Towards orally available immunotherapy – small molecule inhibitors of the PD-1/PD-L1 interaction

The `programmed cell death 1 receptor` (PD-1) is a cell surface protein receptor involved in the regulation of T-cell activation. It is expressed on activated T-cells and is part of the cellular `immune-checkpoints` - a set of signal transduction proteins that can stimulate or inhibit the immune system such as the anti-tumor response. The immune checkpoint receptor PD-1 has two main ligands, `programmed cell death ligand-1 and -2` (PD-L1 and PD-L2) that are highly expressed on vast range of cancer cells. Binding to these ligands modulates PD-1 activity such that it sends the `off-signal` to shut down immune response. Given that PD-L1 and PD-L2 are expressed on the cell surface of many tumors and directly bind PD-1 makes both PD-1 and PD-L1/2 important therapeutic targets. At ‎present there are two approved biological drugs that target PD-1 for cancer therapy- Nivolumab and Pembrolizumab, a fully human anti-PD-1 monoclonal antibody and an anti PD-1 humanized monoclonal antibody, respectively. No other small molecule and/or non-antibody biologics have been approved that target PD-1 or PD-L1/2.

Shown here are new results of small molecule inhibitors targeting PD-1 and inhibiting its interaction with PD-L1. The molecules were filtered by the application of in-house developed in-silico algorithms on a library of approximately 20 million small molecules. Thereafter, a subset of the molecules was selected and tested in-vitro using two orthogonal assays. The first assay directly measured the binding of the small molecules to PD-1 by microscale thermophoresis (MST). The second assay followed the ability of the molecules to interfere with the PD-1/PD-L1 binding via ELISA. For the active hits from the in-vitro assays which represent different chemical families, closely related chemical analogues were evaluated based on the structural model. Ultimately, four novel chemical scaffolds showing promising inhibition of the PD-1/PD-L1 complex were selected for further investigation. For each new scaffold, representative molecules were also tested in cellular assay monitoring the secretion of IL-2 nad IFNg to verify their mode of action.

Our results from both biophysical and cellular assays indicate that the small molecule inhibitors found in our research are capable of inhibiting the PD-1/PD-L1 interaction and may be further developed into orally-available immunotherapeutic drug.