High Throughput Screen to Identify Inhibitors of Mycobacterium tuberculosis H37Rv
Mycobacterium tuberculosis (Mtb) is a notorious pathogen whose increasing resistance to antibiotics and heightened lethality in combination with AIDS makes it a major health concern worldwide. The World Health Organization (WHO) estimates that one-third of the world's population is infected with Mtb; eight million people worldwide develop tuberculosis annually while nearly two million die. Mtb more ..
BioActive Compounds: 2044
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider Lucile White, Southern Research Institute, Birmingham, AL
Mycobacterium tuberculosis (Mtb) is a notorious pathogen whose increasing resistance to antibiotics and heightened lethality in combination with AIDS makes it a major health concern worldwide. The World Health Organization (WHO) estimates that one-third of the world's population is infected with Mtb; eight million people worldwide develop tuberculosis annually while nearly two million die. Mtb causes more deaths than any other infectious agent in the world. Immunocompromised individuals, particularly those infected with human immunodeficiency virus (HIV), are at great risk for infection with Mtb. WHO estimates that 11.4 million people worldwide are infected with both Mtb and HIV. Although infections with drug sensitive strains can be effectively cured with a 6 to 9 month regimen of multiple antibiotics, the inability to deliver and complete appropriate courses of therapy on a global level has led to the selection of resistant strains over the past 50 years. Especially alarming is the upsurge in cases of multidrug-resistant tuberculosis (MDR-TB). The selection and spread of multiple drug resistant Mtb continued for decades leading to selection and spread of two operationally distinct forms, multiple drug resistant (MDR-TB resistant to isoniazid and rifampicin) and extensively drug resistant (XDR-TB resistant to isoniazid, rifampicin, a fluoroquinolone and at least one of the injectable second-line agents). The estimate for global MDR-TB and XDR-TB cases for 2005 were 424,000 and 27,000 respectively, and the situation is worst in areas with high incidences of HIV infection. XDR-TB has been found in 41 countries.
Thus the discovery of new types of anti-TB drugs acting on novel drug targets with no cross-resistance to any existing drugs is urgently needed. Modern high-throughput screening systems provide an immensely powerful strategy to identify new lead compounds in a relatively short amount of time. In this study we have adapted the microdilution Alamar blue assay (PMID: 9145860, Collins and Franzblau 1997) to a 384-well plate format and used it to screen a compound library in a BSL-3 contained high throughput platform for antimicrobial activity. A total of 216,163 compounds were screened initially in a single dose of 10 uM and a final DMSO concentration of 1%. 5,883 compounds were identified that inhibited bacterial growth >80%. Cheminformatics techniques were used to select a diverse set of 2,500 compounds that were structurally different from known TB drugs. Compounds were screened in a dose-response assay using a stacked-plate method at concentrations ranging from 100uM to 0.19uM.
Mycobacterium tuberculosis Assay
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 7H12 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 in two concentrations, 0.13 and 2.5 ug/mL. The low concentration is the approximate MIC and is an indicator of proper assay performance of each plate. The high concentration 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 (320 compounds/plate) and positive control compounds were transferred into our BSL3 facility for bacteria addition and incubation. The bacterial stock was diluted to 1-2x10^5 CFU/ml in the assay medium, Middlebrook 7H12 broth (7H9 broth supplemented with 0.1% casitone, 5.6 ug/mL palmitate, 0.5% bovine serum albumin and 4 ug/mL catalase) and 25 uL was plated over the compounds. Positive and negative control wells were included in each plate. Amikacin was included in one of the compound wells as an internal control in dose response runs. Plates were placed in stacks of two inside double low density polyethylene bags and incubated for 7 days at 37C with approximately 90% 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.
Cell Cytotoxicity Assay
Vero cell line (ATCC CCL-81) was obtained from the American Type Culture Collection (Manassas, VA). The cell line was cultured in ATCC complete growth medium (No. 30-2003) supplemented with 10% fetal bovine serum, 100 IU/ml Penicillin and 100 ug/mL streptomycin (invitrogen/GIBCO cat no. 15140-155) (culture media) and incubated at 37C, 5% CO2 and high humidity. Vero cell stocks were frozen in culture media supplemented with 5% DMSO at a density of 6x10^6 cells /ml. For freezing, two ml aliquots were dispensed to Nunc vials. For use in the assay cells are thawed in 37C water bath. Cells were mixed in the vial by light vortexing and an equal volume of culture media added. Cells were then centrifuged and media removed. Cells were resuspended in culture media, counted and diluted to 125,000 cells per mL.
Twenty uL of cells were added to all wells of the assay plate (corning 3712) contained 5 uL of pre-plated compounds or controls. Plates were then incubated for 72 hours at 37C, with 5% CO2 in a humidified incubator. Cell viability was assessed using CellTiter-Glo reagent from Promega according to manufacturer's protocol. Luminescence was recorded using an integration time of 0.1 second per well. Columns 1 and 2 in the assay plates contained media + 0.4% DMSO for negative control and columns 23 and 24 contained media in 0.4% DMSO and 100 uM hyamine as a positive control. A stacked-plate dose-response method was used and the final test concentrations for the compounds ranged from 40 ug/ml to 0.078 ug/ml in 2-fold dilutions with a final DMSO concentration of 0.4%.
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) 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). The dose response data was analyzed using a four parameter logistic fit to the data (XLFit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC90 and IC50 values were calculated for Mtb and CC50 values calculated for Vero cell cytotoxicity. A Selectivity Index for compounds showing activity against Mtb H37Rv was calculated as SI = CC50/IC90.
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. Possible artifacts in the Vero Cytotoxicity assay include, but are not limited to, compounds that that interfere with the luciferase reaction, absorb at 700nm (the wavelength of light emitted by the luciferase reaction), or precipitate.
Outcome: Compounds that showed >30% inhibition for at least one concentration in the Mtb dose response were defined as "Active". If the inhibition at all doses was <30% in the Mtb assay, the compound was defined as "Inactive". In the primary screen a compound was deemed "Inactive" if it had a Percent Inhibition <80%. Compounds with a Percent Inhibition >80% but were not selected for follow up dose response were labeled "Inconclusive."
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 IC90 result in the Mtb assay while compounds that did not confirm as actives were given the score 0.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)