Journal of Molecular Medicine

, Volume 97, Issue 11, pp 1601–1613 | Cite as

Identification of novel scaffolds targeting Mycobacterium tuberculosis

  • Michael Dal Molin
  • Petra Selchow
  • Daniel Schäfle
  • Andreas Tschumi
  • Thomas Ryckmans
  • Stephan Laage-Witt
  • Peter SanderEmail author
Original Article


Drug resistance in Mycobacterium tuberculosis is relentlessly progressing while only a handful of novel drug candidates are developed. Here we describe a GFP-based high-throughput screening of 386,496 diverse compounds to identify putative tuberculosis drug candidates. In an exploratory analysis of the model organism M. bovis BCG and M. smegmatis and the subsequent screening of the main library, we identified 6354 compounds with anti-mycobacterial activity. These hit compounds were predominantly selective for mycobacteria while dozens had activity in the low μM range. We tested toxicity against the human monocyte/macrophage cell line THP-1 and elaborated activity against M. tuberculosis growing in liquid broth, under unique conditions such as non-replicating persistence or inhibition of M. tuberculosis residing in macrophages. Finally, spontaneous compound-resistant M. tuberculosis mutants were selected and subsequently analyzed by whole genome sequencing. In addition to compounds targeting the well-described proteins Pks13 and MmpL3, we identified two novel scaffolds targeting the bifunctional guanosine pentaphosphate synthetase/ polyribonucleotide nucleotidyltransferase GpsI, or interacting with the aminopeptidase PepB, a probable pro-drug activator.

Key messages

  • A newly identified scaffold targets the bifunctional enzyme GpsI.

  • The aminopeptidase PepB is interacting with a second novel scaffold.

  • Phenotypic screenings regularly identify novel compounds targeting Pks13 and MmpL3.


Mycobacterium tuberculosis High-throughput screening Early drug development GpsI PepB Pks13 MmpL3 



We thank F. Hoffmann-La Roche AG (in particular Martin Brunner, Kenneth Bradley, and Paul Gillespie) for providing the compounds, helpful discussions, and comments on the manuscript. Additionally, we thank Karoline Wagner from the Institute of Medical Microbiology and Siricha Aluri from the Functional Genomics Center Zurich for sequencing the resistant mutants, and Chantal Quiblier and Erik C. Böttger for providing the S. aureus GFP-expressing plasmid and clinical isolates, respectively.

This work was supported by the University of Zurich and the Institue of Medical Microbiology. We achnolwedge financial support from Roche Extending the Innovation Network (EIN-UZ-14/0681), Swiss National Science Foundation (31003A_153349), Lungenliga Schweiz/Georg and Bertha Schwyzer-Winiker Stiftung (SLA-2018-02) and Baugarten Stiftung (STWF-18-011).

Supplementary material

109_2019_1840_MOESM1_ESM.docx (1.8 mb)
ESM 1 (DOCX 1816 kb)


