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34 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYactivate degradation of proteins by ClpPs in many bacteria[19]. However, in M. tuberculosis, ADEPs appear to act primarily by blocking the interaction of ClpP1P2 with its regulatory ATPases. Enhancement of ClpC1-mediated proteolysis has also been proposed as the mode of action of the antibiotic cyclomarin, which also binds to ClpC1 [20,21]. An uncoupling mechanism similar to that observed here has recently been demonstrated for lassomycin, another M. tuberculosis specific cyclic peptide antibiotic that also targets ClpC1 [22]. Although having similar effects, ecumicin differs markedly from lassomycin in containing many noncanonical amino acids, not being synthesized on ribosomes, and not being extremely basic. So it is very likely that the two agents bind to distinct domains on ClpC1. Our findings imply that certain M. tuberculosis proteins that are normally degraded by the ClpC1P1P2 complex are highly toxic and accumulate in the presence of ecumicin. This unique target and mechanism predict no cross-resistance with existing TB drugs, which is consistent with the observed data. 5. CONCLUSIONS Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A highthroughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. Genome mining of lab-generated, spontaneous ecumicin-resistant M.tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test.