THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria

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.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global disease with an estimated 8.7 million new cases and around 1.4 million deaths annually1. TB drug resistance first emerged 40 years ago, but since then has grown to an alarming level, requiring the development of new antibiotics. Although enzyme-screening campaigns have dominated antibiotic discovery for years, their lack of success has prompted a change of strategy. In many instances, target identification of phenotypic hits is initiated by generating spontaneous drug-resistant mutants, with the expectation that resistance-conferring mutations will be revealed by whole genome sequencing (WGS)2,​3,​4,​5. For instance, Bedaquiline was identified as an inhibitor of the M. tuberculosis F0F1 adenosine triphosphate (ATP) synthase through WGS of spontaneous resistant mutants6. Using the same approach, MmpL3 was shown to be targeted by several inhibitors including SQ109, adamantyl ureas, BM212, tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamides (THPPs), N-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] (SPIROs) and indolcarboxamides7,​8,​9,​10,​11,​12,​13. However, spontaneous resistance can occur through mutations not only in the drug target, but also in other proteins linked to interactions between the cell and inhibitor14,15. In this study we were able to exploit the stereoselectivity of ligand binding in a quantitative affinity pull-down to identify the target of THPPs and reveal a novel fatty acid shuttle in mycobacteria.