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Targeting the menaquinol binding loop of mycobacterial cytochrome bd oxidase

  • Amaravadhi Harikishore
  • Sherilyn Shi Min Chong
  • Priya Ragunathan
  • Roderick W. Bates
  • Gerhard GrüberEmail author
Short Communication

Abstract

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H2 inhibited both NADH2- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H2 also inhibited NADH2-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H2, reflecting that MQL-H2 targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies.

Graphic abstract

Characterization of novel cytochrome bd oxidase Menaquinol binding domain inhibitor (MQL-H2) using virtual screening and ATP synthesis inhibition assays.

Keywords

Tuberculosis Mycobacteria Cytochrome bd oxidase OXPHOS pathway Respiration Drug resistance 

Notes

Acknowledgements

This research was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number (NRF–CRP18–2017–01). S. S. M. C. is grateful for an NTU research scholarship at Nanyang Technological University.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11030_2020_10034_MOESM1_ESM.pdf (481 kb)
Supplementary material 1 (PDF 480 kb)

References

  1. 1.
  2. 2.
    Working Group (2019) FDA approves new drug for treatment-resistant forms of tuberculosis in. Working Group on New Drugs, New YorkGoogle Scholar
  3. 3.
    Tang S, Yao L, Hao X, Liu Y, Zeng L, Liu G, Li M, Li F, Wu M, Zhu Y, Sun H, Gu J, Wang X, Zhang Z (2015) Clofazimine for the treatment of multidrug-resistant tuberculosis: prospective, multicenter, randomized controlled study in China. Clin Infect Dis 60:1361–1367.  https://doi.org/10.1093/cid/civ027 CrossRefPubMedGoogle Scholar
  4. 4.
    Pethe K, Bifani P, Jang J, Kang S, Park S, Ahn S, Jiricek J, Jung J, Jeon HK, Cechetto J, Christophe T, Lee H, Kempf M, Jackson M, Lenaerts AJ, Pham H, Jones V, Seo MJ, Kim YM, Seo M, Seo JJ, Park D, Ko Y, Choi I, Kim R, Kim SY, Lim S, Yim SA, Nam J, Kang H, Kwon H, Oh CT, Cho Y, Jang Y, Kim J, Chua A, Tan BH, Nanjundappa MB, Rao SP, Barnes WS, Wintjens R, Walker JR, Alonso S, Lee S, Kim J, Oh S, Oh T, Nehrbass U, Han SJ, No Z, Lee J, Brodin P, Cho SN, Nam K, Kim J (2013) Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat Med 19:1157–1160.  https://doi.org/10.1038/nm.3262 CrossRefPubMedGoogle Scholar
  5. 5.
    FDA (2012) SIRTURO™ (bedaquiline). https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/204384s002lbl.pdf. Accessed 17 Oct 2019
  6. 6.
    Sutherland HS, Tong AST, Choi PJ, Conole D, Blaser A, Franzblau SG, Cooper CB, Upton AM, Lotlikar MU, Denny WA, Palmer BD (2018) Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles. Bioorg Med Chem 26:1797–1809.  https://doi.org/10.1016/j.bmc.2018.02.026 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Blaser A, Sutherland HS, Tong AST, Choi PJ, Conole D, Franzblau SG, Cooper CB, Upton AM, Lotlikar M, Denny WA, Palmer BD (2019) Structure-activity relationships for unit C pyridyl analogues of the tuberculosis drug bedaquiline. Bioorg Med Chem 27:1283–1291.  https://doi.org/10.1016/j.bmc.2019.02.025 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bald D, Villellas C, Lu P, Koul A (2017) Targeting energy metabolism in mycobacterium tuberculosis, a new paradigm in antimycobacterial drug discovery. MBio.  https://doi.org/10.1128/mBio.00272-17 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Safarian S, Hahn A, Mills DJ, Radloff M, Eisinger ML, Nikolaev A, Meier-Credo J, Melin F, Miyoshi H, Gennis RB, Sakamoto J, Langer JD, Hellwig P, Kühlbrandt W, Michel H (2019) Active site rearrangement and structural divergence in prokaryotic respiratory oxidases. Science 366:100.  https://doi.org/10.1126/science.aay0967 CrossRefPubMedGoogle Scholar
  10. 10.
    Belevich I, Borisov VB, Zhang J, Yang K, Konstantinov AA, Gennis RB, Verkhovsky MI (2005) Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site. Proc Natl Acad Sci U S A 102:3657–3662.  https://doi.org/10.1073/pnas.0405683102 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Yang K, Zhang J, Vakkasoglu AS, Hielscher R, Osborne JP, Hemp J, Miyoshi H, Hellwig P, Gennis RB (2007) Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center. Biochemistry 46:3270–3278.  https://doi.org/10.1021/bi061946+ CrossRefPubMedGoogle Scholar
  12. 12.
    Safarian S, Rajendran C, Muller H, Preu J, Langer JD, Ovchinnikov S, Hirose T, Kusumoto T, Sakamoto J, Michel H (2016) Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases. Science 352:583–586.  https://doi.org/10.1126/science.aaf2477 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Aung HL, Berney M, Cook GM (2014) Hypoxia-activated cytochrome bd expression in Mycobacterium smegmatis is cyclic AMP receptor protein dependent. J Bacteriol 196:3091–3097.  https://doi.org/10.1128/JB.01771-14 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Megehee JA, Hosler JP, Lundrigan MD (2006) Evidence for a cytochrome bcc–aa3 interaction in the respiratory chain of Mycobacterium smegmatis. Microbiology 152:823–829.  https://doi.org/10.1099/mic.0.28723-0 CrossRefPubMedGoogle Scholar
