Rosavin is identified as a privileged novel and unexpected scaffold for designing potent PD-1/PD-L1 modulators, offering promising candidates for cancer immunotherapy and exhibiting the unusual small-molecule property of inhibiting PD-L1 without inducing its dimerization.
Abstract
Background
Given the limitations of the existing monoclonal antibody (mAb)-based therapies, more efficient and safer small-molecule-based checkpoint therapies targeting the programmed cell death-1 (PD-1) / programmed cell death ligand-1 (PD-L1) axis are gaining growing attention and urgently required.
Objective
To identify a novel PD-L1 small-molecule inhibitor from natural products and systematically evaluate its antitumor activity, mechanism of action, and potential biomarkers.
Methods
Based on the co-crystal structure of PD-L1 with BMS-202 (PDB ID: 5J89), molecular docking was employed for high-throughput virtual screening of 16,563 natural products. The binding affinity of candidate compounds to PD-L1 protein was validated through microscale thermophoresis (MST), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assays and nuclear magnetic resonance (NMR). The blockade of PD-1/PD-L1 interaction was assessed using homogeneous time-resolved fluorescence (HTRF), NFAT-Luc luciferase reporter gene system, and cell membrane PD-1 binding assays. In vivo antitumor efficacy was systematically assessed in humanized PD-L1 knock-in B16F10 and MC38 tumor-bearing mouse models. The mechanism of action was investigated using RNA-seq transcriptomics, flow cytometry, and immunofluorescence staining. Finally, response mechanisms and potential biomarkers were systematically analyzed by comparing differential responses across distinct tumor models.
Results
Through high-throughput virtual screening, we identified rosavin as a small molecule with a novel scaffold that targets PD-L1, exhibiting the unusual small-molecule property of inhibiting PD-L1 without inducing its dimerization. Rosavin demonstrated significant antitumor activity in vivo by promoting antitumor immunity through enhancing CD8+ T cell activation, consistent with the effects of PD-L1/PD-1 blockade. Notably, rosavin was particularly effective for fighting against tumor progression in microsatellite instability-high (MSI-H) solid tumors and robustly strengthened the expression levels of CXCL9 and CXCL10 in tumors, which may serve as potential biomarkers for predicting responsiveness to rosavin-mediated PD-1/PD-L1 blockade.
Conclusion
Rosavin serves as a privileged novel and unexpected scaffold for designing potent PD-1/PD-L1 modulators, offering promising candidates for cancer immunotherapy.
Background/aim: Targeted therapies with monoclonal antibodies provide cancer patients with better prognosis and diseasefree survival. The blockade of immune checkpoints, including programmed cell death protein-1 (PD-1) and its ligand PD-L1, with monoclonal antibodies may boost immune responses against tumors and is regarded as an effective strategy in cancer immunotherapy. We describe the generation of anti-PD-L1 monoclonal antibodies with high affinity and specificity, and we assess their potential for therapeutic use in cancer.
Materials and methods: Hybridomas were selected for PD-L1 specificity and cross-reactivity with other immune checkpoint proteins and PD-L1 orthologs using indirect ELISA. Immunofluorescence and Western blotting assays were conducted for further characterization of the antibodies. The affinities of the antibodies for PD-L1 were determined using surface plasmon resonance. Receptor blocking activities were examined through competitive ELISA and cell-based luciferase reporter assays. Sequences of variable regions of the selected antibodies were determined by Sanger sequencing and subjected to BLAST analysis.
Results: A total of 25 PD-L1-specific monoclonal antibodies were generated. While most clones reacted with PD-L1 from cynomolgus monkeys, none of the antibodies displayed cross-reactivity with other checkpoint proteins. Immunofluorescence assays showed that the selected clones stained PD-L1-expressing cell membranes specifically, but not those of PD-L1-negative cells. Western blotting revealed that most of the clones recognized both glycosylated and nonglycosylated PD-L1, and a few reacted with the glycosylated form only. Only two clones with subnanomolar affinity for human PD-L1 were effective at blocking PD-1/PD-L1 and CD80/PD-L1 interactions. Sequence analysis of their variable regions revealed their unique specificity.
Conclusion: Of the 25 monoclonal antibodies produced in this study, only one was identified as a potential therapeutic drug candidate thanks to its high capacity for checkpoint blockade and affinity, as well as its unique sequence specificity. These properties are comparable to those of anti-PD-L1 antibodies currently used in clinical practice.
Nurşah Ersezen, Maide Şeker, Arzu Aysan et al.· Turkish Journal of Biology· 0 citations
Inhibiting overexpressed key immunoregulatory proteins, programmed
death-ligand 1 (PD-L1) and V-domain immunoglobulin suppressor of T cell activation (VISTA),
represents a critical strategy for restoring antitumor immunity. These proteins are upregulated under
hypoxic conditions, leading to immune evasion and drug resistance to monotherapy.
In this study, a comprehensive computational workflow, including structure-based pharmacophore
screening and validation, molecular docking, predictive toxicity profiling, and molecular
dynamics (MD) simulations, was used to explore a marine database for compounds that bind
PD-L1 and VISTA simultaneously.