  1. 1.
    WHO (2017) Global tuberculosis report 2017. World Health Organization, GenevaGoogle Scholar
  2. 2.
    Koul A, Arnoult E, Lounis N, Guillemont J, Andries K (2011) The challenge of new drug discovery for tuberculosis. Nature 469:483–490PubMedGoogle Scholar
  3. 3.
    Houben RMGJ, Dodd PJ (2016) The global burden of latent tuberculosis infection: a re-estimation using mathematical modelling. PLoS Med 13:e1002152PubMedPubMedCentralGoogle Scholar
  4. 4.
    Vynnycky E, Fine PEM (2000) Lifetime risks, incubation period, and serial interval of tuberculosis. Am J Epidemiol 152:247–263PubMedGoogle Scholar
  5. 5.
    Lönnroth K, Castro KG, Chakaya JM et al (2010) Tuberculosis control and elimination 2010–50: cure, care, and social development. Lancet 375:1814–1829PubMedGoogle Scholar
  6. 6.
    Gómez-Reino JJ, Carmona L, Valverde VR, Mola EM, Montero MD, BIOBADASER Group (2003) Treatment of rheumatoid arthritis with tumor necrosis factor inhibitors may predispose to significant increase in tuberculosis risk: a multicenter active-surveillance report. Arthritis Rheum 48:2122–2127PubMedGoogle Scholar
  7. 7.
    WHO (2010) Global tuberculosis control 2010. World Health Organization, GenevaGoogle Scholar
  8. 8.
    Hett EC, Rubin EJ (2008) Bacterial growth and cell division: a mycobacterial perspective. Microbiol Mol Biol Rev 72:126–156PubMedPubMedCentralGoogle Scholar
  9. 9.
    Smith I (2003) Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 16:463–496PubMedPubMedCentralGoogle Scholar
  10. 10.
    Gold B, Nathan C (2017) Targeting phenotypically tolerant Mycobacterium tuberculosis. Microbiol Spectr 5:1
  11. 11.
    Balganesh TS, Alzari PM, Cole ST (2008) Rising standards for tuberculosis drug development. Trends Pharmacol Sci 29:576–581PubMedGoogle Scholar
  12. 12.
    Mahajan R (2013) Bedaquiline: first FDA-approved tuberculosis drug in 40 years. Int J Appl Basic Med Res 3:1–2PubMedPubMedCentralGoogle Scholar
  13. 13.
    WHO (2016) Global tuberculosis report 2016. World Health Organization, GenevaGoogle Scholar
  14. 14.
    WHO (2013) The use of bedaquiline in the treatment of multidrug-resistant tuberculosis. World Health Organization, GenevaGoogle Scholar
  15. 15.
    WHO (2014) The use of delamanid in the treatment of multidrug-resistant tuberculosis. World Health Organization, GenevaGoogle Scholar
  16. 16.
    Bloemberg GV, Keller PM, Stucki D, Trauner A, Borrell S, Latshang T, Coscolla M, Rothe T, Hömke R, Ritter C, Feldmann J, Schulthess B, Gagneux S, Böttger EC (2015) Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis. N Engl J Med 373:1986–1988PubMedPubMedCentralGoogle Scholar
  17. 17.
    Hoffmann H, Kohl TA, Hofmann-Thiel S, Merker M, Beckert P, Jaton K, Nedialkova L, Sahalchyk E, Rothe T, Keller PM, Niemann S (2016) Delamanid and bedaquiline resistance in Mycobacterium tuberculosis ancestral Beijing genotype causing extensively drug-resistant tuberculosis in a Tibetan refugee. Am J Respir Crit Care Med 193:337–340PubMedGoogle Scholar
  18. 18.
    Andries K, Verhasselt P, Guillemont J et al (2005) A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307:223–227PubMedGoogle Scholar
  19. 19.
    Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat Rev Drug Discov 6:29–40PubMedGoogle Scholar
  20. 20.
    Tommasi R, Brown DG, Walkup GK, Manchester JI, Miller AA (2015) ESKAPEing the labyrinth of antibacterial discovery. Nat Rev Drug Discov 14:529–542PubMedGoogle Scholar
  21. 21.
    Cole ST (2016) Inhibiting Mycobacterium tuberculosis within and without. Philos Trans R Soc Lond Ser B Biol Sci 371:20150506Google Scholar
  22. 22.
    Raynaud C, Papavinasasundaram KG, Speight RA, Springer B, Sander P, Böttger EC, Colston MJ, Draper P (2002) The functions of OmpATb, a pore-forming protein of Mycobacterium tuberculosis. Mol Microbiol 46:191–201PubMedGoogle Scholar
  23. 23.
