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Tuberculosis Biology Platform Services

Housed at the Institute of Infectious Disease & Molecular Medicine (IDM)

 

Tuberculosis Biology Platform

H3D has a dedicated TB biology team housed at the Institute of Infectious Disease and Molecular Medicine (IDM) with access to BSLII and BSLIII facilities. The TB biology team routinely conducts whole-cell and target-based screening, biology triaging, target identification and validation studies for the TB projects. The BSLII Drug Discovery Lab houses a separate access-controlled tissue culture facility, the TB screening platform; and bench space for assay development and target ID/mechanism of action work.

See the table below for additional details, including average turnaround time and control(s). For assays where an average turnaround time/control(s) are not available, this indicates that turnaround time is dependent on your unique experimental design and conditions; please book a consultation for an estimated turnaround time.

Bacteria Mycobacterium tuberculosis, the causative agent of tuberculosis, 3D illustration

 

H3D's expertise and experience is especially pronounced in the following TB Biology areas (see also chart and references below):

Phenotypic Screening

The H37Rv strain is the most commonly used M. tuberculosis strain in labs worldwide and is used by H3D for many TB biology assays. Screening in both non-resistant and resistant strains is available. "Drug resistant strains" of TB are resistant to at least one first line drug used for TB treatment.

TB is currently treated with several antimicrobial drugs simultaneously, and these include isoniazid (INH), rifampin (Rif), pyrazinamide, and ethambutol (Eth). Investigating a compound's activity and efficacy in relation to multiple of these established treatments is critical to developing a competitive drug candidate for treating tuberculosis.

  • Single Point MIC Assay (3 media*)
  • Routine TB MIC Assay (3 media*)
    • *The inhibitory activity of several compounds has been shown to be impacted by the media composition in which assays are performed. The 3 media compositions utilized allow us to compare the role of defined parameters on compound activity i.e. the effect of detergent and protein/serum binding.
  • Non-replicating
    • TB has both replicating and non-replicating stages, and it is critical to evaluate a drug candidate in both
  • Erdman
    • highly virulent TB strain
  • Screening against single drug resistant (SDR) strains:
    • INH-R: Isoniazid resistant
    • Rif-R: Rifampin resistant
    • DCS-R: D-cycloserine resistant
    • Moxi-R: Moxifloxacin resistant
    • Eth-R: Ethionamide resistant

 

Target Based Screening

  • Target based screening approaches are available for the following M. tuberculosis targets
    • PrecA and PiniB lux assay
      • Luminescence reporter assays reporting on DNA and cell wall damage respectively
    • QcrB MIC assay
      • QcrB is the cytochrome b subunit of the cytochrome bc1 complex
      • The assay utilizes a QcrB mutant strain and the CydA (cytochromebd menaquinol oxidase) mutant strain which is hyper-sensitive to QcrB inhibitors
    • DprEl-mutant
      • pentose-reductase related mutant
    • MMPL3 assay MIC
      • inner membrane transporter from the Mycobacterial Membrane Protein Large (MmpL) family
    • ClpP1P2 Assay
      • ClpP1P2 is a 300-kDa protease from Mycobacterium tuberculosis

 

Additional TB Biology Assays

  • Time-Kill Kinetics
    • anti-TB drugs differ significantly regarding their time-kill kinetics, and time-kill kinetics assays are an important part of preclinical modeling framework for assessing the activity of anti-tuberculosis drugs
  • Spontaneous Resistant Mutants (SRMs)
    •  Drug-resistant TB is one of the leading causes of death worldwide and arises mainly from spontaneous mutations
    • SRM generation aids in the deconvolution of the mechanism of action of compounds and allows for the in vitro determination of the frequency of resistance development to compounds

 

Data from these assays can be used for lead optimization and drug candidate evaluation and results from these assays thereby inform key decision making and experimental design of human and/or animal in vivo studies.

 

Assay Group Assay Specifications Average Turnaround Time Control(s)
Phenotypic Screening      
TB Screening Single Point MIC Assay (3 media) 2 weeks Isoniazid + Moxifloxacin
TB Screening MIC90 Routine TB MIC Assay (3 media) 2 weeks Isoniazid + Moxifloxacin
TB Screening MIC90 Non-replicating 2 weeks Isoniazid + Moxifloxacin
TB Screening MIC90 Erdman MIC    
Screening Against Single-Drug Resistant (SDR) Strains      
TB Screening MIC90 INH-R MIC    
TB Screening MIC90 Rif-R MIC    
TB Screening MIC90 DCS-R MIC    
TB Screening MIC90 Moxi-R MIC    
TB Screening MIC90 CydA-mutant MIC    
TB Screening MIC90 EthA-mutant MIC    
       
