This study aims to identify novel small-molecule inhibitors that target both PD-L1 and EZH2 to enhance colorectal cancer immunotherapy.
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.
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.
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.
Copyright © 2026 Guan, Cheng and Li.

This work aims to design and characterize a novel bifunctional small molecule that simultaneously targets PD-L1 and CD73 to enhance the therapeutic efficacy of tumor immunotherapy.
Multiple methodologies were integrated for compound screening and biological characterization, including computer-aided molecular docking, homogeneous time-resolved fluorescence (HTRF) binding assay, surface plasmon resonance (SPR), and PD-1/PD-L1 NFAT reporter cell assay.
The lead compound CP-1 exhibited potent dual-target inhibitory activities. It blocked the PD-1/PD-L1 interaction with an IC50 of 10.27 nM and suppressed CD73 activity with an IC50 of 300.2 nM. Molecular docking simulations revealed that CP-1 stably binds to the functional domains of PD-L1 and CD73 via specific non-covalent interactions. Cellular functional assays further demonstrated that CP-1 effectively restored T cell function in the PD-1/PD-L1 reporter system, with an EC50 of 0.9 μM.
CP-1 exhibits balanced, dual-nanomolar inhibitory activity against PD-L1 and CD73 and displays potent immunomodulatory effects at the cellular level. It serves as a promising lead candidate for developing novel bifunctional agents to advance tumor immunotherapy.
Copyright © 2026 Du, Wu, Cheng, Zeng and Cheng.

Current PD-L1 degraders, whether antibody-based or small-molecule-mediated, are hindered by limitations in pharmacokinetics (e.g., poor tissue penetration) or pharmacodynamics (e.g., suboptimal degradation efficacy, immunogenicity concerns). These drawbacks highlight the necessity for novel PD-L1 degradation platforms using innovative technologies.
This study aims to design and synthesize bifunctional small molecules as PD-L1 degraders by leveraging the unexplored E3 ligase SPOP, aiming to overcome the limitations of existing degraders and evaluate their potential in cancer immunotherapy.
A series of SPOP-based bifunctional small molecules were designed and synthesized. Their PD-L1 inhibitory and degradation activities were assessed using HTRF and western blot assays, respectively. Mechanistic studies (His pull-down, bio-layer interferometry, western blot) were performed to verify ternary complex formation with PD-L1 and SPOP. In vivo pharmacokinetic properties and antitumor efficacy were evaluated in a B16-F10 tumor model, with analysis of tumor-infiltrating lymphocytes (TILs) to explore immune microenvironment effects.
Compound SPOP9 exhibited potent PD-L1 inhibition (IC50 = 357.2 nM) and degradation (DC50 = 1.0 μM). Mechanistic studies confirmed its assembly into a stable ternary complex with PD-L1 and SPOP. SPOP9 showed favorable in vivo bioavailability (F = 74.8 %) and, at 10 mg/kg (i.p.), reduced tumor weight by 44 % in B16-F10 mice, superior to anti-PD-L1 antibody (TGI = 34.4 %). TIL analysis indicated SPOP9 activated the tumor immune microenvironment and downregulated PD-L1.
SPOP9, as the first SPOP-binding bifunctional PD-L1 degrader, demonstrates promising preclinical efficacy and pharmacokinetic properties, addressing key limitations of existing degraders. It merits further investigation as a potential agent for cancer immunotherapy.
Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.

Small molecule blockade of the programmed death receptor 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway represents a promising approach for tumor immunotherapy. Based on the previously developed 3D-quantitative structure-activity relationship pharmacophore model for PD-L1 inhibitors, virtual screening followed by homogeneous time-resolved fluorescence (HTRF) activity testing identified the compound {1-[(biphenyl-4-yl)methyl]-7-methoxy-1,2,3,4-tetrahydroisoquinolin-6-yl}acetic acid methyl ester (Compound Y6) as a hit. Analysis of computational docking results of compound Y6 with PD-L1 suggested targeting the methyl acetate substituent at position 6 for optimization. Guided by this, 20 new derivatives were designed and synthesized. The synthesized compounds were subjected to HTRF test and 6 compounds with significant protein blocking effects were screened for subsequent surface plasmon resonance (SPR) test. Subsequent SPR analysis confirmed strong binding of these 6 compounds to hPD-L1 protein with K D values ranging from 0.24 to 21.31 μM. Several derivatives displayed improved or comparable PD-L1 binding affinity relative to the lead compound Y6 (K D = 11.3 μM). A co-incubation system (PD-1+ Jurkat T/PD-L1+ HepG2) was established to evaluate functional immune restoration. This evaluation revealed that compound Y7f effectively promoted HepG2 cells death by restoring T cell immune function. MD simulations identify (1R,16R)-Y7f as the most potent PD-L1 dimerization inducer within the compound Y7f stereoisomer series. The results indicated that the biphenyl-tetrahydroisoquinoline scaffold is a promising structural framework for developing novel PD-1/PD-L1 inhibitors and deserves further investigation.
© 2026 The Author(s). Smart Molecules published by John Wiley & Sons Australia, Ltd on behalf of Dalian University of Technology.

This study aims to identify novel small-molecule inhibitors capable of dual targeting of PD-L1 and tubulin, intending to enhance cancer immunotherapy.
A combination of computer-aided virtual screening, molecular docking, homogeneous time-resolved fluorescence (HTRF) assays, tubulin polymerization inhibition assays, and in vivo antitumor assays was utilized to identify compounds with dual-targeting potential.
Compound PP-1 exhibited moderate inhibitory activity against the PD-1/PD-L1 interaction (IC50 = 81.1 µM) and showed dose-dependent inhibition of tubulin polymerization (IC50 = 70.1 µM). Molecular docking analysis further confirmed that PP-1 can effectively bind to both PD-L1 and tubulin at the molecular level, supporting its bifunctional targeting capability. Importantly, compound PP-1 (50 mg/kg, P.O.) demonstrated significant antitumor efficacy in a melanoma model, achieving a tumor growth inhibition rate of 42% without apparent systemic toxicity.
PP-1 demonstrates dual-target inhibitory activity against both the PD-1/PD-L1 immune checkpoint and tubulin polymerization, underscoring its potential as a promising lead compound for the development of next-generation dual-functional anticancer agents.
Copyright © 2026 Zhou, Ding, An, Wang, He, Du, Su and Yao.