Structure-BasedAnalysis of Semisynthetic Anti-TBRufomycin Analogues

This study employed structural information from cocrystals of rufomycin 4 (<b>1a</b>) and caseinolytic protein C1 (ClpC1)-NTD-wt to guide design and semisynthesis of rufomycin analogues, evaluate their antituberculosis (TB) biological profiles, and establish structure–activity relationships (SAR). Covering three regions of interest (ROIs, A–C) as modification sites, 14 of the 30 semisynthetic analogues (<b>2</b>–<b>31</b>) showed similar or improved MICs relative to the main natural precursors, rufomycins 4/6 (<b>1a/b</b>). Compounds <b>5</b> and <b>27</b> exhibited up to 10-fold enhanced potency against <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) in vitro, with MIC values of 1.9 and 1.4 nM, respectively. Evaluation of ClpC1-binding properties used existing ClpC1-NTD complexes with rufomycin 4 (PDB: 6cn8) and ecumicin (PDB: 6pbs) as references. The newly reported X-ray ClpC1-NTD cocrystal structure of <b>11</b> (syn. But4-Cl) revealed significant conformational effects involving the side chains of certain amino acids of the heptapeptide and confirmed the importance of ROIs A–C for medicinal chemistry efforts. Observed interactions of the <i>N</i>-terminal tail of ClpC1 with the rufomycin analogues vs ecumicin explains their different modes of inactivating the ClpC1/P1/P2 homeostatic machinery. Collectively, the observations inform further SAR optimization strategies for the rufomycin class of antibiotics and complement our understanding of their mode of action.