ClpC1-targeting peptide natural products differentially dysregulate the proteome of Mycobacterium tuberculosis

Targeting the protein quality control system in Mycobacterium tuberculosis offers a powerful and largely untapped opportunity for antibiotic development. The ClpC1:ClpP1P2 protease is an essential component of the system and is involved in both regulatory and stress-related protein degradation. Several non-ribosomal peptide natural product families have been discovered, including ecumicin, ilamycins (rufomycins) and cyclomarins, which bind to the ClpC1 chaperone and possess potent antimycobacterial activity. This has led to significant interest in these molecules, and the ClpC1:ClpP1P2 system more generally, as a bona fide target for the development of new tuberculosis drugs. In this study, we apply a systematic and multi-layered approach combining quantitative proteomics, bioinformatics, transcriptomics, CRISPRi gene knockdown, and targeted biochemical and biophysical assays to dissect the mechanisms of ecumicin, ilamycin and cyclomarin in clinically relevant Mycobacterium tuberculosis. Strikingly, despite exhibiting similar binding modes to ClpC1, each natural product elicits distinct substrate-specific effects on protein degradation. We further show that derivatives of ilamycin and ecumicin engage the stress-response chaperones ClpC2 and Hsp20 respectively with varied affinities. The differential disruption of ClpC1 substrates and stress-response chaperones, and distinct reshaping of the Mycobacterium tuberculosis proteome by the three natural products, unveils opportunities for the development of protein quality control-targeted antimycobacterials.