Abstract
We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13F79S mutant conferred high resistance. In vitro, TP inhibited fatty acyl–AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Espinal, M.A. The global situation of MDR-TB. Tuberculosis (Edinb.) 83, 44–51 (2003).
Mondal, R. & Jain, A. Extensively drug-resistant Mycobacterium tuberculosis, India. Emerg. Infect. Dis. 13, 1429–1431 (2007).
Caminero, J.A., Sotgiu, G., Zumla, A. & Migliori, G.B. Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis. Lancet Infect. Dis. 10, 621–629 (2010).
Kroon, A.M. & Van den Bogert, C. Antibacterial drugs and their interference with the biogenesis of mitochondria in animal and human cells. Pharm. Weekbl. Sci. 5, 81–87 (1983).
Kohanski, M.A., Dwyer, D.J. & Collins, J.J. How antibiotics kill bacteria: from targets to networks. Nat. Rev. Microbiol. 8, 423–435 (2010).
Wei, J.R. et al. Depletion of antibiotic targets has widely varying effects on growth. Proc. Natl. Acad. Sci. USA 108, 4176–4181 (2011).
Jovetic, S., Zhu, Y., Marcone, G.L., Marinelli, F. & Tramper, J. β-Lactam and glycopeptide antibiotics: first and last line of defense? Trends Biotechnol. 28, 596–604 (2010).
Khoo, K.H. et al. Truncated structural variants of lipoarabinomannan in ethambutol drug-resistant strains of Mycobacterium smegmatis. Inhibition of arabinan biosynthesis by ethambutol. J. Biol. Chem. 271, 28682–28690 (1996).
Vilchèze, C. et al. Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid. Nat. Med. 12, 1027–1029 (2006).
Slayden, R.A. et al. Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis. Antimicrob. Agents Chemother. 40, 2813–2819 (1996).
Glickman, M.S., Cox, J.S. & Jacobs, W.R. Jr. A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol. Cell 5, 717–727 (2000).
Dubnau, E. et al. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol. Microbiol. 36, 630–637 (2000).
Bhatt, A., Molle, V., Besra, G.S., Jacobs, W.R. Jr. & Kremer, L. The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development. Mol. Microbiol. 64, 1442–1454 (2007).
Kremer, L., Baulard, A.R. & Besra, G.S. Genetics of mycolic acid biosynthesis. in Molecular Genetics of Mycobacteria (eds. Hatfull, G.F. & Jacobs, W.R. Jr.) 173–190 (ASM Press, 2000).
Takayama, K., Wang, C. & Besra, G.S. Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin. Microbiol. Rev. 18, 81–101 (2005).
Barry, C.E. III et al. Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid Res. 37, 143–179 (1998).
Brennan, P.J. Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis (Edinb.) 83, 91–97 (2003).
Ojha, A.K., Trivelli, X., Guerardel, Y., Kremer, L. & Hatfull, G.F. Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms. J. Biol. Chem. 285, 17380–17389 (2010).
Gavalda, S. et al. The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis. J. Biol. Chem. 284, 19255–19264 (2009).
Portevin, D. et al. A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc. Natl. Acad. Sci. USA 101, 314–319 (2004).
Léger, M. et al. The dual function of the Mycobacterium tuberculosis FadD32 required for mycolic acid biosynthesis. Chem. Biol. 16, 510–519 (2009).
Carroll, P., Faray-Kele, M.C. & Parish, T. Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach. Appl. Environ. Microbiol. 77, 5040–5043 (2011).
Sassetti, C.M. & Rubin, E.J. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. USA 100, 12989–12994 (2003).
Alland, D., Steyn, A.J., Weisbrod, T., Aldrich, K. & Jacobs, W.R. Jr. Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition. J. Bacteriol. 182, 1802–1811 (2000).
Maddry, J.A. et al. Antituberculosis activity of the molecular libraries screening center network library. Tuberculosis (Edinb.) 89, 354–363 (2009).
Ananthan, S. et al. High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv. Tuberculosis (Edinb.) 89, 334–353 (2009).
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).
Telenti, A. et al. The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat. Med. 3, 567–570 (1997).
Onodera, Y., Tanaka, M. & Sato, K. Inhibitory activity of quinolones against DNA gyrase of Mycobacterium tuberculosis. J. Antimicrob. Chemother. 47, 447–450 (2001).
Trivedi, O.A. et al. Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature 428, 441–445 (2004).
Freundlich, J.S. et al. Triclosan derivatives: towards potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuberculosis. ChemMedChem 4, 241–248 (2009).
Reddy, V.M., Einck, L., Andries, K. & Nacy, C.A. In vitro interactions between new antitubercular drug candidates SQ109 and TMC207. Antimicrob. Agents Chemother. 54, 2840–2846 (2010).
Siddiqi, S.H., Libonati, J.P. & Middlebrook, G. Evaluation of rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis. J. Clin. Microbiol. 13, 908–912 (1981).
Safi, H., Sayers, B., Hazbon, M.H. & Alland, D. Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin. Antimicrob. Agents Chemother. 52, 2027–2034 (2008).
Middlebrook, G. Sterilization of tubercle bacilli by isonicotinic acid hydrazide and the incidence of variants resistant to the drug in vitro. Am. Rev. Tuberc. 65, 765–767 (1952).
Siddiqi, S., Takhar, P., Baldeviano, C., Glover, W. & Zhang, Y. Isoniazid induces its own resistance in nonreplicating Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 51, 2100–2104 (2007).
Gumbo, T. et al. Isoniazid bactericidal activity and resistance emergence: integrating pharmacodynamics and pharmacogenomics to predict efficacy in different ethnic populations. Antimicrob. Agents Chemother. 51, 2329–2336 (2007).
