Structural Biology Studies Of Peptide G Protein-Coupled Receptors

G protein-coupled receptors（GPCRs）are the largest superfamily of membrane protein receptors in the human and mediate important physiological functions,thus making them the most important targets for drug development.More than 35%of the FDA approved drugs target GPCRs.However,only about 10%of these drugs target peptide receptors of class A GPCRs.To further expand the new drug market,two peptide receptors,SSTR4（somatostatin receptor 4）and NMU2（neuromedin U receptor 2）that recognize cyclic peptide and linear peptide respectively were chosen for structural biology studies.SSTR4 is specifically distributed in the hypothalamus,cerebral cortex and striatum of the central nervous system,involved in the regulation of learning,memory,pain and inflammation-related pathways,becoming a new target for the development of non-opioid analgesic/anti-inflammatory drugs.All marketed drugs of SSTRs have particularly low affinity for SSTR4.SSTR4 was taken as the research object to uncover the ligand recognition and selectivity mechanism using cryo-EM（cryo-electronic microscopy）.After expression vectors screening,G protein screening,purification optimization and specific antibody test,we obtained two cryo-EM structures of endogenous agonist SST-14 and small molecule selective agonist J-2156with SSTR4-G_i1 complex at a resolution of 2.9（?）and 2.8（?）,respectively.Compared with the structure of SST-14-SSTR2 complex,conservative and specific ligand recognition mechanism of the peptide cyclic agonist SST-14 is uncovered.The key pharmacophore of SST-14 takes a similar binding conformation in SSTR2 and SSTR4 and has similar interaction patterns as well,but interactions outside the key pharmacophore are quite diverse.These differences are related with structural specificity of SSTR2 and SSTR4,while the flexible conformation of SST-14 allows it to maintain the high affinity for all somatostatin receptors.Due to the conformational restriction of marketed drugs,they can only selectively bind to specific receptors.The structure of SSTR4 and J-2156 complex shows a unique sub-pocket in SSTR4,and drug design targeting this sub-pocket is expected to improve the selectivity of ligands for SSTR4.NMU2 belongs to neuropeptide receptors,and unlike NMU1,which is widely expressed in peripheral tissues,NMU2 is specifically expressed in specific areas of the brain,especially the hypothalamus,medulla oblongata and pituitary gland.It has been shown that activation of NMU2 would dramatically decrease body weight and food intake in mice while in contrast inhibition of NMU2 would promote weight gain and aspiration for obesogenic food,making NMU2 a new target for the treatment of obesity.Unfortunately,drug development targeting NMU2 was lagged behind partially due to the lack of structural information.Therefore,we carried out construct optimization and ligand screening of NMU2,and conducted large crystallization experiments on NMU2 with the selective antagonists EX9657 or R-PSOP,but did not obtain crystals.Subsequently,we used the structural model of NMU2 of the solved Nm U-25–NMU2–G_i1 complex to perform molecular docking with the selective antagonist R-PSOP.Molecular docking experiment shows that R-PSOP is located at the bottom of ligand binding pocket,forming key polar and hydrophobic interactions with NMU2.In addition,the hydrophobic pocket formed by the third and fourth transmembrane domain of NMU2 also has a tight hydrophobic interaction with R-PSOP.Inositol phosphate accumulation assay verified the binding pattern of R-PSOP and demonstrated that the hydrophobic pocket determined the selectivity of R-PSOP.These structural information elucidates the recognition and selectivity mechanism of R-PSOP and provides new ideas for the design of selective ligands that target NMU2.These structural models of SSTR4 and NMU2 with diverse ligands further expand our understanding of the ligand recognition and selectivity mechanism of class A peptide receptors,providing reliable structural models for structure-based drug design by targeting these receptors.