Cyclin-dependent kinase 2 (CDK2) represents a critical therapeutic target in tumors resistant to CDK4/6 inhibitors or with CCNE1 amplification. However, selective inhibition of CDK2 remains challenging owing to the high structural homology among CDKs. In this study, we identify B10 and B12 as cereblon (CRBN)-based molecular glue degraders that selectively degrade CDK2. Ternary complex structures reveal a noncanonical recruitment mode centered on CDK2 Glu57, which bypasses the canonical G-loop/β-hairpin and kinase glycine-rich loop interactions and is stabilized by an extended CRBN-CDK2 interface. Mechanistically, these degraders inhibit retinoblastoma (Rb) phosphorylation and induce G1/S-phase arrest, suppressing CDK2-dependent cell proliferation. B12 further exhibits improved pharmacokinetics, measurable oral bioavailability, and in vivo target engagement, achieving intratumoral CDK2 degradation following intraperitoneal administration. Collectively, this study provides a structural blueprint for designing selective kinase degraders and establishes B12 as a chemically tractable probe for targeting CDK2-driven malignancies.

The χ-conotoxins are venom-derived peptides that specifically target the noradrenaline transporter (also known as norepinephrine transporter, NET). Regulation of noradrenergic signaling by NET affects neurophysiological processes, including pain. Therefore, the χ-conotoxin MrIA and its synthetic analogs have been previously investigated for their analgesic activity. Here we describe the synthesis and pharmacological characterization of χ-AoIA, a peptide that selectively inhibits NET with a higher potency compared to MrIA in in vitro radiotracer flux assays. Furthermore, we resolved the structure of the human NET:χ-AoIA complex by cryogenic electron microscopy, which revealed an atypical binding mode consisting of both the central binding site and the outer vestibule of the transporter. Lastly, χ-AoIA displays antinociceptive efficacy in a model of inflammatory pain after subcutaneous administration in mice. Our results demonstrate the efficacy of χ-AoIA as a highly selective ligand of NET and provide a mechanistic basis for its potential development as a nonopioid analgesic.

Glycosphingolipids are essential membrane components that organize lipid microdomains and orchestrate cellular signalling, differentiation and neuronal function1-4. In humans, these functions arise from a repertoire of several hundred glycosphingolipid species generated through stepwise glycan elaboration5,6. Entry into this network is controlled by a single committed reaction catalysed by UDP-glucose ceramide glucosyltransferase (UGCG), the gatekeeper that dictates the scale and composition of glycosphingolipid diversity. Despite its biological and therapeutic importance7,8, its mechanism and regulation have remained unknown. Here we report cryogenic electron microscopy structures of full-length human UGCG in eight functional states at 2.9-3.4 Å resolution. UGCG adopts a previously unrecognized triple-pass transmembrane architecture that anchors a GT-A core at the membrane interface and creates a bipartite active site engaging soluble and membrane-embedded substrates. Contrary to canonical GT-A enzymes, UGCG uses a metal-independent catalytic mechanism driven by an arginine network. We identify a primate-specific steric element that tunes lipid affinity and catalytic turnover, modulating glycosphingolipid entry. Structures with clinically used inhibitors reveal how this architecture governs their potency and selectivity. Together, these findings define the structural and evolutionary logic by which one enzyme controls glycosphingolipid diversity and provide a framework for precision modulation of membrane lipid homeostasis in disease.

The vasopressin V2 receptor (V2R), a class A G protein-coupled receptor, is essential for regulating body water homeostasis. V2R antagonists have emerged as promising treatments for hyponatremia; however, the absence of structural information for antagonist-bound V2R hampers our understanding of antagonist recognition and the targeted design of V2R antagonists. In this study, we present two cryo-electron microscopy structures of inactive V2R bound to the clinically approved antagonists tolvaptan and conivaptan. Combined with functional analyses and molecular dynamic simulations, these structures reveal distinct binding poses: tolvaptan is deeply inserted within the binding pocket, whereas conivaptan is positioned at a shallower depth. Integrated analyses further define critical pharmacophoric features governing antagonist activity and unveil a TM7 helical conformation-dependent antagonism mechanism that is distinct from classical GPCR inactivation modes. Our findings deepen understanding of antagonist recognition and antagonism of V2R, providing a foundation for the development of V2R-targeted therapies.

