Structure-Based Discovery of Potent Small-Molecule modulator Targeting the PD-1/PD-L1 interaction

Abstract

The PD-1/PD-L1 immune checkpoint is a central regulator of T-cell activity and a clinically validated target in cancer immunotherapy. While antibody-based therapies have achieved remarkable success, discovering small-molecule inhibitors capable of disrupting the PD-1/PD-L1 interaction remains challenging due to the shallow and dynamic nature of the interface. In this study, structure-based virtual screening and molecular docking identified nine candidate compounds, among which ID23468559 and ID7844239 displayed favorable docking performance and adopted well-positioned poses within the PD-1/PD-L1 interfacial pocket. Surface plasmon resonance (SPR) confirmed their binding affinities, yielding KD values of 0.08 µM and 1.51 µM. Molecular dynamics simulations and MM/GBSA energy decomposition revealed that both molecules anchor within the PD-L1 interfacial pocket and establish stable hydrogen bond and hydrophobic interaction contacts with key residues, including Gln75, Thr76, Leu122, and Glu136, consistent with their stable binding poses. Functionally, both compounds restored T-cell activation and significantly reduced tumor cell viability in vitro, as demonstrated by IL-2 secretion assays, LDH release, and MTT analyses. Collectively, these findings identify ID23468559 and ID7844239 as promising small-molecule modulators capable of disrupting the PD-1/PD-L1 interaction. This work establishes an integrated computational–experimental pipeline for discovering interface-targeted immune checkpoint inhibitors and provides a solid foundation for future structure–activity relationship (SAR) optimization and drug development.