Rational design and biological validation of EZH2/PD-L1 bifunctional inhibitors for colorectal cancer immunotherapy.

OBJECTIVES: This study aims to identify novel small-molecule inhibitors that target both PD-L1 and EZH2 to enhance colorectal cancer immunotherapy. METHODS: A combination of computer-aided molecular simulation, homogeneous time-resolved fluorescence (HTRF) assays, biolayer interferometry (BLI) assays, microscale thermophoresis (MST), and MTase-Glo methyltransferase assays was used to identify compounds with dual-targeting potential. RESULTS: Compound PE-1 exhibited potent inhibitory activity against the PD-1/PD-L1 interaction (IC50 = 48.1 nM) and showed strong EZH2 methyltransferase inhibitory effects, with an IC50 of 101.2 nM. BLI and MST further confirmed that PE-1 can effectively bind to both PD-L1 and EZH2 at the molecular level, supporting its bifunctional targeting capability. Importantly, compound PE-1 demonstrated significant activity in a PD-1/PD-L1 NFAT reporter bioassay, dose-dependently enhancing luciferase activity (EC50 = 0.61 μM), indicating effective blockade of the PD-1/PD-L1 pathway and reactivation of T-cell NFAT signaling. Notably, PE-1 demonstrated favorable in vivo pharmacokinetic properties, including a satisfactory oral bioavailability of 58.4%. In syngeneic tumor models, oral administration of PE-1 elicited substantial anti-tumor efficacy, with tumor growth inhibition (TGI) reaching 69.9% in CT26 tumors. CONCLUSIONS: PE-1 demonstrates dual-target inhibitory activity against the PD-1/PD-L1 immune checkpoint and EZH2, underscoring its potential as a lead compound for the development of next-generation bifunctional anticancer agents.