  15. 15.
    Forte E, Borisov VB, Davletshin A, Mastronicola D, Sarti P, Giuffre A (2013) Cytochrome bd oxidase and hydrogen peroxide resistance in Mycobacterium tuberculosis. MBio 4:e01006–e01013.  https://doi.org/10.1128/mBio.01006-13 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Giuffre A, Borisov VB, Mastronicola D, Sarti P, Forte E (2012) Cytochrome bd oxidase and nitric oxide: from reaction mechanisms to bacterial physiology. FEBS Lett 586:622–629.  https://doi.org/10.1016/j.febslet.2011.07.035 CrossRefPubMedGoogle Scholar
  17. 17.
    Berney M, Hartman TE, Jacobs WR Jr (2014) A Mycobacterium tuberculosis cytochrome bd oxidase mutant is hypersensitive to bedaquiline. MBio 5:e01275–14.  https://doi.org/10.1128/mBio.01275-14 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kalia NP, Hasenoehrl EJ, Ab Rahman NB, Koh VH, Ang MLT, Sajorda DR, Hards K, Grüber G, Alonso S, Cook GM, Berney M, Pethe K (2017) Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection. Proc Natl Acad Sci U S A 114:7426–7431.  https://doi.org/10.1073/pnas.1706139114 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Moosa A, Lamprecht DA, Arora K, Barry CE 3rd, Boshoff HIM, Ioerger TR, Steyn AJC, Mizrahi V, Warner DF (2017) Susceptibility of Mycobacterium tuberculosis cytochrome bd oxidase mutants to compounds targeting the terminal respiratory oxidase, cytochrome c. Antimicrob Agents Chemother.  https://doi.org/10.1128/AAC.01338-17 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mascolo L, Bald D (2019) Cytochrome bd in Mycobacterium tuberculosis: a respiratory chain protein involved in the defense against antibacterials. Prog Biophys Mol Biol.  https://doi.org/10.1016/j.pbiomolbio.2019.11.002 CrossRefPubMedGoogle Scholar
  21. 21.
    Lu P, Asseri AH, Kremer M, Maaskant J, Ummels R, Lill H, Bald D (2018) The anti-mycobacterial activity of the cytochrome bcc inhibitor Q203 can be enhanced by small-molecule inhibition of cytochrome bd. Sci Rep 8:2625.  https://doi.org/10.1038/s41598-018-20989-8 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Madhavi Sastry G, Adzhigirey M, Day T, Annabhimoju R, Sherman W (2013) Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 27:221–234.  https://doi.org/10.1007/s10822-013-9644-8 CrossRefPubMedGoogle Scholar
  23. 23.
    Schrödinger Release 2019-2 (2019) Maestro, Force Fields, MacroModel, prime, protein preparation wizard, Ligprep, ConfGen, Phase, QikProp, Glide. Schrödinger, LLC, New York, NYGoogle Scholar
  24. 24.
    Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486.  https://doi.org/10.1007/bf00228148 CrossRefPubMedGoogle Scholar
  25. 25.
    Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963) Stereochemistry of polypeptide chain configurations. J Mol Biol 7:95–99.  https://doi.org/10.1016/s0022-2836(63)80023-6 CrossRefPubMedGoogle Scholar
  26. 26.
    Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS (2004) Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47:1739–1749.  https://doi.org/10.1021/jm0306430 CrossRefPubMedGoogle Scholar
  27. 27.
    Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, Sanschagrin PC, Mainz DT (2006) Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49:6177–6196.  https://doi.org/10.1021/jm051256o CrossRefPubMedGoogle Scholar
  28. 28.
    Lu P, Heineke MH, Koul A, Andries K, Cook GM, Lill H, van Spanning R, Bald D (2015) The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress. Sci Rep 5:10333.  https://doi.org/10.1038/srep10333 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Hotra A, Suter M, Biukovic G, Ragunathan P, Kundu S, Dick T, Grüber G (2016) Deletion of a unique loop in the mycobacterial F-ATP synthase gamma subunit sheds light on its inhibitory role in ATP hydrolysis-driven H(+) pumping. FEBS J 283:1947–1961.  https://doi.org/10.1111/febs.13715 CrossRefPubMedGoogle Scholar
  30. 30.
    (2019) Dose response inhibition using four parameter varaible slope non-linear regression was performed using GraphPad Prism version 8.00 for Windows, GraphPad Software, La Jolla California USA. www.graphpad.com
  31. 31.
    Levine RL, Mosoni L, Berlett BS, Stadtman ER (1996) Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci U S A 93:15036–15040.  https://doi.org/10.1073/pnas.93.26.15036 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kim G, Weiss SJ, Levine RL (2014) Methionine oxidation and reduction in proteins. Biochim Biophys Acta 1840:901–905.  https://doi.org/10.1016/j.bbagen.2013.04.038 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Division of Chemistry and Biological Chemistry, School of Physical and Mathematical SciencesNanyang Technological UniversitySingaporeRepublic of Singapore
  2. 2.School of Biological SciencesNanyang Technological UniversitySingaporeRepublic of Singapore
  3. 3.Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate SchoolNanyang Technological UniversitySingaporeRepublic of Singapore

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