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Targeting cholecystokinin receptors (CCKRs) signaling has become an attractive therapeutic strategy for many diseases. The description of cryo-EM structures of CCKRs in the active or inactive states reveal the molecular mechanism of ligand recognition and G-protein-coupling promiscuity.

The authors report a 2.6 Å resolution crystal structure of the human CB1 cannabinoid receptor trapped in the inactive conformation and bound to the antagonist taranabant. CB1 cannabinoid receptor structure The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the responses to endocannabinoids and the plant cannabinoid Δ 9 -tetrahydrocannabinol (THC). They are important drug discovery targets because of the therapeutic potential of receptor modulators for controlling disorders such as pain, epilepsy and obesity. Daniel Rosenbaum and colleagues determine a crystal structure of the human CB1 receptor bound to the inhibitor taranabant. The extracellular surface of the receptor is distinct from other lipid-activated GPCRs and forms a critical part of the ligand-binding pocket. Docking studies demonstrate how this pocket might accommodate tetrahydrocannabinol. The structure should aid drug discovery efforts for novel cannabinoid system modulators as potential therapeutics. The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) and to the widely consumed plant phytocannabinoid Δ 9 -tetrahydrocannabinol (THC) 1 . The cannabinoid receptors have been the targets of intensive drug discovery efforts, because modulation of these receptors has therapeutic potential to control pain 2 , epilepsy 3 , obesity 4 , and other disorders. Although much progress in understanding the biophysical properties of GPCRs has recently been made, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. Here we report the use of GPCR engineering and lipidic cubic phase crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6-Å resolution. We found that the extracellular surface of CB1, including the highly conserved membrane-proximal N-terminal region, is distinct from those of other lipid-activated GPCRs, forming a critical part of the ligand-binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function and will aid the design and optimization of therapeutic modulators of the endocannabinoid system.

Dopamine receptors are widely distributed in the central nervous system and are important therapeutic targets for treatment of various psychiatric and neurological diseases. Here, we report three cryo-electron microscopy structures of the D1 dopamine receptor (D1R)-Gs complex bound to two agonists, fenoldopam and tavapadon, and a positive allosteric modulator LY3154207. The structure reveals unusual binding of two fenoldopam molecules, one to the orthosteric binding pocket (OBP) and the other to the extended binding pocket (EBP). In contrast, one elongated tavapadon molecule binds to D1R, extending from OBP to EBP. Moreover, LY3154207 stabilizes the second intracellular loop of D1R in an alpha helical conformation to efficiently engage the G protein. Through a combination of biochemical, biophysical and cellular assays, we further show that the broad conformation stabilized by two fenoldopam molecules and interaction between TM5 and the agonist are important for biased signaling of D1R. D1 dopamine receptor is an important drug target for treatment of hypertension and Parkinson’s disease. Here, authors report three cryo-EM structures of the D1R-Gs complex bound to three distinct D1R-selective drugs.

Much effort has been invested in the investigation of the structural basis of G protein-coupled receptors (GPCRs) activation. Inverse agonists, which can inhibit GPCRs with constitutive activity, are considered useful therapeutic agents, but the molecular mechanism of such ligands remains insufficiently understood. Here, we report a crystal structure of the ghrelin receptor bound to the inverse agonist PF-05190457 and a cryo-electron microscopy structure of the active ghrelin receptor-Go complex bound to the endogenous agonist ghrelin. Our structures reveal a distinct binding mode of the inverse agonist PF-05190457 in the ghrelin receptor, different from the binding mode of agonists and neutral antagonists. Combining the structural comparisons and cellular function assays, we find that a polar network and a notable hydrophobic cluster are required for receptor activation and constitutive activity. Together, our study provides insights into the detailed mechanism of ghrelin receptor binding to agonists and inverse agonists, and paves the way to design specific ligands targeting ghrelin receptors. Ghrelin receptor regulates energy homeostasis through constitutive activity or by the ghrelin. Here the authors report two structures of ghrelin receptor bound to agonist and inverse agonist, providing insights into the mechanism of inverse agonism, which is of interest for specific ligand design.

