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BioAssay: AID 504857

High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (4)

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 ..
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 Tested Compounds
 Tested Compounds
All(59)
 
 
Active(25)
 
 
Inactive(34)
 
 
 Tested Substances
 Tested Substances
All(59)
 
 
Active(25)
 
 
Inactive(34)
 
 
 Related BioAssays
 Related BioAssays
AID: 504857
Data Source: Southern Research Specialized Biocontainment Screening Center (TB_Inh_Sec_DR4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-06-27
Hold-until Date: 2012-04-06
Modify Date: 2012-04-09

Data Table ( Complete ):           Active    All
BioActive Compounds: 25
Depositor Specified Assays
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AIDNameTypeComment
449762High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Mediaconfirmatory
449764A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerolconfirmatory
435019A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Mycobacterium Tuberculosisconfirmatory
449775Summary of Assays used to Identify Novel Compounds That Inhibit Mycobacterium Tuberculosis in 7H9 Media with Glycerolsummary
493198High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compoundsconfirmatory
493181A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compoundsconfirmatory
504335A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis.confirmatory
504556High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (2)confirmatory
504564A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (2)confirmatory
504646High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (3)confirmatory
504645A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (3)confirmatory
504652Antagonist of Human D 1 Dopamine Receptor: qHTSscreening
504684A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (3)confirmatory
504640A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerolconfirmatory
504683A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (2)confirmatory
504897High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (6)confirmatory
504898A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (5)confirmatory
504901A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (6)confirmatory
504903High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (5)confirmatory
504909A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (5)confirmatory
504910A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (4)confirmatory
504911A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (4)confirmatory
588437A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (7)confirmatory
588441A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (5)confirmatory
588443A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (5)confirmatory
588445A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (6)confirmatory
588447High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (8)confirmatory
602431A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (8)confirmatory
602432A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (6)confirmatory
602433A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (6)confirmatory
602435High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (9)confirmatory
602437A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (8)confirmatory
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. William Bishai, Johns Hopkins University Tuberculosis Research Center
Award: 1 R03 MH084877-01A1

