How Polyproline Type II Conformation at P₂ Residues Influences the Success of Proline-Based Peptidyl Inhibitors Against Coronavirus Main Protease

In the wake of the pandemic, peptidyl protease inhibitors with Pro-based rigid Leu mimetics at the P₂ position have emerged as potent drug candidates against the SARS-CoV-2 main protease. This success is intuitively attributed to the enhanced hydrophobic interactions and rigidity of Pro-based rigid Leu mimetics in the literature. However, the tertiary amide of proline P₂ derivatives, which hinders the formation of a critical hydrogen bond with the enzyme active site, and the constrained PP_II conformation, which contradicts the protease preferred β-strand conformation, represent two overlooked disadvantages associated with these inhibitors over traditional inhibitors and, theoretically, should adversely affect their potency. Interestingly, despite these major disadvantages, they maintain or display improved potency compared to traditional peptidyl protease inhibitors. In this study, we uncover a previously unnoticed preference for P₂ residues of the protease inhibitors to adopt the PP_II conformation, regardless of residue identity, in the main protease-bound form of key RNA viruses, deviating from the traditional β-strand conformation. We also demonstrate that Pro-based rigid Leu mimetics at P₂ enhance binding affinity by favoring the enzyme-preferred PP_II conformation and significantly reducing configurational entropy loss upon binding, comparable to that of a typical hydrogen bond. This work also highlights the importance of a multidisciplinary approach to enhance the understanding of structure-activity relationships beyond traditional medicinal chemistry intuition. We believe these findings provide new, deep insights and address a major knowledge gap in the area of peptidyl protease inhibitor design, identifying key drivers behind the success of Pro-based peptidyl protease inhibitors beyond mere rigidity and hydrophobicity.