Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Microbiology volume 1, Article number: 15006 (2016) Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: THPP chemical structures and in vivo anti-tubercular activity.
Figure 2: Chemoproteomics profiling identifies the putative enoyl-CoA hydratase EchA6 as the target of the THPP series.
Figure 3: GSK951A inhibition of mycolic acid biosynthesis, resistance and protein–protein interaction studies.
Figure 4: Saturation binding assay using intrinsic tryptophan fluorescence to quantify the association of EchA6 with acyl-CoAs and THPPs.
Figure 5: Structural features of EchA6 in the free and C20-CoA bound state.
Figure 6: Binding site of GSK951A in EchA6.

Similar content being viewed by others

References

  1. WHO. Global Tuberculosis Report 2013 (World Health Organization, 2013).

  2. Abrahams, K. A. et al. Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis QcrB. PLoS ONE 7, e52951 (2012).

    Article  CAS  Google Scholar 

  3. Gurcha, S. S. et al. Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. PLoS ONE 9, e113568 (2014).

    Article  Google Scholar 

  4. Mugumbate, G. et al. Mycobacterial dihydrofolate reductase inhibitors identified using chemogenomic methods and in vitro validation. PLoS ONE 10, e0121492 (2015).

    Article  Google Scholar 

  5. Wang, F. et al. Identification of a small molecule with activity against drug-resistant and persistent tuberculosis. Proc. Natl Acad. Sci. USA 110, E2510–E2517 (2013).

    Article  CAS  Google Scholar 

  6. Andries, K. et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307, 223–227 (2005).

    Article  CAS  Google Scholar 

  7. Grzegorzewicz, A. E. et al. Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane. Nature Chem. Biol. 8, 334–341 (2012).

    Article  CAS  Google Scholar 

  8. La Rosa, V. et al. MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212. Antimicrob. Agents Chemother. 56, 324–331 (2012).

    Article  CAS  Google Scholar 

  9. Li, K. et al. Multitarget drug discovery for tuberculosis and other infectious diseases. J. Med. Chem. 57, 3126–3139 (2014).

    Article  CAS  Google Scholar 

  10. Li, W. et al. Novel insights into the mechanism of inhibition of MmpL3, a target of multiple pharmacophores in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 58, 6413–6423 (2014).

    Article  Google Scholar 

  11. Rao, S. P. et al. Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis. Sci. Transl. Med. 5, 214ra168 (2013).

    Article  Google Scholar 

  12. Remuinan, M. J. et al. Tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and N-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3. PLoS ONE 8, e60933 (2013).

    Article  CAS  Google Scholar 

  13. Tahlan, K. et al. SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 56, 1797–1809 (2012).

    Article  CAS  Google Scholar 

  14. Banerjee, A. et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263, 227–230 (1994).

    Article  CAS  Google Scholar 

  15. Zhang, Y., Heym, B., Allen, B., Young, D. & Cole, S. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358, 591–593 (1992).

    Article  CAS  Google Scholar 

  16. Bantscheff, M. et al. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nature Biotechnol. 29, 255–265 (2011).

    Article  CAS  Google Scholar 

  17. Brown, A. K. et al. Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex. Microbiology 153, 4166–4173 (2007).

    Article  CAS  Google Scholar 

  18. Rana, A. K. et al. Ppm1-encoded polyprenyl monophosphomannose synthase activity is essential for lipoglycan synthesis and survival in mycobacteria. PLoS ONE 7, e48211 (2012).

    Article  CAS  Google Scholar 

  19. Kremer, L. et al. Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. J. Biol. Chem. 275, 16857–16864 (2000).

    Article  CAS  Google Scholar 

  20. Cantaloube, S., Veyron-Churlet, R., Haddache, N., Daffe, M. & Zerbib, D. The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes. PLoS ONE 6, e29564 (2011).

    Article  CAS  Google Scholar 

  21. Bahnson, B. J., Anderson, V. E. & Petsko, G. A. Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion. Biochemistry 41, 2621–2629 (2002).

    Article  CAS  Google Scholar 

  22. Rullas, J. et al. Fast standardized therapeutic-efficacy assay for drug discovery against tuberculosis. Antimicrob. Agents Chemother. 54, 2262–2264 (2010).

    Article  CAS  Google Scholar 

  23. Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K. & Wierenga, R. K. Crystal structure of enoyl-coenzyme A (CoA) hydratase at 2.5 angstroms resolution: a spiral fold defines the CoA-binding pocket. EMBO J. 15, 5135–5145 (1996).

    Article  CAS  Google Scholar 

  24. Hamed, R. B., Batchelar, E. T., Clifton, I. J. & Schofield, C. J. Mechanisms and structures of crotonase superfamily enzymes—how nature controls enolate and oxyanion reactivity. Cell. Mol. Life Sci. 65, 2507–2527 (2008).

