A new promising family of small molecules regulating the PD-L1/PD-1 signaling pathway

10 min read
July 12, 2022
J for ImmunoTherapy of Cancer

Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment

Rita C Acúrcio et al. J Immunother Cancer. 2022 Jul‍

Immune checkpoint therapy has revolutionized cancer treatment. Inhibiting programmed cell death protein 1 (PD-1) or PD-ligand 1 (PD-L1) has shown exciting clinical outcomes in diverse human cancers. However, only monoclonal antibodies have been approved to achieve immune checkpoint blockade targeting the PD-1 axis and these expansive treatments are financially inaccessible to many. In a study published in Journal for Immunotherapy of Cancer, Rita C. Acurcio et al. identified a new class of PD-L1/PD-1 signaling pathway regulators that promote an extensive infiltration of effector CD8 T cells to the tumor microenvironment.

While tremendous clinical benefits are observed in patients who respond to monoclonal antibodies (mAb), the lack of understanding of the mechanistic basis regulating this immune checkpoint pathway results in low response rates, absence of long-term remission and severe immune related adverse events (IRAEs), which add up to the very high production cost of mAb. In this study, Rita C. Acurcio et al. focused on the identification and validation of small-molecule inhibitors as an alternative approach to therapeutically target PD-L1 or PD-1. Small molecules can indeed provide increased oral bioavailability, bio-efficiency and short half-life activity, which is particularly relevant for IRAEs.

In silico identification of PD-1/PD-L1 small molecules inhibitors

The authors followed a translational strategy initiated by developing a computationally driven approach to identify small-inhibitor candidates. Nearly 900,000 compounds were screened from synthetic compound libraries like the National Cancer Institute, Enamine, Specs, Mu.Ta.Lig Chemoteca, MMV and inhouse databases. A structure-based virtual screening campaign was performed, using molecular docking into the PD-L1 binding site. The top-ranked compounds were then subjected to exhaustive docking analyses that predicted with higher precision the corresponding binding pose and the interactions within the receptor-binding pocket. Finally, the selected compound pool was filtered by applying the Lipinski’s rule of five criteria for enhanced drug-likeness, and only those compounds that presented favorable binding conformations and surface complementarity with the receptor, and exhibited the important interactions with key pocket residues, were retained.


This approach yielded 95 possible PD-L1 binders with chemically diverse structures.


In vitro functional assays to inhibit PD-1/PD-L1 interaction

The authors tested them afterward for their capacity to inhibit the PD-1/PD-L1 interaction using in vitro functional assays with homogeneous time-resolved fluorescence (HTRF). The results show that out of the 95 compounds tested, 16 (17%) chemically diverse compounds were able to lead to a 50% reduction of the HTRF signal, thus indicating a significant effect on the PD-1/PD-L1 inhibition (p<0.001). Among the 16 compounds, 12 revealed dose-response effect and were further analyzed for their ability to bind to PD-L1.

Selection of compounds improving the thermal stability of PD-L1

Looking into their rationale, the authors expected that the validated hits would bind to PD-L1 similar to the BMS inhibitors. To test their hypothesis, they determined the thermal transition of PD-L1 in thermal denaturation assays by differential scanning fluorimetry (DSF) and checked for a shift in the proteins’ Tm in the presence of the PD-L1 inhibitor candidates. Notably, a thermal shift was observed for all molecules. The most promising small-molecule inhibitor was the 69, for which a WaterLOGSY NMR experiment was conducted (to confirm the stabilizing effect observed by DSF).

Hit compounds’ effect on cell viability and modulation of PD-1/PD-L1 interaction

The authors then decided to further evaluate the compounds’ activity exploiting 2D and 3D models based on naturally expressing PD-L1 cells. Two different types of human cancer cell lines (breast cancer MDA-MB-231 and melanoma A375) were selected to perform in vitro studies. The authors noticed that their PD-L1-binding small molecules considerably impacted PD-L1 levels in both cancer cell lines.



To further address the ultimate role of the compound 69, they developed 2D and 3D co-culture studies of paired, matched, patient-derived tumor cells and peripheral blood mononuclear cell (PBMC) and realized that samples treated with their most promising PD-1/PD-L1 inhibitor could activate T cells by inhibiting this pathway.

Ex vivo and in vivo T-cell infiltration promotion

In addition, the co-culture of 3D melanoma spheroids and PBMC demonstrated the capacity of small molecules to promote T-cell infiltration.

Finally, to extend the clinical relevance of their ex vivo findings, the authors tested the small-molecule inhibitor using a human-relevant in vivo model. Of note, this model was a humanized PD-1 mice developed by genOway by inserting a chimeric PD-1 with a human extracellular domain in the mouse PD-1 locus. These mice were implanted with colorectal cancer MC38 cells expressing the human PD-L1, also created by genOway. This study showed that the compound 69 inhibits the PD-1/PD-L1 interaction, leads to the activation of T-cell function, and ultimately recruits cytotoxic T lymphocytes (CTL) to the tumor microenvironment (TME), which resulted in a strong control of tumor growth.

To conclude, Rita C. Acurcio et al. identified a new promising family of small-molecule candidates that are less expensive and that regulate the PD-1/PD-L1 signaling pathway, promoting an extensive infiltration of effector CD8 T cells to the tumor microenvironment. Potential off-the-shelf products to enhance immune checkpoint clinical outcomes!


  1. AcĂşrcio RC, et al. J Immunother Cancer 2022;10:e004695. doi:10.1136/jitc-2022-004695.

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