Conformationally restricted pleuromutilins via a pyridinyl-amide tether bias peptidyl transferase center engagement enabling broad-spectrum potency against multidrug-resistant bacteria.

Drug-resistant bacteria are spreading at an alarming rate, yet the global antimicrobial pipeline remains largely dependent on structural modifications of existing antibiotics. In this study, we explore innovative agents targeting multidrug-resistant (MDR) bacteria, drawing inspiration from recently approved pleuromutilins that feature a novel chemical scaffold. A conformational restriction strategy was employed, cyclizing the flexible C14 thioether-amide chain of valnemulin into a rigid pyridine-amide bridge. This modification preorganized the scaffold into its bioactive conformation at the ribosomal peptidyl transferase center (PTC). Guided by this design, we synthesized a series of amino acid-substituted pyridine-amide pleuromutilin derivatives and systematically assessed their antibacterial properties. Among these, compound 14c exhibited broad-spectrum antibacterial and pronounced antimycoplasmal activity, demonstrated rapid dose-dependent bactericidal effects, and showed low cytotoxicity. Mechanistic studies revealed that 14c binds to the PTC to inhibit protein synthesis, while also suppressing biofilm formation, disrupting preformed biofilms, and damaging bacterial membranes. In a murine model of systemic methicillin-resistant Staphylococcus aureus (MRSA) infection, treatment with 14c (15 mg/kg) resulted in a 90 % survival rate over seven days and significantly reduced bacterial loads in multiple organs, outperforming valnemulin at equivalent doses. Overall, these findings indicate that conformationally restricted pleuromutilins with an intramolecular pyridinyl-amide tether bias PTC engagement, establishing 14c as a promising therapeutic candidate for combating MDR bacterial infections.