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.
Protocol
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.
Comment
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 average IC90 results is used. Compounds that did not confirm as actives were given the score 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Avg I90 ModifierString
2Avg IC90*FloatμM
3Avg IC50 ModifierString
4Avg IC50FloatμM
5IC90 Modifier Rep 1String
6IC90 Rep 1FloatμM
7IC50 Modifier Rep 1String
8IC50 Rep 1FloatμM
9IC50 Std Dev Rep 1Float
10IC50 Hill Slope Rep 1Float
11IC50 NormChi2Float
12IC90 Modifier Rep 2String
13IC90 Rep 2FloatμM
14IC50 Modifier Rep 2String
15IC50 Rep 2FloatμM
16IC50 Std Dev Rep 2Float
17IC50 Hill Slope Rep 2Float
18IC50 NormChi2 Rep 2Float
19IC90 Modifier Rep 3String
20IC90 Rep 3FloatμM
21IC50 Modifier Rep 3String
22IC50 Rep 3FloatμM
23IC50 Std Dev Rep 3Float
24IC50 Hill Slope Rep 3Float
25IC50 NormChi2 Rep 3Float
26% Inhibition @ 100 uM Rep 1 (100μM**)Float%
27% Inhibition @ 50 uM Rep 1 (50μM**)Float%
28% Inhibition @ 25 uM Rep 1 (25μM**)Float%
29% Inhibition @ 12.5 uM Rep 1 (12.5μM**)Float%
30% Inhibition @ 6.25 uM Rep 1 (6.25μM**)Float%
31% Inhibition @ 3.125 uM Rep 1 (3.125μM**)Float%
32% Inhibition @ 1.563 uM Rep 1 (1.563μM**)Float%
33% Inhibition @ 0.781 uM Rep 1 (0.781μM**)Float%
34% Inhibition @ 0.391 uM Rep 1 (0.391μM**)Float%
35% Inhibition @ 0.195 uM Rep 1 (0.195μM**)Float%
36% Inhibition @ 0.098 uM Rep 1 (0.098μM**)Float%
37% Inhibition @ 0.048 uM Rep 1 (0.048μM**)Float%
38% Inhibition @ 0.024 uM Rep 1 (0.024μM**)Float%
39% Inhibition @ 0.012 uM Rep 1 (0.012μM**)Float%
40% Inhibition @ 0.006 uM Rep 1 (0.006μM**)Float%
41% Inhibition @ 0.003 uM Rep 1 (0.003μM**)Float%
42% Inhibition @ 0.0015 uM Rep 1 (0.0015μM**)Float%
43% Inhibition @ 0.0008 uM Rep 1 (0.0008μM**)Float%
44% Inhibition @ 0.0004 uM Rep 1 (0.0004μM**)Float%
45% Inhibition @ 100 uM Rep 2 (100μM**)Float%
46% Inhibition @ 50 uM Rep 2 (50μM**)Float%
47% Inhibition @ 25 uM Rep 2 (25μM**)Float%
48% Inhibition @ 12.5 uM Rep 2 (12.5μM**)Float%
49% Inhibition @ 6.25 uM Rep 2 (6.25μM**)Float%
50% Inhibition @ 3.125 uM Rep 2 (3.125μM**)Float%
51% Inhibition @ 1.563 uM Rep 2 (1.563μM**)Float%
52% Inhibition @ 0.781 uM Rep 2 (0.781μM**)Float%
53% Inhibition @ 0.391 uM Rep 2 (0.391μM**)Float%
54% Inhibition @ 0.195 uM Rep 2 (0.195μM**)Float%
55% Inhibition @ 0.098 uM Rep 2 (0.098μM**)Float%
56% Inhibition @ 0.048 uM Rep 2 (0.048μM**)Float%
57% Inhibition @ 0.024 uM Rep 2 (0.024μM**)Float%
58% Inhibition @ 0.012 uM Rep 2 (0.012μM**)Float%
59% Inhibition @ 0.006 uM Rep 2 (0.006μM**)Float%
60% Inhibition @ 0.003 uM Rep 2 (0.003μM**)Float%
61% Inhibition @ 0.0015 uM Rep 2 (0.0015μM**)Float%
62% Inhibition @ 0.0008 uM Rep 2 (0.0008μM**)Float%
63% Inhibition @ 0.0004 uM Rep 2 (0.0004μM**)Float%
64% Inhibition @ 100 uM Rep 3 (100μM**)Float%
65% Inhibition @ 50 uM Rep 3 (50μM**)Float%
66% Inhibition @ 25 uM Rep 3 (25μM**)Float%
67% Inhibition @ 12.5 uM Rep 3 (12.5μM**)Float%
68% Inhibition @ 6.25 uM Rep 3 (6.25μM**)Float%
69% Inhibition @ 3.125 uM Rep 3 (3.125μM**)Float%
70% Inhibition @ 1.563 uM Rep 3 (1.563μM**)Float%
71% Inhibition @ 0.781 uM Rep 3 (0.781μM**)Float%
72% Inhibition @ 0.391 uM Rep 3 (0.391μM**)Float%
73% Inhibition @ 0.195 uM Rep 3 (0.195μM**)Float%
74% Inhibition @ 0.098 uM Rep 3 (0.098μM**)Float%
75% Inhibition @ 0.048 uM Rep 3 (0.048μM**)Float%
76% Inhibition @ 0.024 uM Rep 3 (0.024μM**)Float%
77% Inhibition @ 0.012 uM Rep 3 (0.012μM**)Float%
78% Inhibition @ 0.006 uM Rep 3 (0.006μM**)Float%
79% Inhibition @ 0.003 uM Rep 3 (0.003μM**)Float%
80% Inhibition @ 0.0015 uM Rep 3 (0.0015μM**)Float%
81% Inhibition @ 0.0008 uM Rep 3 (0.0008μM**)Float%
82% Inhibition @ 0.0004 uM Rep 3 (0.0004μM**)Float%
83VerificationString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084877-01A1

Data Table (Concise)
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