Discovery and Crystallographic Study of Water-Soluble Quaternary Ammonium-Based PD-1/PD-L1 Inhibitors with...

Programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) represents a critical immune checkpoint in cancer immunotherapy; however, most small-molecule PD-1/PD-L1 inhibitors suffer from poor aqueous solubility. Here, we designed a series of quaternary ammonium-based PD-1/PD-L1 inhibitors. Among them, QA9 exhibited potent PD-1/PD-L1 inhibition (IC50 = 18.7 nM), outperforming the lead compound NP19, and demonstrated a 600-fold improvement in water solubility (0.642 vs 0.001 mg/mL). X-ray crystallography revealed that QA9 stabilizes the PD-L1 dimer through multiple interactions, providing a structural basis for its high affinity. In MC38/Jurkat and HCT116/Jurkat coculture models, QA9 enhanced immune-mediated tumor cell death. Additionally, QA9 suppressed VEGF-A-induced tube formation and migration in HUVECs, which was associated with reduced FAK phosphorylation. Furthermore, in an MC38 mouse colon cancer model, QA9 showed robust antitumor activity without overt toxicity. Collectively, the quaternary ammonium strategy represents an effective approach to developing water-soluble PD-1/PD-L1 inhibitors with dual immunomodulatory and antiangiogenic functions.