The norepinephrine transporter (NET) plays a crucial role in synaptic neurotransmission and is implicated in major depression and attention-deficit/hyperactivity disorders, yet our understanding of its allosteric, conformation-selective regulation-crucial for developing targeted therapeutics-remains limited. Through cryo-electron microscopy analysis of NET complexes with levomilnacipran, vanoxerine, and vilazodone, we identify a previously undefined allosteric site within NET's inner vestibule that enables conformation-selective regulation. This discovery introduces a "valve model," in which specific residues partition the cytoplasmic cavity into distinct chambers, determining inhibitor binding specificity. Leveraging this structural insight through virtual screening, we identify a set of inhibitors with potent NET inhibitory activity and demonstrate their antidepressant effects. Moreover, our structural identification of inhibitor occupancy at this conformation-selective site defines a mechanistic framework for targeted therapeutic intervention. These findings advance our understanding of NET allosteric modulation, providing a structure-guided framework for developing next-generation antidepressants targeting the inward-open conformation of NET for the treatment of neuropsychiatric disorders.

Histamine H3 receptor (H3R) and H4 receptor (H4R) are key members of the histamine receptor family, with H3R as a potential target for narcolepsy treatments and H4R as a candidate for next-generation antihistamines for inflammatory and allergic diseases. Although progress has been made in understanding the structure of histamine receptors, the detailed mechanisms of ligand recognition and receptor antagonism for H3R and H4R remain unclear. In this study, using cryo-electron microscopy, we present an inactive structure of H4R bound to a selective antagonist, adriforant, and two Gi-coupled structures of H3R and H4R in complex with histamine. Our structural and mutagenesis analyses provide insights into the selective binding of adriforant to H4R and the recognition of histamine across histamine receptors. Our findings also uncovered distinct antagonistic mechanisms for H3R and H4R and identified the role of aromatic amino acids on extracellular loop 2 in modulating the constitutive activity of H3R and H4R. These findings advance our knowledge of the functional modulation of histamine receptors, providing a foundation for the development of targeted therapeutics for neurological and immune-related disorders.

In cardiology, the classification of electrocardiograms (ECGs) or heartbeats serves as a vital instrument. Techniques grounded in deep learning for ECG signal examination support medical professionals in swiftly identifying heart ailments, thereby aiding in life preservation. The present investigation endeavors to convert a dataset comprising ECG record images into time-series signals, followed by the implementation of deep learning (DL) methodologies on this transformed dataset. Cutting-edge DL methodologies are introduced for categorizing ECG signals across diverse cardiac categories. This work examines and juxtaposes various DL architectures, encompassing a convolutional neural network (CNN), a long short-term memory (LSTM) network, and a self-supervised learning framework leveraging autoencoders. Training of these models occurs on a dataset derived from ECG tracings of individuals at multiple medical facilities in Pakistan. Initially, the ECG images undergo digitization with segmentation of lead II heartbeats, after which the resulting signals are inputted into the advocated DL models for categorization. Within the array of DL models evaluated herein, the advocated CNN architecture attains the peak accuracy of > 90%. This architecture exhibits superior precision and expedited inference, facilitating instantaneous and unmediated surveillance of ECG signals acquired via electrodes (sensors) positioned on various bodily regions. Employing the digitized variant of ECG signals, as opposed to pictorial representations, for cardiac arrhythmia categorization empowers cardiologists to deploy DL models directly onto signals emanating from ECG apparatus, enabling contemporaneous and precise ECG oversight.

