Mutations in ClpC1 or ClpX subunit of the caseinolytic protease confer resistance to natural product ilamycins in mycobacteria

The mycobacterial caseinolytic protease (Clp) system has been recognized as a promising therapeutic target. In this study, we identified two novel ilamycin analogs, ilamycin E (ILE) and ilamycin F (ILF), both targeting the ClpC1 component of the ClpC1P1P2 proteasome. ILE potently disrupts ClpC1P1P2-mediated proteolysis, leading to delayed bactericidal activity, while ILF also binds ClpC1, albeit with lower affinity. Notably, we discovered a unique mutation in clpX and a novel insertion in clpC1 , both conferring resistance to ILE and ILF in mycobacterium, validated by gene editing. Furthermore, ILE can also inhibit the proteolytic activity of ClpXP1P2 in a manner dependent on the substrate’s tag sequence and adaptor. This first demonstration of clpX - and clpC1 -mediated ilamycin resistance underscores the potential of ilamycins to target multiple components of the Clp protease system, offering a novel dual-target strategy for combating mycobacterial infections.