1. THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria
- Author
-
Jonathan A. G. Cox, Joaquín Rullas, Katherine A. Abrahams, David Barros, Sonja Ghidelli-Disse, Klaus Fütterer, Sudagar S. Gurcha, Marcus Bantscheff, Lourdes Encinas, Ulrich Kruse, Carlos Alemparte, Stephen Bethell, Gerard Drewes, Peter J. Jervis, Lluis Ballell, Iñigo Angulo-Barturen, Nicholas Cammack, Apoorva Bhatt, Vijayashankar Nataraj, Modesto J. Remuiñán, Albel Singh, and Gurdyal S. Besra
- Subjects
0301 basic medicine ,Microbiology (medical) ,030106 microbiology ,Immunology ,Antitubercular Agents ,Mutation, Missense ,Microbial Sensitivity Tests ,Biology ,Fatty Acid-Binding Proteins ,Applied Microbiology and Biotechnology ,Microbiology ,Mycobacterium tuberculosis ,Mice ,03 medical and health sciences ,Minimum inhibitory concentration ,Bacterial Proteins ,Essential fatty acid ,In vivo ,Two-Hybrid System Techniques ,Protein Interaction Mapping ,Genetics ,Animals ,Point Mutation ,Tuberculosis ,chemistry.chemical_classification ,Genes, Essential ,Fatty Acids, Essential ,Point mutation ,Cell Biology ,biology.organism_classification ,Small molecule ,In vitro ,3. Good health ,Disease Models, Animal ,Pyrimidines ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Pyrazoles ,Protein Binding - Abstract
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.
- Published
- 2016