Discovery and development of new small-molecule immune system modulators

Modulation of immune modulatory pathways has emerged as one of the most successful and explored approaches for cancer immunotherapy. Current therapeutics include monoclonal antibodies, which have shown impressive clinical outcomes in the treatment of several types of tumors. However, low response rate, patient acquired resistance and induction of severe immune-related adverse effects represent critical drawbacks. To overcome these limitations, complementary strategies that inhibit tumor immunosuppressive pathways and enhance immunity are urgently needed. We hypothesized that inhibition of PD-1/PD-L1 interaction, for immunosuppression blockade, could be improved by the development of immune modulatory small molecules. For this purpose, we developed a computer-assisted drug design approach that allowed the identification of potential PD-1/PD-L1 small-molecule inhibitors. These potential hits were confirmed as true hits by in vitro and ex vivo assessment of its antitumor immune response. The compounds identified were able to activate T cell function by increasing tumor-associated antigen recognition, processing and presentation to effector T cells. The use of computational-aided drug design tools was decisive to accelerate the identification of new immune small molecules modulators which, certainly, will play a key role in the next generation of cancer immunotherapy approaches. Hits were identified by a structure based virtual screening campaign based on molecular docking. In addition to hit discovery, these methods were essential in providing a strong structural elucidation. Over biochemical and in vitro studies three PD-1/PD-L1 small-molecule inhibitors by direct PD-L1-binding were identified at nanomolar scale. Two different types of cancer, a highly immunogenic (melanoma) and a traditionally non-responder to immune checkpoint modulation (breast cancer) were studied in vitro. Finally, co-culture experiments were developed and the treatment with the identified inhibitors led to T cell activation and antigen-specific cytotoxic T-cell activity against melanoma cells ex vivo. Altogether, these findings reveal important aspects on the use of small molecules as immune checkpoint modulators, as well as their effect on the modulation of tumor-infiltrating immune cells. This approach offers numerous advantages over the use of monoclonal antibodies, predominantly on pharmacokinetics properties. In addition, they represent a more affordable and accessible alternative. Therefore, small-molecule immune system modulators emerge as a potential strategy to improve clinical outcomes of cancer treatment