title: THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria.
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.
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Jonathan A G
authors: JA
authors: Cox
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Katherine A
authors: KA
authors: Abrahams
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Carlos
authors: C
authors: Alemparte
authors: Cellzome, Meyerhofstrasse 1, Heidelberg 69117, Germany.
authors: Sonja
authors: S
authors: Ghidelli-Disse
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Joaquín
authors: J
authors: Rullas
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Iñigo
authors: I
authors: Angulo-Barturen
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Albel
authors: A
authors: Singh
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Sudagar S
authors: SS
authors: Gurcha
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Vijayashankar
authors: V
authors: Nataraj
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Stephen
authors: S
authors: Bethell
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Modesto J
authors: MJ
authors: Remuiñán
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Lourdes
authors: L
authors: Encinas
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Peter J
authors: PJ
authors: Jervis
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Nicholas C
authors: NC
authors: Cammack
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Apoorva
authors: A
authors: Bhatt
authors: Cellzome, Meyerhofstrasse 1, Heidelberg 69117, Germany.
authors: Ulrich
authors: U
authors: Kruse
authors: Cellzome, Meyerhofstrasse 1, Heidelberg 69117, Germany.
authors: Marcus
authors: http://orcid.org/0000-0002-8343-8977
authors: M
authors: Bantscheff
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Klaus
authors: K
authors: Fütterer
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: David
authors: D
authors: Barros
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, Madrid 28760, Spain.
authors: Lluis
authors: L
authors: Ballell
authors: Cellzome, Meyerhofstrasse 1, Heidelberg 69117, Germany.
authors: Gerard
authors: G
authors: Drewes
authors: Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors: Gurdyal S
authors: http://orcid.org/0000-0002-5605-0395
authors: GS
authors: Besra
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005228:Fatty Acids, Essential / Q000378:metabolism*; D020043:Genes, Essential; D051379:Mice; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D014376:Tuberculosis / Q000382:microbiology / Q000473:pathology; D020798:Two-Hybrid System Techniques
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D050556:Fatty Acid-Binding Proteins; D005228:Fatty Acids, Essential; D011720:Pyrazoles; D011743:Pyrimidines