Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.
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G.S.B. acknowledges support in the form of a Personal Research Chair from J. Bardrick and a Royal Society Wolfson Research Merit Award. The research leading to these results has received funding from the European Union's 7th Framework Programme (FP7-2007-2013) under grant agreement no. 261378 and the BBSRC through an Industrial CASE studentship. The authors thank N. Zinn and T. Mathieson for mass spectrometry and database design, and S. Batt, P. Moynihan and L. Alderwick for technical support. The authors also acknowledge the TB Alliance for their discussions and expertise in the field.
The authors declare no competing financial interests.
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Cox, J., Abrahams, K., Alemparte, C. et al. THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria. Nat Microbiol 1, 15006 (2016). https://doi.org/10.1038/nmicrobiol.2015.6
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