Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Snapshot of the cannabinoid receptor 1-arrestin complex unravels the biased signaling mechanism.

Cannabis activates the cannabinoid receptor 1 (CB1), which elicits analgesic and emotion regulation benefits, along with adverse effects, via Gi and ß-arrestin signaling pathways. However, the lack of understanding of the mechanism of ß-arrestin-1 (ßarr1) coupling and signaling bias has hindered drug development targeting CB1. Here, we present the high-resolution cryo-electron microscopy structure of CB1-ßarr1 complex bound to the synthetic cannabinoid MDMB-Fubinaca (FUB), revealing notable differences in the transducer pocket and ligand-binding site compared with the Gi protein complex. ßarr1 occupies a wider transducer pocket promoting substantial outward movement of the TM6 and distinctive twin toggle switch rearrangements, whereas FUB adopts a different pose, inserting more deeply than the Gi-coupled state, suggesting the allosteric correlation between the orthosteric binding pocket and the partner protein site. Taken together, our findings unravel the molecular mechanism of signaling bias toward CB1, facilitating the development of CB1 agonists.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

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.

Structures of the human dopamine D3 receptor-Gi complexes.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Structural basis of GABAB receptor-Gi protein coupling.

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Structural basis of GPBAR activation and bile acid recognition.

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.

Structural basis and molecular mechanism of biased GPBAR signaling in regulating NSCLC cell growth via YAP activity.

The G protein-coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for ß-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR-ß-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.

The recent advance and prospect of poly(ADP-ribose) polymerase inhibitors for the treatment of cancer.

Chemotherapies are commonly used in cancer therapy, their applications are limited to low specificity, severe adverse reactions, and long-term medication-induced drug resistance. Poly(ADP-ribose) polymerase (PARP) inhibitors are a novel class of antitumor drugs developed to solve these intractable problems based on the mechanism of DNA damage repair, which have been widely applied in the treatment of ovarian cancer, breast cancer, and other cancers through inducing synthetic lethal effect and trapping PARP-DNA complex in BRCA gene mutated cancer cells. In recent years, PARP inhibitors have been widely used in combination with various first-line chemotherapy drugs, targeted drugs and immune checkpoint inhibitors to expand the scope of clinical application. However, the intricate mechanisms underlying the drug resistance to PARP inhibitors, including the restoration of homologous recombination, stabilization of DNA replication forks, overexpression of drug efflux protein, and epigenetic modifications pose great challenges and desirability in the development of novel PARP inhibitors. In this review, we will focus on the mechanism, structure-activity relationship, and multidrug resistance associated with the representative PARP inhibitors. Furthermore, we aim to provide insights into the development prospects and emerging trends to offer guidance for the clinical application and inspiration for the development of novel PARP inhibitors and degraders.

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1.

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-Gi complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of ß-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine-receptor complexes, providing new insights into the mode of chemokine recognition.

The mechanism of action and chemical synthesis of FDA newly approved drug molecules.

In 2023, the U.S. Food and Drug Administration has approved 29 small molecule drugs. These newly approved small molecule drugs possess the distinct scaffolds, thereby exhibiting diverse mechanisms of action and binding modalities. Moreover, the marketed drugs have always been an important source of new drug development and creative inspiration, thereby fostering analogous endeavors in drug discovery that potentially extend to the diverse clinical indications. Therefore, conducting a comprehensive evaluation of drug approval experience and associated information will facilitate the expedited identification of highly potent drug molecules. In this review, we comprehensively summarized the relevant information regarding the clinical applications, mechanisms of action and chemical synthesis of 29 small molecule drugs, with the aim of providing a promising structural basis and design inspiration for pharmaceutical chemists.

New drug approvals for 2022: Synthesis and clinical applications.

The U.S. Food and Drug Administration has approved a total of 37 new drugs in 2022, which are composed of 20 chemical entities and 17 biologics. In particular, 20 chemical entities, including 17 small molecule drugs, 1 radiotherapy, and 2 diagnostic agents, provide privileged scaffolds, breakthrough clinical benefits, and a new mechanism of action for the discovery of more potent clinical candidates. The structure-based drug development with clear targets and fragment-based drug development with privileged scaffolds have always been the important modules in the field of drug discovery, which could easily bypass the patent protection and bring about improved biological activity. Therefore, we summarized the relevant valuable information about clinical application, mechanism of action, and chemical synthesis of 17 newly approved small molecule drugs in 2022. We hope this timely and comprehensive review could bring about creative and elegant inspiration on the synthetic methodologies and mechanism of action for the discovery of new drugs with novel chemical scaffolds and extended clinical indications.

LSD1: an emerging face in altering the tumor microenvironment and enhancing immune checkpoint therapy.

