Antimycobacterial cyclic peptide natural products bind and dysregulate stress response chaperones

The protein quality control system is a promising and largely untapped mycobacterial target for the discovery of novel antibiotics. The ClpC1:ClpP1P2 protease is an essential component of the system and is involved in both regulatory and stress-related protein degradation. Several cyclic peptide natural products have been discovered, including ecumicin, the ilamycins and cyclomarin, which bind the ClpC1 chaperone component of the system 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 classes of tuberculosis drugs. Utilising cutting edge mass-spectrometry-based proteomics on virulent Mycobacterium tuberculosis H37Rv, we reveal key differences in the mechanism of action for ecumicin, ilamycin E and cyclomarin, with each natural product having distinct effects on the proteome and different consequences for ClpC1 substrate recognition. An additional Clp protein, ClpC2, safeguards the ClpC1:ClpP1P2 complex from obstruction by chaperoning excess unfolded proteins in the event of proteotoxic stress. We show that all three natural products bind ClpC2, with varying affinities, in addition to ClpC1. Notably, we report the significant accumulation of the small heat shock protein, Hsp20, in the presence of ecumicin. A direct binding interaction between Hsp20 and ecumicin is observed that is proposed to stimulate this upregulation. Together, our data reveal that these natural products bind and dysregulate chaperones beyond ClpC1 and dysregulate ClpC1 substrate recognition via distinct mechanisms. This highlights the potential for the development of antimycobacterials that disrupt protein quality control beyond a single protein target.