Gout, a common and painful disease, stems from hyperuricemia, where elevated blood urate levels lead to urate crystal formation in joints and kidneys. The human urate transporter 1 (hURAT1) plays a critical role in urate homeostasis by facilitating urate reabsorption in the renal proximal tubule, making it a key target for gout therapy. Pharmacological inhibition of hURAT1 with drugs such as dotinurad, benzbromarone, lesinurad, and verinurad promotes urate excretion and alleviates gout symptoms. Here, we present cryo-electron microscopy structures of native hURAT1 bound with these anti-gout drugs in the inward-open state, and with urate in inward-open, outward-open, and occluded states. Complemented by mutagenesis and cell-based assays, these structures reveal the mechanisms of urate reabsorption and hURAT1 inhibition. Our findings elucidate the molecular basis of urate transport and anti-gout medication action and provide a structural framework for the rational design of next-generation therapies for hyperuricemia and gout.

Background: Following SARS-CoV-2 infection, the necessity of vaccination after natural infection remains uncertain. However, many asymptomatic individuals who test negative virologically may nevertheless receive vaccination without being aware of their prior infection. Investigating the implications for vaccine safety and efficacy is crucial. Methods: We analyzed the daily fluctuations in anti-SARS-CoV-2 IgG antibody levels during the enrollment period of a phase 3 randomized, double-blinded, placebo-controlled clinical trial of a tetravalent COVID-19 protein vaccine, SCTV01E. Additionally, we investigated the relationship between baseline IgG levels and their protection against COVID-19 in participants who received placebo. Results: The daily enrolled participants with different baseline IgG levels (<338 BAU/mL, 338–1000 BAU/mL, >1000 BAU/mL) showed dynamic changes with the enrollment date. Among participants with baseline IgG levels < 338 BAU/mL, vaccination conferred a relative protective efficacy of 69.15% (95% CI: 51.14–80.52%) against symptomatic SARS-CoV-2 infection compared with the control group. Conversely, in those with higher baseline IgG levels (≥338 BAU/mL), vaccination did not confer additional benefit. In the placebo group, the relative protection in participants with baseline IgG levels ≥ 338 BAU/mL was 93.79% (87.60%, 96.89%) compared to that of those with baseline IgG levels < 338 BAU/mL. The safety profile of SCTV01E in participants with baseline IgG ≥ 338 BAU/mL was comparable to that in participants with <338 BAU/mL, with favorable safety profiles. Conclusions: During the SCTV01E phase 3 clinical trial, an anti-SARS-CoV-2 IgG antibody IgG level of 338 BAU/mL was suitable for screening individuals in the early phase post-infection alongside virological tests. Vaccinating the infected population was safe and did not compromise efficacy. Clinical Trial: NCT05308576.

The motilin receptor (MTLR) is a key target for treating gastrointestinal (GI) disorders like gastroparesis, yet developing effective agonists remains challenging due to drug tolerance and signaling bias. We present cryoelectron microscopy (cryo-EM) structures of MTLR bound to azithromycin, a macrolide antibiotic, and DS- 3801b, a non-macrolide agonist. Distinct ligand recognition mechanisms are revealed, with azithromycin binding deeply within the orthosteric pocket and DS-3801b adopting a special clamp-like conformation stabilized by a water molecule. We also highlight the critical role of extracellular loop 2 (ECL2) in ligand specificity and signaling pathway activation, affecting both G-protein and b-arrestin signaling. Additionally, the "D2.60R2.63S3.28"motif and interactions around transmembranes 6/7 (TM6/7) are identified as key drivers of signaling selectivity. These findings offer insights into the structural dynamics of MTLR, laying the groundwork for the rational design of next-generation GI prokinetic drugs with enhanced efficacy and safety.

