Zhe, YangAlshammari, Eid Salem2025-08-112025https://hdl.handle.net/20.500.14154/76126Ubiquitin-specific proteases USP10 and USP7 play crucial roles in regulating diverse cellular processes and have emerged as attractive therapeutic targets in cancer. In this dissertation, we present a multi-scale structural and molecular dynamics investigation of these deubiquitinases, combining predictive modeling, crystallographic data, molecular dynamics (MD) simulations. For USP10, which lacks a crystal structure, AlphaFold model and MD simulations revealed a stable and highly conserved USP domain, with some flexible loops surrounding the active site and the Ub-binding pocket, suggesting auto-regulatory structural elements that may control conformational regulation, substrate access, and enzymatic activity. USP10’s flexible N-terminal region contains regulatory loops that transiently interact with the catalytic domain, suggesting another layer of regulation and substrate control. Compound GL462 is a potential USP10 inhibitor, exhibiting a binding mode near the active site. Protein stability and inhibition assays confirmed GL462’s destabilizing effect on USP10 through a selective protein unfolding mechanism. Its potent inhibitory activity, positioning it as a promising lead for further development. For USP7, comparative analyses of the apo and ubiquitin-bound structures revealed that proper alignment of the catalytic triad and opening of the switching loop are key elements for USP7 activation. Classical and metadynamics simulations showed that certain mutations in the switching loop, such as H294E, stabilize an active-like state with minimal energy cost, while others, like V302K, remain energetically trapped in either an intermediate or inactive conformation, highlighting the critical role of switching loop dynamics in USP7 activation. Analysis of sixteen USP7-inhibitor complexes identified two distinct binding modes. Mode 1 compounds appear to rigidly block activation by binding to a critical region within the catalytic cleft. In contrast, Mode 2 inhibitors bind at the Thumb-Fingers interface, located at the edge of the ubiquitin-binding pocket, thereby interfering with ubiquitin binding while permitting reversible regulation. Metadynamics simulations revealed distinct energy barriers associated with these inhibitors, correlating with their conformational flexibility and mechanisms of action. Taken together, these findings reveal key structural features regulating USP10 and USP7 conformational activation, regulation, and inhibition, providing a framework for developing targeted inhibitors with therapeutic potential.152enUbiquitin-specific proteases Enzymes Proteins Inhibitors Cancer TherapyThesis