 1 µM. Hence, CLZ may affect the exposure of RIS by inhibiting its metabolism and P-glycoprotein-mediated transport. These findings highlighted that APP with RIS and CLZ might increase the plasma concentrations of RIS and 9-hydroxy-RIS beyond the safety ranges and cause toxic side effects.