Engineering Pleuromutilin Epimers to Engage an Unexploited Ribosomal Binding Pocket

Optimizing the bioactivity of natural product–derived antibiotics is an inherently challenging process, particularly when targeting complex macromolecular assemblies like the bacterial ribosome. Yet such efforts are essential for the rational design and development of next-generation antibiotics. Pleuromutilin derivatives are of particular value given their recent approval for systemic human use and low frequency of resistance development. While most efforts have focused on C22-substituted thioglycolates, identifying new functionalization sites is critical for advancing this antibiotic class. Leveraging structural insights from computational modeling, we identified C12 as a novel derivatization site amenable to divergent semisynthetic modification. To access this site, we epimerized C12 and applied an optimized anti-Markovnikov hydroazidation strategy to activate C20 for rapid diversification into a triazole library using CuAAC click chemistry. Biological evaluation confirmed that aromatic triazole substituents exhibited the best activity. Docking analysis guided structure-based refinements, resulting in derivatives with sub-ug/mL antimicrobial potency. X-ray crystallography revealed that the novel triazole arm binds in a previously unexploited region of the ribosomal A-site, with mechanism-of-action and toxicity studies confirming selectivity. Notably, C22 functionality did not impact activity. Collectively, these findings define a new ribosomal binding region for pleuromutilin and provide a framework for further structurally and computationally guided semisynthetic derivatization.