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.
Wei Zou, Tianle Li, Jacek Plewka et al.· Phytomedicine· 0 citations
Protein–protein interactions dominated by large, flat interfaces are widely considered challenging drug targets. The programmed cell death protein-1/programmed death ligand-1 (PD-1/PD-L1) immune checkpoint exemplifies this problem, as the interaction is mediated by an extended β-sheet surface lacking deep pockets. Despite this, PD-L1 has been successfully inhibited by chemically distinct modalities, including antibodies, macrocyclic peptides, and small molecules. Here, we present a comparative, structure-driven analysis of PD-L1 complexes deposited in the Protein Data Bank and demonstrate a striking convergence: all effective inhibitors engage the same CC′FG β-sheet face of PD-L1. Antibodies directly occlude this surface, macrocyclic peptides such as pAC65 reproduce antibody-like surface coverage in a compact and preorganized scaffold, and biphenyl small molecules neutralize the same epitope indirectly by inducing PD-L1 homodimerization. This unified structural framework reveals modality-agnostic design principles for targeting flat immune checkpoint PPIs. This Perspective provides a unified structural framework for understanding PD-L1 inhibition across clinically tested antibodies, macrocyclic peptides, and small molecules. Both visualizing and quantitatively comparing interface overlap, hotspot conservation, and buried surface area, the work demonstrates that distinct inhibitory modalities converge on the same functional CC′FG hotspot region while employing fundamentally different neutralization mechanisms. These findings establish structure-guided principles for the rational design of next-generation PD-L1 modulators across diverse therapeutic modalities.
Imma Capriello, T. Moreira Pereira, Gustavo Barbosa Reis et al.· Journal of Medicinal Chemist...· 0 citations
Novel selenium-containing small molecule PD-L1 inhibitors were designed and synthesized for the first time to explore their potential as antitumor agents. By computer-aided structural optimization, HTRF and SPR techniques, compound SA13 was identified as the most potent blocker of the PD-1/PD-L1 interaction, exhibiting an IC50 value of 5.2 ± 0.5 nM, a KD value of 9.06 ± 1.25 nM, respectively. Study on the SA13/hPD-L1 cocrystal structure (2.9 Å) revealed a unique selenomethyl-involved binding mode, which may interpret its superior inhibitory activity compared to other analogs. Cell-based assays showed that SA13 can mediate the internalization of PD-L1 and strongly block hPD-1 and hPD-L1 interaction, demonstrating its effectiveness in biological events. Notably, in the Hu-PD-L1 MC38 mouse model, SA13 significantly inhibited tumor growth, with a tumor growth inhibition (TGI) rate of 77.79% (60 mg/kg, administrated intragastrically) with no observable toxicity. These data indicate that SA13 is a promising and safe novel antitumor agent worthy of further development.
Jianwei Wang, Shenwei Yu, Liang Qian et al.· Journal of Medicinal Chemist...· 0 citations