    Matt U, Selchow P, Dal Molin M, Strommer S, Sharif O, Schilcher K, Andreoni F, Stenzinger A, Zinkernagel AS, Zeitlinger M, Sander P, Nemeth J (2017) Chloroquine enhances the antimycobacterial activity of isoniazid and pyrazinamide by reversing inflammation-induced macrophage efflux. Int J Antimicrob Agents 50:55–62PubMedGoogle Scholar
  24. 24.
    Pethe K, Sequeira PC, Agarwalla S et al (2010) A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy. Nat Commun 1:57PubMedGoogle Scholar
  25. 25.
    Kubica GP, Kim TH, Dunbar FP (1972) Designation of strain H37Rv as the neotype of Mycobacterium tuberculosis. Int J Syst Bacteriol Copyr Int Assoc Microbiol Soc 22:99–106Google Scholar
  26. 26.
    Prammananan T, Sander P, Springer B, Böttger EC (1999) RecA-mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA. Antimicrob Agents Chemother 43:447–453PubMedPubMedCentralGoogle Scholar
  27. 27.
    Gold B, Warrier T, Nathan C (2015) A multi-stress model for high throughput screening against non-replicating Mycobacterium tuberculosis. Methods Mol Biol 1285:293–315PubMedGoogle Scholar
  28. 28.
    Rampini SK, Selchow P, Keller C, Ehlers S, Böttger EC, Sander P (2008) LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest. Microbiology 154:2991–3001PubMedGoogle Scholar
  29. 29.
    R Core Team (2014) R: a language and environment for statistical computing. . R Foundation for Statistical Computing, ViennaGoogle Scholar
  30. 30.
    Ritz C, Streibig JC (2005) Bioassay analysis using R. J Stat Softw 12:1–22Google Scholar
  31. 31.
    Stanley SA, Grant SS, Kawate T, Iwase N, Shimizu M, Wivagg C, Silvis M, Kazyanskaya E, Aquadro J, Golas A, Fitzgerald M, Dai H, Zhang L, Hung DT (2012) Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening. ACS Chem Biol 7:1377–1384PubMedPubMedCentralGoogle Scholar
  32. 32.
    Ballell L, Bates RH, Young RJ et al (2013) Fueling open-source drug discovery: 177 small-molecule leads against tuberculosis. ChemMedChem 8:313–321PubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73PubMedGoogle Scholar
  34. 34.
    Maddry JA, Ananthan S, Goldman RC, Hobrath JV, Kwong CD, Maddox C, Rasmussen L, Reynolds RC, Secrist JA 3rd, Sosa MI, White EL, Zhang W (2009) Antituberculosis activity of the molecular libraries screening center network library. Tuberculosis 89:354–363PubMedGoogle Scholar
  35. 35.
    Ananthan S, Faaleolea ER, Goldman RC et al (2009) High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv. Tuberculosis (Edinb) 89:334–353Google Scholar
  36. 36.
    Luthra S, Rominski A, Sander P (2018) The role of antibiotic-target-modifying and antibiotic-modifying enzymes in mycobacterium abscessus drug resistance. Front Microbiol 9:2179PubMedPubMedCentralGoogle Scholar
  37. 37.
    Rice LB (2010) Progress and challenges in implementing the research on ESKAPE pathogens. Infect Control Hosp Epidemiol 31:S7–S10PubMedGoogle Scholar
  38. 38.
    Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M, Spellberg B, Bartlett J (2009) Bad bugs, no drugs: no ESKAPE! An update from the infectious diseases society of America. Clin Infect Dis 48:1–12PubMedGoogle Scholar
  39. 39.
    Segall MD, Barber C (2014) Addressing toxicity risk when designing and selecting compounds in early drug discovery. Drug Discov Today 19:688–693PubMedGoogle Scholar
  40. 40.
    Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84PubMedGoogle Scholar
  41. 41.
    Jones GH, Bibb MJ (1996) Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase. J Bacteriol 178:4281–4288PubMedPubMedCentralGoogle Scholar
  42. 42.
    Njire M, Tan Y, Mugweru J, Wang C, Guo J, Yew W, Tan S, Zhang T (2016) Pyrazinamide resistance in Mycobacterium tuberculosis: review and update. Adv Med Sci 61:63–71PubMedGoogle Scholar
  43. 43.
    Bergval IL, Schuitema ARJ, Klatser PR, Anthony RM (2009) Resistant mutants of Mycobacterium tuberculosis selected in vitro do not reflect the in vivo mechanism of isoniazid resistance. J Antimicrob Chemother 64:515–523PubMedPubMedCentralGoogle Scholar
