Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus

Abstract

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 μM) and Mycobacterium abscessus (MIC, 0.4 μM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

“…Moreover, both ecumicin and lassomycin have been tested for activity against susceptible, MDR and XDR clinical M tuberculosis isolates, with MICs of 0.26 to 1.0, 0.5, and 0.5 to 1.0 μg/mL obtained for ecumicin, respectively . Anti‐TB activity analyses of ecumicin, rufomycin I, and cyclomarin A on M tuberculosis H37Rv strain, virulent strains, and drug‐resistant strains have shown that rufomycin I, cyclomarin A, and ecumicin are more active than capreomycin and the first‐line TB‐drug isoniazid . For lassomycin, the MICs against susceptible and drug‐resistant M tuberculosis strains were found to be between 0.78 and 3.1 μg/mL .…”

“…Studies on the mechanism of action of rufomycin, ecumicin, cyclomarin, and lassomycin identified the ClpC1 ATPase complex as the drug target of these peptides. Ecumicin and lassomycin act through enhancing ATPase activity while blocking ClpC1p1p2‐catalysed proteolysis while cyclomarin A does not affect the ATPase activity and increasing proteolysis . The in vivo study carried out by Gao and colleagues found that 12 doses of 20 or 32 mg/kg ecumicin were enough to completely inhibit the growth of the pathogens from the lungs of mice infected by M tuberculosis Erdman strain .…”

“…The in vivo study carried out by Gao and colleagues found that 12 doses of 20 or 32 mg/kg ecumicin were enough to completely inhibit the growth of the pathogens from the lungs of mice infected by M tuberculosis Erdman strain . Moreover, rufomycin I, ecumicin, and cyclomarin A are nontoxic to human cells at 50 μg/mL . However, poor plasma stability of cyclomarin A makes this molecule less interesting than ecumicin, which has good plasma stability .…”

“…106 Anti-TB activity analyses of ecumicin, rufomycin I, and cyclomarin A on M tuberculosis H37Rv strain, virulent strains, and drug-resistant strains have shown that rufomycin I, cyclomarin A, and ecumicin are more active than capreomycin and the first-line TB-drug isoniazid. 139 For lassomycin, the MICs against susceptible and drug-resistant M tuberculosis strains were found to be between 0.78 and 3.1 μg/mL. 104 Studies on the mechanism of action of rufomycin, ecumicin, cyclomarin, and lassomycin identified the ClpC1…”

“…Ecumicin and lassomycin act through enhancing ATPase activity while blocking ClpC1p1p2-catalysed proteolysis while cyclomarin A does not affect the ATPase activity and increasing proteolysis. 104,106,139,142 The in vivo study carried out by Gao and colleagues found that 12 doses of 20 or 32 mg/kg ecumicin were enough to completely inhibit the growth of the pathogens from the lungs of mice infected by M tuberculosis Erdman strain. 106 Moreover, rufomycin I, ecumicin, and cyclomarin A are nontoxic to human cells at 50 μg/mL.…”