Structure-guided identification and functional evaluation of small-molecule candidates predicted to target the...

Small-molecule modulators may complement antibody therapy, offering potential advantages such as optimizable chemical structures, improved tumor accessibility, and better flexibility in pharmacokinetic control. Lymphocyte activation gene-3 (LAG-3) is an inhibitory immune checkpoint receptor that suppresses T-cell function by interacting with MHC-II molecules; however, systematic research on small-molecule modulators targeting LAG-3 remains lacking. In this study, a multi-stage structure-guided screening process was employed to identify candidate compounds targeting a predicted binding pocket adjacent to the LAG-3/MHC-II interface. Starting with approximately 3.9 million compounds database, the study employed a sequential screening and prioritization process involving AutoDock-GPU docking, Glide hierarchical docking, physicochemical and ADMET screening, 500 ns molecular dynamics simulations, and MM/PBSA binding free energy analysis. Based on predicted binding conformations, interface stability, estimated binding energies, and drug-like properties, ligand160490 and ligand223516, hereafter termed Z21 and Z28, were ultimately prioritized for functional evaluation. In activated human peripheral blood mononuclear cells, Z28 increased the frequency of CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ T cells, whereas Z21 did not exhibit a similar effect. In the MC38 syngeneic tumor model, both Z21 and Z28, when used in combination with anti-PD-1, reduced tumor growth, accompanied by increased frequency of tumor-infiltrating CD8⁺ T cells. Together, the in vitro and in vivo results support Z21 and Z28 as prioritized small-molecule candidates for further investigations. Subsequent studies should focus on determining whether Z21 and Z28 exert their biological effects by directly binding to LAG-3 and interfering with LAG-3/MHC-II interaction. Our findings support further mechanistic and pharmacological evaluation of Z21 and Z28.