  44. 44.
    Portevin D, De Sousa-D’Auria C, Houssin C et al (2004) A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc Natl Acad Sci 101:314–319PubMedGoogle Scholar
  45. 45.
    Ioerger TR, O’Malley T, Liao R et al (2013) Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis. PLoS One 8:e75245PubMedPubMedCentralGoogle Scholar
  46. 46.
    Wilson R, Kumar P, Parashar V, Vilchèze C, Veyron-Churlet R, Freundlich JS, Barnes SW, Walker JR, Szymonifka MJ, Marchiano E, Shenai S, Colangeli R, Jacobs WR Jr, Neiditch MB, Kremer L, Alland D (2013) Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat Chem Biol 9:499–506PubMedPubMedCentralGoogle Scholar
  47. 47.
    Aggarwal A, Parai MK, Shetty N et al (2017) Development of a novel lead that targets M. tuberculosis polyketide synthase 13. Cell 170:249–259PubMedPubMedCentralGoogle Scholar
  48. 48.
    Baell JB, Holloway GA (2010) New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 53:2719–2740PubMedGoogle Scholar
  49. 49.
    Xie Y, Liu Q, Jiang H, Ni J (2003) Novel complexes of ligands containing phenol and alcohol groups: from polynuclear cluster, 1D coordination polymer to 2D supramolecular assemblies. Eur J Inorg Chem 2003:4010–4016Google Scholar
  50. 50.
    McLean LR, Zhang Y, Li H, Li Z, Lukasczyk U, Choi YM, Han Z, Prisco J, Fordham J, Tsay JT, Reiling S, Vaz RJ, Li Y (2009) Discovery of covalent inhibitors for MIF tautomerase via cocrystal structures with phantom hits from virtual screening. Bioorg Med Chem Lett 19:6717–6720PubMedGoogle Scholar
  51. 51.
    Weinert EE, Dondi R, Colloredo-Melz S, Frankenfield KN, Mitchell CH, Freccero M, Rokita SE (2006) Substituents on quinone methides strongly modulate formation and stability of their nucleophilic adducts. J Am Chem Soc 128:11940–11947PubMedPubMedCentralGoogle Scholar
  52. 52.
    Gilberg E, Gütschow M, Bajorath J (2018) X-ray structures of target–ligand complexes containing compounds with assay interference potential. J Med Chem 61:1276–1284PubMedGoogle Scholar
  53. 53.
    Belardinelli JM, Yazidi A, Yang L, Fabre L, Li W, Jacques B, Angala S, Rouiller I, Zgurskaya HI, Sygusch J, Jackson M (2016) Structure-function profile of MmpL3, the essential mycolic acid transporter from Mycobacterium tuberculosis. ACS Infect Dis 2:702–713PubMedPubMedCentralGoogle Scholar
  54. 54.
    Su C-C, Klenotic PA, Bolla JR, Purdy GE, Robinson CV, Yu EW (2019) MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine. Proc Natl Acad Sci 116:11241–11246PubMedGoogle Scholar
  55. 55.
    Li W, Upadhyay A, Fontes FL, North EJ, Wang Y, Crans DC, Grzegorzewicz AE, Jones V, Franzblau SG, Lee RE, Crick DC, Jackson M (2014) Novel insights into the mechanism of inhibition of MmpL3, a target of multiple pharmacophores in Mycobacterium tuberculosis. Antimicrob Agents Chemother 58:6413–6423PubMedPubMedCentralGoogle Scholar
  56. 56.
    Zhang B, Li J, Yang X et al (2019) Crystal structures of membrane transporter MmpL3, an anti-TB drug target. Cell 176:636–648PubMedGoogle Scholar
  57. 57.
    Boeree MJ, Heinrich N, Aarnoutse R et al (2017) High-dose rifampicin, moxifloxacin, and SQ109 for treating tuberculosis: a multi-arm, multi-stage randomised controlled trial. Lancet Infect Dis 17:39–49PubMedPubMedCentralGoogle Scholar
  58. 58.
    Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schäberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K (2015) A new antibiotic kills pathogens without detectable resistance. Nature 517:455–459PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institut für Medizinische MikrobiologieUniversität ZürichZürichSwitzerland
  2. 2.Roche Pharma Research and Early Development, Infectious DiseasesRoche Innovation Center BaselBaselSwitzerland
  3. 3.Nationales Zentrum für MykobakterienZürichSwitzerland

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