Maintaining pH homeostasis is critical for cellular function across all living organisms. Proton-sensing G protein-coupled receptors (GPCRs), particularly GPR4, play a pivotal role in cellular responses to pH changes. Yet, the molecular mechanisms underlying their proton sensing and activation remain incompletely understood. Here we present high-resolution cryo-electron microscopy structures of GPR4 in complex with G proteins under physiological and acidic pH conditions. Our structures reveal an intricate proton-sensing mechanism driven by a sophisticated histidine network in the receptor's extracellular domain. Upon protonation of key histidines under acidic conditions, a remarkable conformational cascade is initiated, propagating from the extracellular region to the intracellular G protein-coupling interface. This dynamic process involves precise transmembrane helix rearrangements and conformational shifts of conserved motifs, mediated by strategically positioned water molecules. Notably, we discovered a bound bioactive lipid, lysophosphatidylcholine, which has positive allosteric effects on GPR4 activation. These findings provide a comprehensive framework for understanding proton sensing in GPCRs and the interplay between pH sensing and lipid regulation, offering insights into cellular pH homeostasis and potential therapies for pH-related disorders.

Neuropeptide FF receptors 1 and 2 (NPFFR1 and NPFFR2) are RF-amide peptide receptors that couple to Gi/o proteins and regulate pain, opioid tolerance, and metabolism. Despite their physiological significance, their ligand selectivity and activation mechanisms remain unclear. Using cryoelectron microscopy, we resolved four NPFFR1 and NPFFR2 structures bound to NPFF or NPVF, revealing conserved C-terminal RF-amide interactions within the orthosteric pocket and N-terminal variations driving subtype specificity. Structural and mutagenesis analyses identified ECL2 and the receptor N terminus as key determinants of NPVF-NPFFR1 and NPFF-NPFFR2 selectivity. Additionally, the structures elucidate the activation mechanism and uncover distinct Gi-coupling features between NPFFR subtypes. These findings provide molecular insights into peptide recognition and receptor activation within the RF-amide family, offering a structural framework for designing selective NPFFR modulators to treat pain, addiction, and metabolic disorders with enhanced specificity and reduced off-target effects.

The growth hormone secretagogue receptor (GHSR) plays a critical role in regulating growth hormone release and metabolic homeostasis. Understanding the molecular mechanisms of ligand-GHSR recognition is essential for developing therapeutic interventions. In this study, we investigated the molecular recognition mechanisms of two clinically approved drugs: Macimorelin (used for diagnosing adult growth hormone deficiency) and Anamorelin (approved in Japan for cancer cachexia). Using high-resolution cryo-electron microscopy, we determined the structures of GHSR bound to Macimorelin and Anamorelin in complex with Gq proteins at resolutions of 2.63 Å and 2.52 Å, respectively. We revealed that both drugs occupied a bifurcated binding pocket divided by a conserved salt bridge between E1243.33 and R2836.55. Through systematic mutagenesis and functional studies, we identified the key residues underlying the higher binding affinity of Anamorelin compared to Macimorelin. In addition, structural comparison of GHSR in complex with different G protein subtypes elucidated the mechanisms driving G protein selectivity. Our results provide crucial insights into GHSR-drug interactions and offer valuable guidance for designing more selective and potent GHSR agonists.

The G protein–coupled cysteinyl leukotriene receptor CysLT2R plays intricate roles in the physiology and pathogenesis of inflammation-related processes. It has garnered increasing attention as a potential therapeutic target for atopic asthma, brain injury, central nervous system disorders, and various types of cancer. In this study, we present the cryo-electron microscopy structure of the cysteinyl leukotriene D4 (LTD4)-bound human CysLT2R in complex with a Gα q protein, adopting an active conformation at a resolution of 3.15 Å. The structure elucidates a spacious polar pocket designed to accommodate the two branched negative ends of LTD4 and reveals a lateral ligand access route into the orthosteric pocket located on transmembrane domain helix (TM) 4 and 5. Furthermore, our findings highlight the crucial role of transmembrane domain helix 3 in sensing agonist moieties, representing the pivotal mechanism of receptor activation for both CysLT1R and CysLT2R. Collectively, the insights derived from our structural investigation establish a foundation for comprehending CysLT2R activation by its endogenous ligand LTD4, offering a rational basis for the design of drugs targeting CysLT2R.

