Stereo-selectivity of enantiomeric inhibitors to ubiquitin-specific protease 7 (USP7) dissected by molecular docking, molecular dynamics simulations, and binding free energy calculations.

The ubiquitin-specific protease 7 (USP7), as a member of deubiquitination enzymes, represents an attractive therapeutic target for various cancers, including prostate cancer and liver cancer. The change of the inhibitor stereocenter from the S to R stereochemistry (S-ALM → R-ALM34) markedly improved USP7 inhibitory activity. However, the molecular mechanism for the stereo-selectivity of enantiomeric inhibitors to USP7 is still unclear. In this work, molecular docking, molecular dynamics (MD) simulations, molecular mechanics/Generalized-Born surface area (MM/GBSA) calculations, and free energy landscapes were performed to address this mystery. MD simulations revealed that S-ALM34 showed a high degree of conformational flexibility compared to the R-ALM34 counterpart, and S-ALM34 binding led to the enhanced intradomain motions of USP7, especially the BL1 and BL2 loops and the two helices α4 and α5. MM/GBSA calculations showed that the binding strength of R-ALM34 to USP7 was stronger than that of S-ALM34 by - 4.99 kcal/mol, a similar trend observed by experimental data. MM/GBSA free energy decomposition was further performed to differentiate the ligand-residue spectrum. These analyses not only identified the hotspot residues interacting with R-ALM34, but also revealed that the hydrophobic interactions from F409, K420, H456, and Y514 play the major determinants in the binding of R-ALM34 to USP7. This result is anticipated to shed light on energetic basis and conformational dynamics information to aid in the design of more potent and selective inhibitors targeting USP7.