In search of novel PD1 inhibitor from natural products by high-throughput virtual screening and molecular dynamics simulation.

Squamous cell carcinoma (SCC) represents a significant oncological challenge. While immune checkpoint inhibitors targeting PD-1/PD-L1 have revolutionized the treatment of SCC, current monoclonal antibody approaches face limitations, including poor tissue penetration, high costs, and immune-related adverse events in patients. Most existing small-molecule efforts target PD-L1, leaving PD-1/PD-L2 interactions intact and enabling immune escape. This study represents the first systematic identification of natural product-derived direct PD-1 inhibitors, offering broader pathway blockade compared to PD-L1-selective approaches. While current therapeutic limitations highlight the need for alternative methods, this computational study lays a foundation for experimental validation and potential advancement of a drug development pipeline. Through integrated computational screening of 17,967 phytochemicals from the IMPPAT database, we employed consensus molecular docking across seven algorithms, 300-ns molecular dynamics simulations, density functional theory calculations, and comprehensive ADME profiling. IMPHY004834 (Mahuannin D) from Ephedra sinica emerged as a lead compound with exceptional free binding energy, forming stable interactions with key PD-1. Molecular dynamics analysis revealed remarkable stability with consistent RMSD, lowest RMSF, and sustained hydrogen bonding throughout the simulation. The biflavonoid structure exhibits a favorable HOMO-LUMO gap, indicating chemical stability, while ADME profiling confirms drug-like properties, albeit requiring parenteral administration due to low GI absorption. This work establishes the first evidence for Mahuannin D's PD-1 inhibitory mechanism, which was previously known only for its cytotoxic effects. It provides a validated computational framework for discovering natural product-based immune checkpoint inhibitors with superior pathway coverage compared to existing PD-L1-selective therapeutics.