Not only ClpC1 but also ClpX are drug targets of Ilamycins

Mycobacterial Clp protease system has emerged as a promising therapeutic target. Numerous natural cyclic peptides, like ilamycins, have been reported to possess antitubercular activities by targeting ClpC1 component of the ClpC1P1P2 proteasome, albeit without strong genetic evidence. Here, we discovered that ilamycin E and F (ILE/F) exhibit more potent delayed mycobactericidal activities than other ilamycin components. Novel insertion mutations in clpC1 and a unique mutation in clpX have been identified. These mutations have been proven to cause resistance to ILE/F in mycobacteria through genome point mutation by gene editing, marking the first instance of such findings. ILE significantly impedes the proteolytic function of both the ClpC1P1P2 complex and ClpXP1P2 complex, representing the inaugural report on killing mycobacteria by targeting ClpX, highlighting the Clp complexes as promising potent targets, particularly through a dual-targeting strategy.