Structure-based discovery of potent small-molecule inhibitors 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 modulators capable of targeting 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 within a PD-1-associated pocket located at the PD-1/PD-L1 interaction interface. Surface plasmon resonance (SPR) analysis confirmed direct binding of the compounds to PD-1 with measurable binding affinity. Molecular dynamics simulations and MM/GBSA energy decomposition suggested that both molecules interact with key residues in the interface-relevant region, including Tyr68, Thr76, and Ile126 of PD-1, and may induce local changes in PD-1/PD-L1 interfacial contacts, supporting their binding in the modeled systems. Functionally, both compounds enhanced T-cell activation and increased tumor cell killing in a T cell-tumor co-culture system in vitro, as demonstrated by IL-2 secretion assays, LDH release, and CCK8 analyses. Collectively, these findings identify ID23468559 and ID7844239 as potential small-molecule modulators associated with PD-1-mediated immune regulation. This work presents an integrated computational–experimental pipeline for discovering PD-1-directed small molecules and provides a basis for future structure–activity relationship (SAR) optimization.