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.

ClpC1:ClpP1P2 蛋白酶是分枝杆菌蛋白质稳态系统的核心组成部分。为了提高靶向 Clp 蛋白酶的抗结核药物的疗效，我们对抗生素 cyclomarin A 和 ecumicin 的作用机制进行了表征。定量蛋白质组学表明，抗生素会导致大量蛋白质组失衡，包括两个未注释但保守的应激反应因子 ClpC2 和 ClpC3 的上调。这些蛋白质可能保护 Clp 蛋白酶免受过量错误折叠蛋白质或 cyclomarin A 的影响，我们证明它可以模拟受损蛋白质。为了克服 Clp 安全系统，我们开发了一种 BacPROTAC，它可以诱导 ClpC1 与其 ClpC2 看管者一起降解。双 Clp 降解器，由连接的 cyclomarin A 头组成，在杀死致病性方面非常有效结核分枝杆菌，效力比母体抗生素高 100 倍以上。总之，我们的数据揭示了 Clp 清道夫蛋白作为重要的蛋白质稳态保障措施，并突出了 BacPROTAC 作为未来抗生素的潜力。