Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosis
Assay Rationale and Summary: Over one third of the world's population is infected with tuberculosis. In 1993, the World Health Organization declared a global health emergency with the resurgence of tuberculosis in the setting of HIV infection and with the emergence of multi-drug resistant TB. In 2004, 14.6 million people had active TB, 8.9 million new cases were documented, and 1.7 million more ..
BioActive Compounds: 1277
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Deborah Hung, Massachusetts Institute of Technology
Award: 1 RO3 MH087444-01
Assay Rationale and Summary: Over one third of the world's population is infected with tuberculosis. In 1993, the World Health Organization declared a global health emergency with the resurgence of tuberculosis in the setting of HIV infection and with the emergence of multi-drug resistant TB. In 2004, 14.6 million people had active TB, 8.9 million new cases were documented, and 1.7 million deaths were attributed to TB (1). Future projections are even more alarming, due to the catastrophic synergy between TB and HIV. In this setting however, we have been forced to rely on suboptimal drugs that were developed in the 1950-60's, with TB drug development vanishing after the 1970's. At present, chemotherapy for TB requires at least six months of a complex multi-drug regimen. Treatment length greatly hinders effective TB control due to the challenges of compliance, which contribute to the development of multi-drug resistance. Despite the alarming implications for public health, research funding has lagged far behind the problem, and the pharmaceutical industry has all but ignored TB (2). New agents that achieve more rapid cures would transform the current pandemic; however, major barriers exist to developing such novel therapeutics including the lack of understanding of the in vivo physiologic states of TB bacilli as they adapt to the host microenvironment in order to survive for extended periods of time.
TB pathogenesis manifests in many forms from active disease to clinical latency that extends for decades requiring long, inefficient antibiotic treatment. The latent form of TB has presented researchers with challenges to understanding this non-replicative, metabolically inactive and drug-tolerant form of TB. Recently hypoxia-induced models that recapitulate the characteristics of the non-replicating, antibiotic resistant state have offered insight into TB lifecycle. Several groups have screened libraries in HTS with hypoxia induced non-replicative TB, but to this date, no one has utilized a carbon starvation model to study TB non-replicative dormancy.
Dr. Hung has developed and validated a successful high throughput screen utilizing a carbon-starvation assay using non-replicating, antibiotic-tolerant TB to screen small molecule candidates that can be identified as targeting essential functions in TB during different physiologic states. For this assay, TB H37Rv strain expressing constitutive, episomal GFP was used. The screen uses a GFP fluorescent outgrowth readout for measuring cell survival upon exposure to a diverse chemical library. Cells are starved for 5 weeks and then plated into 384 well plates where they are exposed to a chemical library for 5 days. Survival of cells is determined by measuring outgrowth in each well after addition of 5x rich media for 4 days. This outgrowth screen identifies several classes of compounds: 1. inhibitors of outgrowth, 2. inhibitors of viability under carbon-starvation, 3. inhibitors of the transition from carbon-starved to replicating state, and 4. quenchers of GFP fluorescence used to measure outgrowth.
Pre-Screening Assay Set-up:
40 days prior to the beginning of the assay, a starter culture of the M. tuberculosis H37Rv from frozen stock in 40mL 7H9 complete medium is started in the BSL3 laboratory (agitation should not exceed 65 rpm in the water bath). This culture is grown for five days to an OD600of .3 to .9. The culture is then pelleted and resuspended in 7H9/Tyloxapol at an OD600of .2 in sealed containers. These containers are left in a humidified incubator at 37 C for 5 weeks, without agitation to prevent gas exchange.
Compound plates are drugged outside of the BSL3 lab and on the day of the screen, the FX to add compounds/controls/media to Corning 384-well black clear bottom plates (catalog #3712); 25ul/well. The plates are transferred into the BSL3 for TB culture addition. The 25microl of culture in 7H9/Tyloxapol, at an OD600 of .005, is added to the plates with the compounds with the Matrix WellMate. The plates are returned to the incubator for an additional 4 days at 370C.
After the 4 days at 370C, the plates are removed from the incubator and an additional 12ul of concentrated supplemental media is added to the assay plates and they are returned to the incubator for an additional 3 days at 370C.
For Alamar Blue assay detection, a solution of 3 parts 18.2% Tween-80 to 4 parts Alamar Blue (3/7th Tween-80 to 4/7th Alamar Blue) is made and 9ul added to each well in the plate. The plates are incubated (stacked 2-3 high) overnight at 370C in humidified incubator.
The plates are removed from the incubator and sealed with aluminium seals. The fluorescence is read using the Envision plate reader (bottom read) with an excitation wavelength of 531nm and an emission wavelength of 595nm (Excitation filter = BODIPY TMR FP 531, barcode 105; Emission filter = Photometric 595, barcode 315; Mirror = BODIPY TMR, barcode 405).
7H9 complete medium (1L):
Middlebrook 7H9 powder (BD, catalog 271310) - 4.7 g
50% Glycerol (Fisher, catalog G33) - 4 ml
20% Tween-80 (Sigma, catalog P1754) - 2.5 ml
Water up to 900 ml
Autoclave, cool to 50oC and add
OADC (BD/Difco) - 100 ml
Middlebrook 7H9 powder (BD, catalog 271310) - 4.7 g
20% Tyloxapol - 2.5ml
Water up to 1L
Concentrated Supplemental Media (1L):
Middlebrook 7H9 powder (BD, catalog 271310) - 4.78 g
Glycerol (Fisher, catalog G33) - 20 ml
20% Tyloxapol - 2.25ml
Water up to 500 ml
Autoclave, cool to 50oC and add
OADC (BD/Difco) - 500 ml
Active compounds selected from the primary screen were tested in ten point stacked-plate dose response in concentrations diluted 1:2 beginning at 50 uM and ending at 0.1 uM.
Data Analysis: Thirty two control wells containing DMSO only, 24 wells containing Mtb and 2.5 ug/mL amikacin and 8 wells containing Mtb and 2.5 ug/mL amikacin/5.0 ug/mL isoniazid were included on each assay plate and used to calculate Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Median Cell Ctrl-High Dose Ctrl Drug) - (Test well-High Dose Ctrl Drug))/(Median Cell Ctrl-High Dose Ctrl Drug). IC50 values were calculated using a four parameter logistic fit to the data (XLFit equation 205) with the maximum and minimum locked at 100 and 0.
Possible artifacts in the Mtb assay include, but are not limited to, compounds that auto fluoresce (false negatives) and compounds that absorb in the 500-600 nm range (false positives), or precipitate.
Outcome: In the primary screen, compound activity is based on a cutoff of 80%. The compounds requested for confirmatory tested were equally divided between compounds thought to inhibit the carbon starved state and the transition between non-replicating and replicating. Of those, 2,294 compounds were available for subsequent testing. 1277 compounds showed an IC50 < 10 and were considered active in the confirmatory screen.
Score: The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the IC50 result while compounds that did not confirm as actives were given the score of 0.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)