Dysregulation of various cells in the tumor microenvironment (TME) causes immunosuppressive functions and aggressive tumor growth. In combination with immune checkpoint blockade (ICB), epigenetic modification-targeted drugs are emerging as attractive cancer treatments. Lysine-specific demethylase 1 (LSD1) is a protein that modifies histone and non-histone proteins and is known to influence a wide variety of physiological processes. The dysfunction of LSD1 contributes to poor prognosis, poor patient survival, drug resistance, immunosuppression, etc., making it a potential epigenetic target for cancer therapy. This review examines how LSD1 modulates different cell behavior in TME and emphasizes the potential use of LSD1 inhibitors in combination with ICB therapy for future cancer research studies.

Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness.

Several studies have examined the functions of nucleic acids in small extracellular vesicles (sEVs). However, much less is known about the protein cargos of sEVs and their functions in recipient cells. This study demonstrates the presence of lysine-specific demethylase 1 (LSD1), which is the first identified histone demethylase, in the culture medium of gastric cancer cells. We show that sEVs derived from gastric cancer cells and the plasma of patients with gastric cancer harbor LSD1. The shuttling of LSD1-containing sEVs from donor cells to recipient gastric cancer cells promotes cancer cell stemness by positively regulating the expression of Nanog, OCT4, SOX2, and CD44. Additionally, sEV-delivered LSD1 suppresses oxaliplatin response of recipient cells in vitro and in vivo, whereas LSD1-depleted sEVs do not. Taken together, we demonstrate that LSD1-loaded sEVs can promote stemness and chemoresistance to oxaliplatin. These findings suggest that the LSD1 content of sEV could serve as a biomarker to predict oxaliplatin response in gastric cancer patients.

Generation, secretion and degradation of cancer immunotherapy target PD-L1.

Cancer immunotherapy is a rapidly developing and effective method for the treatment of a variety of malignancies in recent years. As a significant immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) play the most significant role in cancer immune escape and cancer immunotherapy. Though PD-L1 have become an important target for drug development and there have been various approved drugs and clinic trials targeting it, and various clinical response rate and adverse reactions prevent many patients from benefiting from it. In recent years, combination trials have become the main direction of PD-1/PD-L1 antibodies development. Here, we summarized PD-L1 biofunctions and key roles in various cancers along with the development of PD-L1 inhibitors. The regulators that are involved in controlling PD-L1 expression including post-translational modification, mRNA level regulation as well as degradation and exosome secretory pathway of PD-L1 were focused. This systematic summary may provide comprehensive understanding of different regulations on PD-L1 as well as a broad prospect for the search of the important regulator of PD-L1. The regulatory factors of PD-L1 can be potential targets for immunotherapy and increase strategies of immunotherapy in combination.

An automatic and accurate deep learning-based neuroimaging pipeline for the neonatal brain.

BACKGROUND: Accurate segmentation of neonatal brain tissues and structures is crucial for studying normal development and diagnosing early neurodevelopmental disorders. However, there is a lack of an end-to-end pipeline for automated segmentation and imaging analysis of the normal and abnormal neonatal brain. OBJECTIVE: To develop and validate a deep learning-based pipeline for neonatal brain segmentation and analysis of structural magnetic resonance images (MRI). MATERIALS AND METHODS: Two cohorts were enrolled in the study, including cohort 1 (582 neonates from the developing Human Connectome Project) and cohort 2 (37 neonates imaged using a 3.0-tesla MRI scanner in our hospital).We developed a deep leaning-based architecture capable of brain segmentation into 9 tissues and 87 structures. Then, extensive validations were performed for accuracy, effectiveness, robustness and generality of the pipeline. Furthermore, regional volume and cortical surface estimation were measured through in-house bash script implemented in FSL (Oxford Centre for Functional MRI of the Brain Software Library) to ensure reliability of the pipeline. Dice similarity score (DSC), the 95th percentile Hausdorff distance (H95) and intraclass correlation coefficient (ICC) were calculated to assess the quality of our pipeline. Finally, we finetuned and validated our pipeline on 2-dimensional thick-slice MRI in cohorts 1 and 2. RESULTS: The deep learning-based model showed excellent performance for neonatal brain tissue and structural segmentation, with the best DSC and the 95th percentile Hausdorff distance (H95) of 0.96 and 0.99 mm, respectively. In terms of regional volume and cortical surface analysis, our model showed good agreement with ground truth. The ICC values for the regional volume were all above 0.80. Considering the thick-slice image pipeline, the same trend was observed for brain segmentation and analysis. The best DSC and H95 were 0.92 and 3.00 mm, respectively. The regional volumes and surface curvature had ICC values just below 0.80. CONCLUSIONS: We propose an automatic, accurate, stable and reliable pipeline for neonatal brain segmentation and analysis from thin and thick structural MRI. The external validation showed very good reproducibility of the pipeline.