Gonadotrophin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone, is the main regulator of the reproductive system, acting on gonadotropic cells by binding to the GnRH1 receptor (GnRH1R). The GnRH-GnRH1R system is a promising therapeutic target for maintaining reproductive function; to date, a number of ligands targeting GnRH1R for disease treatment are available on the market. Here, we report the crystal structure of GnRH1R bound to the small-molecule drug elagolix at 2.8 Å resolution. The structure reveals an interesting N-terminus that could co-occupy the enlarged orthosteric binding site together with elagolix. The unusual ligand binding mode was further investigated by structural analyses, functional assays and molecular docking studies. On the other hand, because of the unique characteristic of lacking a cytoplasmic C-terminal helix, GnRH1R exhibits different microswitch structural features from other class A GPCRs. In summary, this study provides insight into the ligand binding mode of GnRH1R and offers an atomic framework for rational drug design. The human gonadotropin-releasing hormone receptor GnRH1R is a GPCR with an important role in the human reproductive system and of interest as a drug target. Here, the authors present the 2.8 Å crystal structure of human GnRH1R with the high affinity antagonist drug Elagolix and the observed unusual ligand binding mode was further analysed with functional assays and molecular docking studies.

Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.The crystallographic and cryo-EM structures of CB1 bound to the positive allosteric modulator ZCZ011, combined with molecular dynamics simulations and mutagenesis experiments, reveal allosteric modulation of CB1 by rearrangement of the TM2 and TM3 transmembrane domains.

Allosteric drugs offer a new avenue for modern drug design. However, the identification of cryptic allosteric sites presents a formidable challenge. Following the allostery nature of residue-driven conformation transition, we propose a state-of-the-art computational pipeline by developing a residue-intuitive hybrid machine learning (RHML) model coupled with molecular dynamics (MD) simulation, through which we can efficiently identify the allosteric site and allosteric modulator as well as reveal their regulation mechanism. For the clinical target β2-adrenoceptor (β2AR), we discover an additional allosteric site located around residues D79 2.50 , F282 6.44 , N318 7.45 and S319 7.46 and one putative allosteric modulator ZINC5042. Using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and protein structure network (PSN), the allosteric potency and regulation mechanism are probed to further improve identification accuracy. Benefiting from sufficient computational evidence, the experimental assays then validate our predicted allosteric site, negative allosteric potency and regulation pathway, showcasing the effectiveness of the identification pipeline in practice. We expect that it will be applicable to other target proteins. The identification of cryptic allosteric sites presents a formidable challenge. Here, the authors develop a residue-intuitive hybrid ML model coupled with MD simulation to successfully identify a novel allosteric site of β2AR and a negative allosteric modulator.

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.

Ferroptosis is a new type of programmed cell death characterized by iron-dependent lipid peroxidation. Ferroptosis inhibition is thought as a promising therapeutic strategy for a variety of diseases. Currently, a majority of known ferroptosis inhibitors belong to either antioxidants or iron-chelators. Here we report a new ferroptosis inhibitor, termed YL-939, which is neither an antioxidant nor an iron-chelator. Chemical proteomics revealed the biological target of YL-939 to be prohibitin 2 (PHB2). Mechanistically, YL-939 binding to PHB2 promotes the expression of the iron storage protein ferritin, hence reduces the iron content, thereby decreasing the susceptibility to ferroptosis. We further showed that YL-939 could substantially ameliorate liver damage in a ferroptosis-related acute liver injury model by targeting the PHB2/ferritin/iron axis. Overall, we identified a non-classical ferroptosis inhibitor and revealed a new regulation mechanism of ferroptosis. These findings may present an attractive intervention strategy for ferroptosis-related diseases. Ferroptosis is a promising therapeutic target for a variety of diseases, but a majority of known ferroptosis inhibitors belong to either antioxidants or iron chelators. Here, the authors discover a new non-classical small molecule inhibitor that is a PHB2 binder and show it ameliorates liver damage in an acute liver injury model.