The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders. Cryo-electron microscopy structures of the noradrenaline transporter in the apo state, bound to noradrenaline and bound to various antidepressants shed light on the substrate transport, molecular recognition and dimeric architecture of this protein.

IntroductionThe coronavirus disease 2019 (COVID-19) global pandemic has been the most severe public health emergency since 2019. Currently, the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the most dominant. The most prominent symptom of SARS-CoV-2 infection is respiratory. Meanwhile, the fatality of COVID-19 was mainly from pneumonia. However ,in patients with SARS-CoV-2 infection who have pneumonia and those who do not, the differences in the immune repertoire still require further investigation.MethodsWe conducted seven-chain adaptome immune repertoire analyses on patients with SARS-CoV-2 Omicron infection, both with and without pulmonary infiltration.ResultsPatients with pulmonary infiltration exhibit lymphopenia, a decreased proportion of the overall TCR repertoire alongside an increased BCR repertoire, reduced IGHD and IGHM isotype expression, a shorter mean CDR3 length for TRG, and a longer mean length for TRD, as well as diminished clonality and diversity in the TCR/BCR repertoire. Meanwhile, patients with pulmonary infiltration have distinct V-J gene usage and unique CDR3 signature, as well as BCR class switch recombination pattern. Finally, prior vaccination triggered less BCR IGHM/IGHD somatic hypermutation response, preserved the diversity of the entire adaptive immune repertoire, and provided clinical protection against severe or critical conditions following Omicron infection.DiscussionWe report a unique, comprehensive adaptive immune system signature in patients with pulmonary infiltration, which may serve as potential immunological biomarkers and therapeutic targets.

Type 2 Diabetes (T2D) is characterized by peripheral insulin resistance, deteriorating beta-cell function and eventual beta-cell failure. The role of Peroxisome proliferator-activated receptor γ (PPAR-γ) in beta-cells, as opposed to its role in peripheral cells in glucose metabolism, is not well understood during T2D progression. Here, we utilized S961, a specific insulin receptor inhibitor, to induce glucose intolerance and hyperglycemia in mice, thereby simulating the stages of T2D progression. Using beta-cell-specific knockouts of PPAR-γ, WNT5A and SMAD7, we explored the molecular mechanisms through which PPAR-γ and related signaling pathways affect beta-cell proliferation, insulin secretion, and dysfunction in T2D. Our results indicated an initial increase in PPAR-γ expression in beta-cells of pre-diabetic patients, followed by a reduction in diabetic patients. In mice, PPAR-γ expression in beta-cells rose after 7 days of S961 treatment (paralleling pre-diabetic conditions) but fell after 14 days (reflecting diabetic conditions). Conversely, SMAD7 levels in beta-cells were stably minimal initially but increased in later stages in both mice and diabetic patients. Assisted with single-cell RNA sequencing, we found that S961-treated mice experienced an upregulation in the TGF-β signaling cascade in beta-cells, leading to the activation of pSMAD3, which subsequently upregulated PPAR-γ, enhancing beta-cell proliferation and secretion. However, prolonged S961 exposure and the direct effects of hyperglycemia augmented SMAD7 levels, which inhibited SMAD signaling, resulting in reduced PPAR-γ, compromised beta-cell functionality and the onset of diabetes. This late upregulation of SMAD7 was likely mediated through WNT5A/Smarf2/ubiquitin signaling, which was progressively altered during T2D progression. Our findings underscore the intricate interplay of PPAR-γ and related pathways in beta-cells during T2D pathogenesis. Disclosure Y. Xiao: None. J. Shi: None. M.R. Adama: None. G.J. Kim: None. M. Welch: None. Y. Jiang: None. Funding Internal grants