Unique Interactions of Novel Rufomycin “Click Chemistry” Analogs with <i>Mtb</i> ClpC1 and Implications

Disrupting protein homeostasis in <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) by targeting the ClpC1P1P2 proteolytic complex is a promising anti-TB strategy. We synthesized conformationally constrained monomeric and dimeric rufomycin 4/6 (RUF) analogs via click chemistry. While most monomeric analogs were inactive, dimeric analogs displayed potent anti-<i>Mtb</i> activity. Surface plasmon resonance revealed tight, slow-dissociating binding of dimers to the ClpC1 N-terminal domain (ClpC1_NTD), indicating prolonged residency time. X-ray crystallography and size exclusion chromatography demonstrated that dimeric analogs induce NTD dimerization, likely shifting the equilibrium toward enzymatically active hexamers rather than inactive decamers. Dimers enhanced ATPase activity over 10-fold, surpassing that of ecumicin (8-fold) and far exceeding RUF (<2-fold). Notably, our dimers bind two NTDs per molecule (1:2), contrasting with RUF (1:1) and ecumicin (2:1), suggesting a distinct activation mechanism. These RUF-based click chemistry dimers represent potent ClpC1 modulators with extended residency and novel stoichiometry, offering promising tools for studying protein degradation.