Abstract
<title>Abstract</title> <p>Objective Pyruvate dehydrogenase subunit B (PDHB) constitutes a pivotal regulatory element within the cuproptosis pathway. This study utilized PDHB as the central target, integrating multi-omics analysis with dual virtual screening methodologies, to identify lead compounds capable of targeting and modulating PDHB to ameliorate type 2 diabetes mellitus (T2DM). In vitro experiments were conducted to substantiate the intervention effects and underlying mechanisms, providing empirical evidence for precision therapy targeting cuproptosis in T2DM and for the advancement of novel drug candidates. Methods This study adopted an integrated approach that combined multi-omics analysis, computational virtual screening, and in vitro cellular functional assays to systematically explore the regulatory role of PDHB in cuproptosis within pancreatic β-cells in T2DM, as well as to screen and validate novel lead compounds targeting PDHB. Initially, through network pharmacology, bulk transcriptomics, and single-cell transcriptomics analysis, in conjunction with gene virtual knockout technology, the function and significance of PDHB as a core target gene for cuproptosis associated with T2DM were elucidated. Subsequently, structural preprocessing of the PDHB protein and small molecules within the compound library was executed, followed by ADME/T drug-likeness assessment, active site identification, multi-level molecular docking, MM-GBSA binding energy calculations, and molecular dynamics simulations to identify potential lead compounds that stably bind to PDHB. Finally, an in vitro injury model of pancreatic β-cells induced by high glucose and high fat was constructed to verify the intervention effects of the target compounds on glucose metabolism disorders, copper homeostasis imbalance, and β-cell cuproptosis. Results Multi-omics analysis combined with virtual knockout results corroborated that PDHB is a core target gene that regulates cuproptosis and improves T2DM, exhibiting specific high expression in pancreatic β-cells, and serving as a critical molecule for maintaining β-cell metabolic homeostasis and cell survival. Through multi-level virtual screening, 30 potential small molecules capable of specifically binding to PDHB were identified from compound libraries, among which F984-0883 demonstrated the lowest binding energy and strongest affinity. Molecular dynamics simulations further confirmed the stable and robust conformation of the compound when bound to PDHB was stable and robust. In vitro cellular experiments revealed that, compared with the model group, F984-0883 intervention significantly enhanced pancreatic β-cell viability and insulin secretion levels, substantially increased cellular ATP content and stabilized mitochondrial membrane potential, effectively improving mitochondrial function. Additionally, F984-0883 significantly reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels, increased glutathione (GSH) content and superoxide dismutase (SOD) activity, and significantly upregulated PDHB gene and protein expression. Conclusion Through an integrated multi-omics and dual virtual screening system, this study successfully identified a novel lead compound F984-0883 targeting PDHB. This compound can effectively alleviate T2DM-related pathological injuries by upregulating PDHB expression, inhibiting β-cell cuproptosis, and protecting mitochondrial and metabolic functions. These findings provide a novel target, candidate drug, and experimental evidence for the development of cuproptosis-targeted inhibitors and precision therapy for T2DM.</p>