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Rational design and biological validation of EZH2/PD-L1 bifunctional inhibitors for colorectal cancer immunotherapy
Bingsheng Guan1, Binbin Cheng2, Hongqiao Li3
1Department of Gastrointestinal Surgery, First Affiliated Hospital of Jinan University, Guangzhou, China.
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.
Insights
A novel compound, PE-1, effectively inhibits both PD-1/PD-L1 and EZH2, showing promise for enhancing colorectal cancer immunotherapy and demonstrating significant anti-tumor effects in vivo.
Area of Science:
- Oncology
- Immunotherapy
- Drug Discovery
Background:
- Colorectal cancer (CRC) remains a significant health challenge.
- Current immunotherapies often face limitations in efficacy.
- Targeting immune checkpoints like PD-1/PD-L1 and epigenetic regulators like EZH2 offers potential therapeutic strategies.
Purpose of the Study:
- To identify novel small-molecule inhibitors with dual-targeting capabilities against PD-L1 and EZH2.
- To evaluate the efficacy of these dual inhibitors in enhancing colorectal cancer immunotherapy.
Main Methods:
- Utilized computer-aided molecular simulation for compound identification.
- Employed homogeneous time-resolved fluorescence (HTRF), biolayer interferometry (BLI), and microscale thermophoresis (MST) assays.
- Assessed methyltransferase activity using MTase-Glo assays and evaluated in vivo anti-tumor efficacy in syngeneic models.
Main Results:
- Compound PE-1 demonstrated potent inhibition of PD-1/PD-L1 interaction (IC50 = 48.1 nM) and EZH2 methyltransferase (IC50 = 101.2 nM).
- BLI and MST confirmed PE-1's molecular binding to both PD-L1 and EZH2.
- PE-1 showed significant anti-tumor efficacy in vivo, achieving 69.9% tumor growth inhibition in CT26 models with good oral bioavailability (58.4%).
Conclusions:
- PE-1 exhibits dual-target inhibitory activity against the PD-1/PD-L1 immune checkpoint and EZH2.
- This bifunctional activity suggests PE-1's potential as a lead compound for next-generation cancer therapeutics.
- PE-1 holds promise for advancing colorectal cancer immunotherapy.
