Structure-Based Analysis of Semisynthetic Anti-TB Rufomycin 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.