Not only ClpC1 but also ClpX are drug targets of Ilamycins

Abstract

Combatting drug-resistantMycobacterium tuberculosis(Mtb) necessitates the discovery of novel anti-Mtb agents targeting unexploited molecular pathways. The Clp protease system, integral to protein homeostasis in Mtb, has emerged as a promising target. We report that ilamycins exhibit potent antimycobacterial activity by specifically targeting ClpX and ClpC1. Spontaneous resistance to ilamycin E (ILE) and ilamycin F (ILF) in Mtb mutants correlated with mutations in ClpC1 and ClpX. Molecular docking simulations suggest these mutations occur at critical residues in the N-terminal domain (NTD) of MtbClpC1 and MtbClpX. Functional analyses, including gene over-expression inMycobacterium marinumand targeted gene editing inMycobacterium abscessus(Mab) andMycobacterium smegmatis(Msm), substantiated the significance of these mutations in conferring ILE and ILF resistance. Gene silencing heightened the susceptibility of recombinant Mab and Msm strains to ILE and ILF, concurrently impairing their growth. Differential scanning fluorimetry confirmed the stabilization of MtbClpC1-NTD by ILE and ILF. Moreover, ILE markedly impeded the proteolytic function of the ClpC1P1P2 complex without diminishing ClpC1 ATPase activity. Collectively, our findings demonstrate that ilamycins exert mycobactericidal effects by disrupting ClpC1 and ClpX functions, leading to proteostasis dysregulation. This study underscores the therapeutic potential of targeting the Clp protease system in the treatment of tuberculosis.