A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (2)
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: 37
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.
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. 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 (320 compounds/plate) 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 dilution, 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.
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".
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. The confirmatory assay ranks compounds on a scale of 41-80 based on IC50 results. In this dose response which used purified and synthesized compounds, a scale of 81-100 based on the IC50 results is used. Compounds that did not confirm as actives were given the score 0.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)