Discovery of bifunctional small molecules targeting PD-L1/VISTA with favorable pharmacokinetics for cancer immunotherapy.

In this work, we designed and synthesized a series of bifunctional PD-L1/VISTA (V-domain immunoglobulin suppressor of T-cell activation) small molecule inhibitors. Among them, S8 showed acceptable PD-L1 inhibitory effects (IC = 1.4 μM, HTRF assay) and VISTA binding activity (K = 2.1 μM, ITC assay). BLI, ITC, and DSF assays further confirmed its dual action mode. Notably, S8 exhibited desirable in vivo pharmacokinetic properties, featuring a respectable oral bioavailability of 34.2 %. Moreover, oral administration of S8 led to a 40 % reduction in tumor weight and a 51 % decrease in tumor volume in a B16-F10 tumor model, better than the positive control an anti-PD-L1 antibody, and CA-170. PK-PD studies show that the plasma level of unbound S8 covered the biochemical IC concentration determined by ITC and HTRF assays, which is consistent with the strong antitumor activity observed in vivo. Analysis of tumor-infiltrating lymphocytes (TILs) via flow cytometry suggested that S8 activated the tumor immune microenvironment to exert its anti-cancer effects. In summary, S8 represents a dual PD-L1/VISTA inhibitor with potential for further investigation as a dual-function immunotherapeutic agent.