The hydroxycarboxylic acid receptor 2 (HCA2) agonist niacin has been used as treatment for dyslipidemia for several decades albeit with skin flushing as a common side-effect in treated individuals. Extensive efforts have been made to identify HCA2 targeting lipid lowering agents with fewer adverse effects, despite little being known about the molecular basis of HCA2 mediated signalling. Here, we report the cryo-electron microscopy structure of the HCA2-G_i signalling complex with the potent agonist MK-6892, along with crystal structures of HCA2 in inactive state. These structures, together with comprehensive pharmacological analysis, reveal the ligand binding mode and activation and signalling mechanisms of HCA2. This study elucidates the structural determinants essential for HCA2 mediated signalling and provides insights into ligand discovery for HCA2 and related receptors.

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.

The quest for effective virtual screening algorithms is hindered by the scarcity of training data, calling for innovative approaches. This study presents the use of experimental electron density (ED) data for improving active compound enrichment in virtual screening, supported by ED’s ability to reflect the time-averaged behavior of ligands and solvents in the binding pocket. Experimental ED-based grid matching score (ExptGMS) was developed to score compounds by measuring the degree of matching between their binding conformations and a series of multi-resolution experimental ED grids. The efficiency of ExptGMS was validated using both in silico tests with the Directory of Useful Decoys-Enhanced dataset and wet-lab tests on Covid-19 3CLpro-inhibitors. ExptGMS improved the active compound enrichment in top-ranked molecules by approximately 20%. Furthermore, ExptGMS identified four active inhibitors of 3CLpro, with the most effective showing an IC₅₀ value of 1.9 µM. We also developed an online database containing experimental ED grids for over 17,000 proteins to facilitate the use of ExptGMS for academic users.

Introduction: The remarkable commercial success of mRNA vaccines against COVID-19 and tumors, along with their potential as therapeutic drugs, has significantly boosted enthusiasm for circular RNAs (circRNA) as a promising next-generation therapeutic platform. The development of novel circRNA cyclization technologies represents a significant leap forward in RNA engineering and therapeutic applications. Recent advancements in group I and IIB self-splicing intron-based ribozymes have enabled precise cyclization of RNA molecules. However, this approach faces significant limitations, including low cyclization efficiency and the requirement for additional additives, which restrict its broader application. Group IIC self-splicing introns represent the shortest known selfsplicing ribozymes and employ a splicing mechanism that is fundamentally distinct from that of group IIB self-splicing introns. However, the potential of group IIC self-splicing introns to carry exogenous sequences for the development of circular RNA-based platforms remains an open question and warrants further investigation.Methods: Here, we demonstrate that group IIC self-splicing introns can efficiently circularize and express exogenous proteins of varying lengths, as evidenced by luciferase and GFP reporter systems. Leveraging structural biology-based design, we engineered the RSV pre-F protein and validated the potential of IIC self-splicing introns as a vaccine platform for preventing infectious diseases.Results: In mouse models, the novel nucleic acid vaccine developed using IIC self-splicing introns elicited superior immunogenicity and in vivo protective efficacy compared to protein-adjuvant vaccines.Discussion: The development of the novel circular RNA vaccine platform holds significant promise for advancing next-generation therapeutics for disease treatment and prevention.

Human stem cell-derived β (SC-β) cells still exhibit limited glucose response required for insulin secretion due to glycolytic bottlenecks, yet how these metabolic abnormalities impact glucose response and functional maturation of SC-β cells remains unclear. In this study, we identify a metabolic checkpoint located at PEP accumulation that impedes the functional maturation, which is rescued by restoration of pyruvate kinase M1 (PKM1). Glucose-tracing metabolomics in human stem cell-derived islets reveal abnormal glycolytic PEP accumulation at resting condition, associated with impaired calcium response and insulin secretion upon high glucose or glycolytic metabolite stimulation. Mechanistically, elevated PEP significantly raises intracellular basal calcium levels and downregulates expression of genes involved in TCA cycle as elucidated by single cell transcriptomics. Furthermore, the activity of pyruvate kinase, which metabolizes PEP, is notably reduced due to low PKM1 expression. Overexpression of PKM1 alleviates PEP accumulation, restores TCA-related gene expression, and enhances glucose-stimulated calcium responses and insulin secretion. Together, our findings reveal a critical role of PKM1-regulated PEP metabolism in SC-β cell functional maturation and highlight the importance of metabolic reprogramming for advancing stem cell-based therapies for diabetes.