Integrated computational pipeline for advanced sampling-based investigation and alternative inhibition of the...

Cancer represents a major public health challenge, demanding the continuous search for therapeutic strategies. The PD-1/PD-L1 pathway is crucial in tumor immune evasion, and its inhibition imposes difficulties such as antibody-related adverse effects and reduced affinity of small molecules to these checkpoint proteins. Previously, we uncovered a novel binding site within the C'D loop of PD-1. The 1508 ligand interaction induced a structural modification of this loop, which had not been explored in the PD-1/PD-L1 complex. In this work, we employed a robust computational pipeline to validate C'D loop modulation by small ligands as a promising strategy to impair the PD-1/PD-L1 interaction. Thus, we characterized the molecular recognition and binding mechanisms of 1508, revealing critical interaction dynamics within the C'D loop cavity through Supervised Molecular Dynamics (SuMD) and Umbrella Sampling (US). Using Gaussian Accelerated Molecular Dynamics (GaMD), we identified new conformations of PD-1 potentially unfavorable to the complexation to PD-L1. We specifically identified conformational constraints in the backbone of the C'D loop, particularly involving residues S87-P89. These constraints were accompanied by simultaneous structural modifications in the FG loop (A129-I134), resulting in a combined destabilizing effect as evidenced by molecular docking analysis of the ternary complex comprising PD-1, 1508, and PD-L1. These findings highlight the C'D loop as a promising druggable hotspot and validate the effectiveness of the computational pipeline in characterizing PD-1/PD-L1 pathway inhibitors.