Harnessing the druggability at orthosteric and allosteric sites of PD-1 for small molecule discovery by an integrated in silico pipeline.

The PD-1/PD-L1 interaction is a promising target for small molecule inhibitors in cancer immunotherapy, but targeting this interface has been challenging. While efforts have been made to identify compounds that target the orthosteric sites, no reports have explored the potential of small molecules to target the allosteric region of PD-1. Therefore, our study aims to establish a pipeline to identify small molecules that can effectively bind to either the orthosteric or allosteric pockets of PD-1. We categorized the PD-1 interface into two hot-spot zones (P-and N-zones) based on extensive analysis of its structural, dynamical, and energetic properties. These zones correspond to the orthosteric and allosteric PPI sites, respectively, targeted by monoclonal antibodies. We used a guided virtual screening workflow to identify hits from ∼7 million compounds library, which were then clustered based on structural similarity and assessed by interaction fingerprinting. The selective and diverse chemical representatives were subjected to MD simulations and binding energetics calculations to filter out false positives and identify actual binders. Binding poses metadynamics calculations confirmed the stability of the final hits in the pocket. This study emphasizes the need for an integrated pipeline that uses molecular dynamics simulations and binding energetics to identify potential binders for the dynamic PD-1/PD-L1 interface, due to the lack of small molecule co-crystals. Only a few potential binders were discovered from a large pool of molecules targeting both the allosteric and orthosteric zones. Our results suggest that the allosteric site has more potential than the orthosteric site for inhibitor design. The identified "computational hits" hold potential as starting points for in vitro evaluations followed by hit-to-lead optimization. Overall, this study represents an effort to establish a computational pipeline for exploring and enriching both the allosteric and orthosteric sites of PPI interfaces, "a tough but indispensable nut to crack".