    Article  CAS  Google Scholar 

  25. Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  Google Scholar 

  26. Hazbon, M. H. et al. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 50, 2640–2649 (2006).

    Article  CAS  Google Scholar 

  27. Cole, S. T. et al. Massive gene decay in the leprosy bacillus. Nature 409, 1007–1011 (2001).

    Article  CAS  Google Scholar 

  28. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 48, 77–84 (2003).

    Article  CAS  Google Scholar 

  29. Franceschini, A. et al. STRING v9.1: protein–protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 41, D808–D815 (2013).

    Article  CAS  Google Scholar 

  30. Kremer, L. et al. Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis. Biochem. J. 364, 423–430 (2002).

    Article  CAS  Google Scholar 

  31. Besra, G. S. et al. Identification of the apparent carrier in mycolic acid synthesis. Proc. Natl Acad. Sci. USA 91, 12735–12739 (1994).

    Article  CAS  Google Scholar 

  32. Hu, Y. et al. 3-Ketosteroid 9α-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis. Mol. Microbiol. 75, 107–121 (2010).

    Article  CAS  Google Scholar 

  33. Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl Acad. Sci. USA 95, 5752–5756 (1998).

    Article  CAS  Google Scholar 

  34. Savitski, M. M. et al. Targeted data acquisition for improved reproducibility and robustness of proteomic mass spectrometry assays. J. Am. Soc. Mass Spectr. 21, 1668–1679 (2010).

    Article  CAS  Google Scholar 

  35. Savitski, M. M. et al. Measuring and managing ratio compression for accurate iTRAQ/TMT quantification. J. Proteome Res. 12, 3586–3598 (2013).

    Article  CAS  Google Scholar 

  36. Savitski, M. M. et al. Delayed fragmentation and optimized isolation width settings for improvement of protein identification and accuracy of isobaric mass tag quantification on Orbitrap-type mass spectrometers. Anal. Chem. 83, 8959–8967 (2011).

    Article  CAS  Google Scholar 

  37. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  38. CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

    Article  Google Scholar 

  39. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  40. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  41. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  42. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).

    Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Author notes

  1. Jonathan A. G. Cox and Katherine A. Abrahams: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, B15 2TT, Birmingham, UK

    Jonathan A. G. Cox, Katherine A. Abrahams, Albel Singh, Sudagar S. Gurcha, Vijayashankar Nataraj, Stephen Bethell, Peter J. Jervis, Apoorva Bhatt, Klaus Fütterer & Gurdyal S. Besra

  2. Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, 28760, Madrid, Spain

    Carlos Alemparte, Joaquín Rullas, Iñigo Angulo-Barturen, Modesto J. Remuiñán, Lourdes Encinas, Nicholas C. Cammack, David Barros & Lluis Ballell

  3. Cellzome, Meyerhofstrasse 1, 69117, Heidelberg, Germany

    Sonja Ghidelli-Disse, Ulrich Kruse, Marcus Bantscheff & Gerard Drewes

Authors
  1. Jonathan A. G. Cox
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katherine A. Abrahams
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carlos Alemparte
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sonja Ghidelli-Disse
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joaquín Rullas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Iñigo Angulo-Barturen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Albel Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sudagar S. Gurcha
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Vijayashankar Nataraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stephen Bethell
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Modesto J. Remuiñán
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Lourdes Encinas
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Peter J. Jervis
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Nicholas C. Cammack
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Apoorva Bhatt
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Ulrich Kruse
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Marcus Bantscheff
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Klaus Fütterer
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. David Barros
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Lluis Ballell
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Gerard Drewes
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Gurdyal S. Besra
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.A.G.C., K.A.A., S.G.D., U.K., M.B., G.D., N.C.C., A.B., L.B., D.B., K.F. and G.S.B. conceived and designed the experiments. J.A.G.C., K.A.A., I.A.B., S.B., C.A., A.B., A.S., P.J.J., S.G.D., V.N., S.S.G., M.J.R., L.E. and J.R. performed the experiments. K.A.A., J.A.G.C., J.R., S.B., S.G.D., U.K., M.B., S.G.D., D.B., L.B., K.F. and G.S.B. analysed the data. L.B., M.J.P. and G.S.B. contributed reagents, materials and analysis tools. J.A.G.C., K.A.A., L.B., D.B., S.G.D., G.D., C.A., A.B., K.F. and G.S.B. wrote the paper.

Corresponding authors

Correspondence to Klaus Fütterer, Lluis Ballell or Gurdyal S. Besra.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–6 and Tables 1–7 (PDF 19717 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2015.6

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing