Main Proteases of Key RNA Viruses, Including Coronavirus, Recognize P2 Residues in the Polyproline Type II Conformation: Harnessing Proline's Unique Conformation for Protease Inhibitors

Positive-sense RNA viruses, particularly those in the Picornavirus-like superfamily and the Coronaviridae family, significantly impact public health, necessitating the development of focused antiviral agents. The main proteases of these viruses are promising targets for antiviral development. Peptidyl protease inhibitors containing Pro derivatives, mimicking rigid Leu at the P2 position, have emerged as potent drug candidates against SARS-CoV-2 3CLpro, despite several theoretical disadvantages compared to non-Pro rigid Leu mimetics: loss of a critical hydrogen bond due to the Pro tertiary amide and the unique PPII conformation associated with Pro, contrasting the protease-preferred β-strand conformation in the solution phase. Interestingly, despite these potential drawbacks, Pro derivatives that mimic a rigid Leu at the P2 position manage to either enhance or maintain inhibitory activities when compared to non-Pro derivatives. In this study, we analyzed over 200 crystal structures of main protease-bound peptidyl inhibitors of major RNA viruses. Our analysis revealed a previously unnoticed preference of these main proteases for the P2 residues of peptidyl inhibitors to adopt the PPII conformation, contrasting the traditional β-strand conformation. Furthermore, our unbiased all-atom classical MD simulations of literature examples confirm that using Pro-based rigid Leu mimetics constrains the P2 residue to the enzyme-preferred PPII conformation in the solution phase. This leads to a significant reduction in configurational entropy, comparable in magnitude to a typical strong hydrogen bonding interaction when compared to non-Pro P2 residues. Despite the loss of a critical hydrogen bond, this phenomenon accounts for the enhanced binding affinities of peptidyl protease inhibitors that feature Pro-based rigid Leu mimetics as the P2 residue. These findings provide new insights into the binding dynamics of peptidyl protease inhibitors with viral main proteases and highlight the importance of new strategies to constrain the P2 residue of peptidyl inhibitors in the PPII conformation, without sacrificing the P2 amide hydrogen, which will further enhance the binding affinities.