Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics. [Display omitted] •Cyclomarin A and ecumicin hijack ClpC1P1P2 proteases by mimicking damaged proteins•ClpC2 and ClpC3 function as isolated receptor proteins safeguarding the Clp protease•Homo-BacPROTACs induce degradation of the essential ClpC1 and its ClpC2 security•BacPROTACs present potent antibiotics that efficiently kill M. tuberculosis Homo-dimeric BacPROTACs induce the self-degradation of essential Clp components of the mycobacterial proteostasis system, introducing a potent antibiotic strategy against M. tuberculosis.