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.

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.

Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death protein 1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesised that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death.
Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry and ELISA. EV PD-L1 binding to PD-1 was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8+ T cells. Plasma EV and soluble PD-L1 were assayed in the plasma of islet autoantibody-positive (Ab+) individuals or individuals with recent-onset type 1 diabetes and compared with levels in non-diabetic control individuals.
PD-L1 protein co-localised with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. Treatment with IFN-α or IFN-γ for 24 h induced a twofold increase in EV PD-L1 cargo without a corresponding increase in the number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8+ T cells. Plasma EV PD-L1 levels were increased in Ab+individuals, particularly in those positive for a single autoantibody. Additionally, in Ab+ individuals or those who had type 1 diabetes, but not in control individuals, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV PD-L1 could be protective for residual beta cell function.
IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8+ T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in Ab+ individuals than in control individuals. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.
© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.