Gumbo, T. et al. Isoniazid's bactericidal activity ceases because of the emergence of resistance, not depletion of Mycobacterium tuberculosis in the log phase of growth. J. Infect. Dis. 195, 194–201 (2007).
Wallis, R.S. et al. Drug tolerance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 43, 2600–2606 (1999).
Falzari, K. et al. In vitro and in vivo activities of macrolide derivatives against Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 49, 1447–1454 (2005).
Boechat, N. et al. Novel 1,2,3-triazole derivatives for use against Mycobacterium tuberculosis H37Rv (ATCC 27294) strain. J. Med. Chem. 54, 5988–5999 (2011).
Morris, G.M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).
Šali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).
Phetsuksiri, B. et al. Antimycobacterial activities of isoxyl and new derivatives through the inhibition of mycolic acid synthesis. Antimicrob. Agents Chemother. 43, 1042–1051 (1999).
Kremer, L. et al. Inhibition of InhA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria. J. Biol. Chem. 278, 20547–20554 (2003).
Roujeinikova, A. et al. X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site. Structure 10, 825–835 (2002).
Parris, K.D. et al. Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure 8, 883–895 (2000).
Lee, R.E. et al. Combinatorial lead optimization of [1,2]-diamines based on ethambutol as potential antituberculosis preclinical candidates. J. Comb. Chem. 5, 172–187 (2003).
Jackson, M., Crick, D.C. & Brennan, P.J. Phosphatidylinositol is an essential phospholipid of mycobacteria. J. Biol. Chem. 275, 30092–30099 (2000).
Veyron-Churlet, R., Zanella-Cleon, I., Cohen-Gonsaud, M., Molle, V. & Kremer, L. Phosphorylation of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis. J. Biol. Chem. 285, 12714–12725 (2010).
Stover, C.K. et al. New use of BCG for recombinant vaccines. Nature 351, 456–460 (1991).
Kim, P. et al. Structure-activity relationships of antitubercular nitroimidazoles. 2. Determinants of aerobic activity and quantitative structure-activity relationships. J. Med. Chem. 52, 1329–1344 (2009).
van Soolingen, D., Hermans, P.W., de Haas, P.E., Soll, D.R. & van Embden, J.D. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29, 2578–2586 (1991).
Slayden, R.A. & Barry, C.E. III. Analysis of the lipids of Mycobacterium tuberculosis. Methods Mol. Med. 54, 229–245 (2001).
Seeliger, J.C. et al. Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis. J. Biol. Chem. 287, 7990–8000 (2012).
Domenech, P. & Reed, M.B. Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies. Microbiology 155, 3532–3543 (2009).
Alibaud, L. et al. A Mycobacterium marinum TesA mutant defective for major cell wall–associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos. Mol. Microbiol. 80, 919–934 (2011).
DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).
Acknowledgements
This work was supported in part by US National Institutes of Health (NIH) grant R01 AI080653 to D.A., a United Negro College Fund–Merck Postdoctoral Science Research Fellowship to R.W., a grant from the University of Medicine and Dentistry of New Jersey (UMDNJ) foundation to R.C. and NIH grant R01 AI081736 to M.B.N. W.R.J. acknowledges generous support from the NIH Centers for AIDS Research grant AI-051519 at the Albert Einstein College of Medicine and by NIH grant AI26170. The compounds initially screened in this work were supplied as part of NIH grants AI-95364 and AI-15449. We thank R.C. Goldman (formally Department of Health and Human Services, NIH, and Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), currently RCG Consulting) for his support obtaining Molecular Libraries Probe Centers Network (MLPCN) compounds for the piniBAC screen, R. Reynolds (Southern Research Institute) for his advice on the initial analysis of the MLPCN library and C.E. Barry, III and H.I.M. Boshoff (Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH) for their kind gift of TDM and TMM standards. The MS data were obtained from an Orbitrap instrument funded in part by NIH grant NS046593 for the support of the UMDNJ Neuroproteomics Core Facility.
Author information
Authors and Affiliations
Contributions
R.W., P.K., C.V., W.R.J., L.K. and D.A. conceived and designed experiments. J.S.F. synthesized compound JSF-1735, and M.J.S. and J.S.F. synthesized FAME standards. V.P. and M.B.N. performed computational docking studies. S.W.B. and J.R.W. performed whole-genome sequencing and analysis. R.W., P.K., C.V., R.V.-C., E.M., S.S., R.C. and L.K. performed whole-cell screening; performed MIC testing; selected resistant mutants; constructed recombinant strains; performed mycolic acid analyses; overexpression studies; and bactericidal, intracellular and synergy assays. R.W., P.K. and D.A. wrote the manuscript. All of the authors discussed the results and commented and contributed to sections of the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Results (PDF 1458 kb)
Rights and permissions
About this article
Cite this article
Wilson, R., Kumar, P., Parashar, V. et al. Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat Chem Biol 9, 499–506 (2013). https://doi.org/10.1038/nchembio.1277
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.1277
This article is cited by
-
The evolution of antibiotic resistance is associated with collateral drug phenotypes in Mycobacterium tuberculosis
Nature Communications (2023)
-
An integrated computational approach towards novel drugs discovery against polyketide synthase 13 thioesterase domain of Mycobacterium tuberculosis
Scientific Reports (2023)
-
Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis
BMC Biology (2022)
-
Identification of novel inhibitors for mycobacterial polyketide synthase 13 via in silico drug screening assisted by the parallel compound screening with genetic algorithm-based programs
The Journal of Antibiotics (2022)
-
Tuberculosis: current scenario, drug targets, and future prospects
Medicinal Chemistry Research (2021)