Target Based Screening      
TB Screening MIC90 PrecA & PiniB lux MIC Assay   Isoniazid (PiniB) + Ciprofloxacin (PrecA)
TB Screening MIC90 QcrB assay MIC    
TB Screening MIC90 DprEl assay MIC    
TB Screening MIC90 MMPL3 assay MIC    
       
Additional TB Biology Assays      
Time-Kill Kinetics     Rifampin
Spontaneous Resistant Mutants      
ClpP1P2 Assay      
Tuberculosis Biology Studies
Select Relevant H3D Authored Publications

van der Westhuyzen R, Winks S, Wilson CR, Boyle GA, Gessner RK, Soares de Melo C, Taylor D, de Kock C, Njoroge M, Brunschwig C, Lawrence N, Rao SP, Sirgel F, van Helden P, Seldon R, Moosa A, Warner DF, Arista L, Manjunatha UH, Smith PW, Street LJ, Chibale K. Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A Novel Antimycobacterial Class Targeting Mycobacterial Respiration. J Med Chem. 2015 Dec 10;58(23):9371-81. doi: 10.1021/acs.jmedchem.5b01542. Epub 2015 Nov 20. PMID: 26551248.

Murugesan D, Ray PC, Bayliss T, Prosser GA, Harrison JR, Green K, Soares de Melo C, Feng TS, Street LJ, Chibale K, Warner DF, Mizrahi V, Epemolu O, Scullion P, Ellis L, Riley J, Shishikura Y, Ferguson L, Osuna-Cabello M, Read KD, Green SR, Lamprecht DA, Finin PM, Steyn AJC, Ioerger TR, Sacchettini J, Rhee KY, Arora K, Barry CE 3rd, Wyatt PG, Boshoff HIM. 2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization. ACS Infect Dis. 2018 Jun 8;4(6):954-969. doi: 10.1021/acsinfecdis.7b00275. Epub 2018 Mar 26. PMID: 29522317; PMCID: PMC5996347.

Soares de Melo C, Feng TS, van der Westhuyzen R, Gessner RK, Street LJ, Morgans GL, Warner DF, Moosa A, Naran K, Lawrence N, Boshoff HI, Barry CE 3rd, Harris CJ, Gordon R, Chibale K. Aminopyrazolo[1,5-a]pyrimidines as potential inhibitors of Mycobacterium tuberculosis: Structure activity relationships and ADME characterization. Bioorg Med Chem. 2015 Nov 15;23(22):7240-50. doi: 10.1016/j.bmc.2015.10.021. Epub 2015 Oct 22. Erratum in: Bioorg Med Chem. 2016 Jan 15;24(2):314. Candice, Soares de Melo [corrected to Soares de Melo, Candice]. PMID: 26522089.

Chacko S, Boshoff HIM, Singh V, Ferraris DM, Gollapalli DR, Zhang M, Lawson AP, Pepi MJ, Joachimiak A, Rizzi M, Mizrahi V, Cuny GD, Hedstrom L. Expanding Benzoxazole-Based Inosine 5'-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure-Activity As Potential Antituberculosis Agents. J Med Chem. 2018 Jun 14;61(11):4739-4756. doi: 10.1021/acs.jmedchem.7b01839. Epub 2018 May 30. PMID: 29746130; PMCID: PMC6166404.

Sahu NU, Singh V, Ferraris DM, Rizzi M, Kharkar PS. Hit discovery of Mycobacterium tuberculosis inosine 5'-monophosphate dehydrogenase, GuaB2, inhibitors. Bioorg Med Chem Lett. 2018 Jun 1;28(10):1714-1718. doi: 10.1016/j.bmcl.2018.04.045. Epub 2018 Apr 18. PMID: 29699922.

 
Additional References supporting H3D’s materials & methods:

Singh, V.; Donini, S.; Pacitto, A.; Sala, C.; Hartkoorn, R. C.; Dhar, N.; Keri, G.; Ascher, D. B.; Mondésert, G.; Vocat, A.; Lupien, A.; Sommer, R.; Vermet, H.; Lagrange, S.; Buechler, J.; Warner, D. F.; McKinney, J. D.; Pato, J.; Cole, S. T.; Blundell, T. L.; Rizzi, M.; Mizrahi, V., The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis. ACS Infectious Diseases 2017, 3 (1), 5-17. DOI: 10.1021/acsinfecdis.6b00102.

Naran, K.; Moosa, A.; Barry, C. E.; Boshoff, H. I. M.; Mizrahi, V.; Warner, D. F. Bioluminescent reporters for rapid mechanism of action assessment in tuberculosis drug discovery. Antimicrobial Agents and Chemotherapy 2016, 60, 6748-6757.