How Polyproline\nType II Conformation at P<sub>2</sub> Residues Influences the Success\nof Proline-Based Peptidyl Inhibitors\nAgainst Coronavirus Main Protease

In the wake of the pandemic, peptidyl protease inhibitors\nwith\nPro-based rigid Leu mimetics at the P<sub>2</sub> position have emerged\nas potent drug candidates against the SARS-CoV-2 main protease. This\nsuccess is intuitively attributed to the enhanced hydrophobic interactions\nand rigidity of Pro-based rigid Leu mimetics in the literature. However,\nthe tertiary amide of proline P<sub>2</sub> derivatives, which hinders\nthe formation of a critical hydrogen bond with the enzyme active site,\nand the constrained PP<sub>II</sub> conformation, which contradicts\nthe protease preferred β-strand conformation, represent two\noverlooked disadvantages associated with these inhibitors over traditional\ninhibitors and, theoretically, should adversely affect their potency.\nInterestingly, despite these major disadvantages, they maintain or\ndisplay improved potency compared to traditional peptidyl protease\ninhibitors. In this study, we uncover a previously unnoticed preference\nfor P<sub>2</sub> residues of the protease inhibitors to adopt the\nPP<sub>II</sub> conformation, regardless of residue identity, in the\nmain protease-bound form of key RNA viruses, deviating from the traditional\nβ-strand conformation. We also demonstrate that Pro-based rigid\nLeu mimetics at P<sub>2</sub> enhance binding affinity by favoring\nthe enzyme-preferred PP<sub>II</sub> conformation and significantly\nreducing configurational entropy loss upon binding, comparable to\nthat of a typical hydrogen bond. This work also highlights the importance\nof a multidisciplinary approach to enhance the understanding of structure–activity\nrelationships beyond traditional medicinal chemistry intuition. We\nbelieve these findings provide new, deep insights and address a major\nknowledge gap in the area of peptidyl protease inhibitor design, identifying\nkey drivers behind the success of Pro-based peptidyl protease inhibitors\nbeyond mere rigidity and hydrophobicity.