The retrospective pharmacophore validation of known actives and decoys confirmed the
discriminatory reliability of both PD-L1 and VISTA models. From the common pharmacophore
alignment of PD-L1 and VISTA models, 235 marine compounds exhibited RMSD values <2.0 Å.
Based on the molecular docking screening of these common hits, CMNPD14606, CMNPD20027,
and CMNPD20697 were found to have higher affinities than the reference P17 and S8. Additionally,
these hits demonstrated favorable physicochemical profiles; however, the in-silico toxicity profiling
revealed modest-to-high genotoxicity and mutagenicity liabilities for all three compounds,
which represent key safety limitations. The MD simulations, performed in triplicate for up to 100
ns, confirmed MD stability across independent replicates.
The identification of marine-derived compounds addresses the need to overcome resistance
to single-checkpoint immunotherapy. Accordingly, these compounds are regarded as earlystage
lead scaffolds requiring structural optimization rather than hits with acceptable overall safety
profiles.
These results suggest the development of dual-targeting PD-L1/VISTA compounds
that warrant lead optimization to mitigate toxicity risks before further experimental investigation.
Jeevan Patra, Surya Nath Pandey, Harishchandra Verma et al.· Current Chemical Biology· 0 citations
CP-1 exhibits balanced, dual-nanomolar inhibitory activity against PD-L1 and CD73 and displays potent immunomodulatory effects at the cellular level and serves as a promising lead candidate for developing novel bifunctional agents to advance tumor immunotherapy.
Jing-Jing Du, Sen Wu, Shiyun Cheng et al.· Frontiers in Immunology· 0 citations
Vascular endothelial growth factor receptor 2 (VEGFR2) is a master regulator of angiogenesis and cancer progression. However, current VEGFR2 modulator face significant challenges, including off-target toxicity and acquired resistance, underscoring the urgent need for novel therapeutic agents with improved efficacy and safety profiles. Here, we reported that virtual screening of 39,442 natural products from the ZINC natural product library, coupled with molecular docking and molecular dynamics (MD) simulations to evaluate the binding stability of candidate compounds, identified NAT-6-321056 as a highly promising modulator of VEGFR2 signaling. Biological evaluations demonstrated that NAT-6-321056 exerted potent inhibition on the growth of a broad spectrum of cancer cells, including both solid tumors and hematological malignancies. In EA.hy 926 endothelial cells and SK-N-DZ neuroblast cells, the compound significantly suppressed proliferation, migration, and invasion. Microscale thermophoresis (MST) confirmed direct binding of NAT-6-321056 to VEGFR2 with favorable affinity. Kinase profiling against a panel of 33 kinases indicated that NAT-6-321056 exhibited a multi-kinase modulation profile. Mechanistic studies revealed that NAT-6-321056 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and was associated with reduced VEGFR2 phosphorylation and attenuation of the downstream ERK/JNK/AKT signaling pathways. Moreover, NAT-6-321056 exhibited robust in vivo anti-angiogenic effects in both the chick chorioallantoic membrane (CAM) assay and transgenic zebrafish vascular fluorescence imaging models. Computational absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction suggested acceptable drug-like properties. Collectively, these findings demonstrated that NAT-6-321056 is a promising modulator of VEGFR2 signaling with potent anti-angiogenic activity and represents a viable candidate for cancer therapy.
Xiao Lei, W. Gong, Nadine Wiesmann-Imilowski et al.· Biochemical Pharmacology· 0 citations
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.
B. Guan, Binbin Cheng, Hongqiao Li· Frontiers in Immunology· 0 citations
This study combines ligand- and structure-based in silico strategies to predict the inhibitory activity of natural flavonoids on the Polo-Like Kinase-1 (PLK-1) enzyme as candidate anticancer agents. This enzyme participates in mitosis and is overexpressed in cancer cells. Furthermore, it has been shown to have important implications for tumor metastasis, and its inhibitors are attractive starting points for drug development. First, classification models are developed using linear discriminant analysis and a multilayer perceptron neural network. Models with accuracy greater than 80%, validated using standard statistical performance metrics and applicability domain, are used for virtual screening identifying four compounds as potential antitumor drugs. Subsequently, the identified compounds are evaluated using a molecular docking methodology to verify their binding mode and interactions with the catalytic domain of PLK-1. Finally, the integration of molecular dynamics simulations, at 300 ns, with Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) thermodynamic calculations demonstrates that the hydroxylation pattern of ring B in the flavonol scaffold is the fundamental chemical-structural determinant of electrostatic interactions and the architecture of water-mediated networks. Among the evaluated flavonoids, myricetin showed the most favorable overall computational profile, including the highest virtual-screening score and the most favorable mean MM/GBSA estimate, supporting its prioritization for experimental evaluation as a potential PLK-1 inhibitor. The integration of these approaches offers a robust methodological framework for proposing candidates with a higher probability of success, in subsequent stages of experimental validation, reducing time and costs in the early stages of drug development.
Y. Cañizares-Carmenate, E. Hernández-Rodríguez, Y. Perera-Sardiña et al.· International Journal of Mol...· 0 citations