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

Jonathan A.G. Cox, Katherine A. Abrahams, Carlos Alemparte, Sonja Ghidelli-Disse, Joaquín Rullas, Iñigo Angulo-Barturen, Albel Singh, Sudagar S. Gurcha, Vijayashankar Nataraj, Stephen Bethell, Modesto J. Remuiñán, Lourdes Encinas, Peter J. Jervis, Nicholas C. Cammack, Apoorva Bhatt, Ulrich Kruse, Marcus Bantscheff, Klaus Fütterer, David Barros, Lluis Ballell & 2 others Gerard Drewes, Gurdyal S. Besra

Research output: Contribution to journalArticle

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.

LanguageEnglish
Article number15006
JournalNature Microbiology
Volume1
DOIs
Publication statusPublished - 18 Jan 2016

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Essential Fatty Acids
Type II Fatty Acid Synthase
Mycobacterium
Coenzyme A
Mycolic Acids
Hydro-Lyases
Multidrug-Resistant Tuberculosis
Two-Hybrid System Techniques
Microbial Sensitivity Tests
Mycobacterium tuberculosis
Point Mutation
Tuberculosis
Fatty Acids
Mutation
Enzymes
Infection
flopropione

Cite this

Cox, J. A. G., Abrahams, K. A., Alemparte, C., Ghidelli-Disse, S., Rullas, J., Angulo-Barturen, I., ... Besra, G. S. (2016). THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria. 1, [15006]. https://doi.org/10.1038/nmicrobiol.2015.6
Cox, Jonathan A.G. ; Abrahams, Katherine A. ; Alemparte, Carlos ; Ghidelli-Disse, Sonja ; Rullas, Joaquín ; Angulo-Barturen, Iñigo ; Singh, Albel ; Gurcha, Sudagar S. ; Nataraj, Vijayashankar ; Bethell, Stephen ; Remuiñán, Modesto J. ; Encinas, Lourdes ; Jervis, Peter J. ; Cammack, Nicholas C. ; Bhatt, Apoorva ; Kruse, Ulrich ; Bantscheff, Marcus ; Fütterer, Klaus ; Barros, David ; Ballell, Lluis ; Drewes, Gerard ; Besra, Gurdyal S. / THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria. 2016 ; Vol. 1.
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Cox, JAG, Abrahams, KA, Alemparte, C, Ghidelli-Disse, S, Rullas, J, Angulo-Barturen, I, Singh, A, Gurcha, SS, Nataraj, V, Bethell, S, Remuiñán, MJ, Encinas, L, Jervis, PJ, Cammack, NC, Bhatt, A, Kruse, U, Bantscheff, M, Fütterer, K, Barros, D, Ballell, L, Drewes, G & Besra, GS 2016, 'THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria' vol. 1, 15006. https://doi.org/10.1038/nmicrobiol.2015.6

THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria. / Cox, Jonathan A.G.; Abrahams, Katherine A.; Alemparte, Carlos; Ghidelli-Disse, Sonja; Rullas, Joaquín; Angulo-Barturen, Iñigo; Singh, Albel; Gurcha, Sudagar S.; Nataraj, Vijayashankar; Bethell, Stephen; Remuiñán, Modesto J.; Encinas, Lourdes; Jervis, Peter J.; Cammack, Nicholas C.; Bhatt, Apoorva; Kruse, Ulrich; Bantscheff, Marcus; Fütterer, Klaus; Barros, David; Ballell, Lluis; Drewes, Gerard; Besra, Gurdyal S.

Vol. 1, 15006, 18.01.2016.

Research output: Contribution to journalArticle

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AU - Cox, Jonathan A.G.

AU - Abrahams, Katherine A.

AU - Alemparte, Carlos

AU - Ghidelli-Disse, Sonja

AU - Rullas, Joaquín

AU - Angulo-Barturen, Iñigo

AU - Singh, Albel

AU - Gurcha, Sudagar S.

AU - Nataraj, Vijayashankar

AU - Bethell, Stephen

AU - Remuiñán, Modesto J.

AU - Encinas, Lourdes

AU - Jervis, Peter J.

AU - Cammack, Nicholas C.

AU - Bhatt, Apoorva

AU - Kruse, Ulrich

AU - Bantscheff, Marcus

AU - Fütterer, Klaus

AU - Barros, David

AU - Ballell, Lluis

AU - Drewes, Gerard

AU - Besra, Gurdyal S.

PY - 2016/1/18

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N2 - 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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Cox JAG, Abrahams KA, Alemparte C, Ghidelli-Disse S, Rullas J, Angulo-Barturen I et al. THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria. 2016 Jan 18;1. 15006. https://doi.org/10.1038/nmicrobiol.2015.6