TB Biology Services

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.

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 Inhibition Assay 
• 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

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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
Phenotypic Screening
TB Screening	Single Point MIC Assay (3 media)
TB Screening MIC90	Routine TB MIC Assay (3 media)
TB Screening MIC90	Non-replicating
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
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
Spontaneous Resistant Mutants
ClpP1P2 Assay

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 Diseases2017,3(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.