Structure-Guided Virtual Screening of Small Peptide Inhibitors Targeting PD-L1 for Cancer Immunotherapy

The programmed cell death-1 (PD-1)/PD-L1 immune checkpoint plays a critical role in tumor immune evasion. While monoclonal antibodies targeting this pathway have demonstrated clinical benefit, their use is often limited by poor tumor penetration and immunogenicity. Peptides offer advantages such as tunable structures and improved tissue penetration. Here, we applied a structure-guided virtual screening strategy to identify small peptides that disrupt the PD-1/PD-L1 interaction. Alanine scanning of BMS-57 (PDB ID: 5O4Y) identified key binding residues, which informed the design of a focused virtual library of 24,423 small peptides. Top hits were selected by docking and molecular dynamics simulations. Experimental validation using ELISA and immune cell cocultures confirmed potent inhibitors. Lead peptide FJ15596 blocked PD-1/PD-L1 binding with an IC₅₀of 570 nM, showed 92.2 ± 1.0% inhibition at 10 μM, and restored CD3⁺T cell viability in a DU145 coculture model. This strategy supports the development of small peptide scaffolds for modulating other therapeutically relevant protein–protein interactions.