A Cell Based Secondary Assay to Explore Compounds that Modulate Non-Replicating, Drug-tolerant Compounds in Replicating H37Rv TB of 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 (TB) 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: 1453
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: 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 (TB) 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.
The purpose of this screen was to differentiate between active compounds in both the non-replicative carbon starved TB, the replicative TB and the transition from the non-replicative carbon starved to the replicative TB. Active compounds selected from the non-replicating TB screen (AID 488890) were screened in against replicating M. tuberculosis H37Rv.
Positive control drugs was Amikacin (both at 2.5 ug/mL and 0.13 ug/ml).
All test compounds were screened in a dose-response assay using a stacked-plate method at concentrations ranging from 50uM to 0.1uM.
Compounds and controls are added to the plates at 2X concentration and given to the BSL3 personnel in charge of the assay execution.
Assay in 384 well plate format contains 25 uL of media and drugs, 25 uL of Mycobacterium tuberculosis H37Rv and 9 uL of Alamar Blue.
Frozen stocks were prepared from Mtb H37Rv (ATCC 27294) obtained from the American Type Culture Collection (Manassas, VA). The Mtb HTS assay was modified from that described by (PMID: 9145860, Collins and Franzblau 1997) using black, clear-bottom, 384-well microtiter plates and 7H9 broth. Compounds stocks of 1 mg/mL in 100% DMSO were diluted in assay media and 25 ul of these diluted compounds were transferred to 384-well plates. Amikacin was included in the positive control wells in every assay plate at 2.5 ug/mL and 0.13ug/ml. The high concentration of amikacin completely inhibits growth and is used in lieu of uninoculated medium (background) to calculate percent inhibition by the test compounds for each plate. Plates containing test compounds and positive control compounds were transferred into our BSL3 facility for bacteria addition and incubation. The bacterial stock was diluted to 2-4x10^4 CFU/ml in the assay medium, Middlebrook 7H9 broth with glycerol and Tween 80 and the cultures were pre-grown for 72h with agitation at 37 C before plating at an OD615 of 0.001. After the bacteria were diluted to the correct OD, 25 uL was plated over the compounds. Positive and negative control wells were included in each plate. Plates were placed in stacks of two inside actively humidified incubators and incubated for 7 days at 37C with >= 95% humidity. After 7 days of incubation, end point reagent (2 parts Alamar blue (Trek diagnostics no. 00-100) + 1.5 parts 18.2% Tween 80 (Difco no. 231181) diluted in milli Q water) was added to all wells in a volume of 9 ul per well. The plates were returned to the incubator for an additional 18-20 hours. The plates were sealed and bottom read for fluorescence using a Perkin Elmer Envision plate reader at 535 nm excitation and 590 nm emission.
Data Analysis: Thirty two control wells containing Mtb only and 24 wells containing Mtb and 2.5 ug/mL amikacin 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) which 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: Compounds that showed >30% inhibition for at least one concentration were defined as Active. If the inhibition at all doses was <30% the compound was defined as Inactive.
The following tiered system has been implemented at Southern Research Institute for use with the the PubChem Score. Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity and a score of 40 corresponds to 100% inhibition. In this 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 0.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)