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BioAssay: AID 602225

Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)

This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the PFM18AAP screen. ..more
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 Tested Compounds
 Tested Compounds
All(15)
 
 
Active(8)
 
 
Inactive(6)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(15)
 
 
Active(8)
 
 
Inactive(6)
 
 
Inconclusive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 602225
Data Source: Southern Research Specialized Biocontainment Screening Center (PfM18_Tox_02)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-01-25
Hold-until Date: 2012-12-30
Modify Date: 2013-01-03

Data Table ( Complete ):           Active    All
BioActive Compounds: 8
Depositor Specified Assays
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AIDNameTypeComment
588678QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)confirmatoryPFM18AAP Main Screen
588679Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP)confirmatoryPFM17LAP Counter Screen
588680Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP)confirmatoryPFM1AAP Counter Screen
588714Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)confirmatoryVero Cytotox
1855Summary of probe development efforts to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP)summary
624174Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the human M18 Aspartyl Aminopeptidase (hM18AAP) (3)confirmatory
624175Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)confirmatory
624176Counterscreen for inhibitors of PFM18AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1AAP (PFM1AAP) (3)confirmatory
624177QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)confirmatory
624205Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (3)confirmatory
720736Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCs, Set2other
743024Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (M18AAP): radiolabel-based cell-based dose response assay to identify compounds that inhibit P. falciparum growth in RBCs, Set 2.confirmatory
Description:
Southern Research Specialized Biocontainment Screening Center (SRSBSC)
Assay Provider: Donald Gardiner
Award: 1 R03 MH082342-01A1
This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the PFM18AAP screen.

In this assay, we treated Vero E6 cells with compounds selected as hits in the PFM18AAP assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.19uM to 100 uM. Following 72 hours of treatment, relative viable cell number was determined using Cell Titer Glo from Promega. Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Protocol
Cell Culture: Vero E6 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide, and maintained for no more than 20 passages.

Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume).

Cell Plating: Twenty uL of complete growth medium containing 3000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection.

Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature for 10 min. Twenty-five uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader in luminescence mode with an integration time of 0.1 s.
Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle and were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.
Comment
Outcome: Compounds that showed <80% cell viability for at least one concentration were defined as "Active" (toxic). If the % viability at all doses was <80%, the compound was defined as "Inactive" (non-toxic). Instances where replicate data sets conflicted on this criteria are listed as "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 % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on CC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on CC50 result. Inactive compounds are given the score 0.
Categorized Comment
Phenotypic Screen: Yes

Multiplexing: No

BSL: BSL1

Screening Concentration
Screening Concentration Range Max: 50

Screening Concentration Range Min: 0.098

Assay Format: Cell-based

Assay Type: Viability/Toxicity

Assay Method: End-point

Assay Detection: Bio-luminescence

Used for Hit Validation?: Yes

Used during SAR?: Yes

Secondary Assay Sub-type: Counter-screen Assay

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average CC50 ModifierString
2Average CC50*FloatμM
3CC50 Modifier Rep 1String
4CC50 Rep 1FloatμM
5CC50 Modifier Rep 2String
6CC50 Rep 2FloatμM
7CC50 Modifier Rep 3String
8CC50 Rep 3FloatμM
9StDev CC50 Rep 1Float
10CC50 Hill Slope Rep 1Float
11CC50 NormChi2 Rep 1Float
12StDev CC50 Rep 2Float
13CC50 Hill Slope Rep 2Float
14CC50 NormChi2 Rep 2Float
15StDev CC50 Rep 3Float
16CC50 Hill Slope Rep 3Float
17CC50 NormChi2 Rep 3Float
18% Viability @ 50 uM Rep 1 (50μM**)Float%
19% Viability @ 25 uM Rep 1 (25μM**)Float%
20% Viability @ 12.5 uM Rep 1 (12.5μM**)Float%
21% Viability @ 6.25 uM Rep 1 (6.25μM**)Float%
22% Viability @ 3.13 uM Rep 1 (3.13μM**)Float%
23% Viability @ 1.56 uM Rep 1 (1.56μM**)Float%
24% Viability @ 0.78 uM Rep 1 (0.78μM**)Float%
25% Viability @ 0.39 uM Rep 1 (0.39μM**)Float%
26% Viability @ 0.19 uM Rep 1 (0.19μM**)Float%
27% Viability @ 0.09 uM Rep 1 (0.09μM**)Float%
28% Viability @ 50 uM Rep 2 (50μM**)Float%
29% Viability @ 25 uM Rep 2 (25μM**)Float%
30% Viability @ 12.5 uM Rep 2 (12.5μM**)Float%
31% Viability @ 6.25 uM Rep 2 (6.25μM**)Float%
32% Viability @ 3.13 uM Rep 2 (3.13μM**)Float%
33% Viability @ 1.56 uM Rep 2 (1.56μM**)Float%
34% Viability @ 0.78 uM Rep 2 (0.78μM**)Float%
35% Viability @ 0.39 uM Rep 2 (0.39μM**)Float%
36% Viability @ 0.19 uM Rep 2 (0.19μM**)Float%
37% Viability @ 0.09 uM Rep 2 (0.09μM**)Float%
38% Viability @ 50 uM Rep 3 (50μM**)Float%
39% Viability @ 25 uM Rep 3 (25μM**)Float%
40% Viability @ 12.5 uM Rep 3 (12.5μM**)Float%
41% Viability @ 6.25 uM Rep 3 (6.25μM**)Float%
42% Viability @ 3.13 uM Rep 3 (3.13μM**)Float%
43% Viability @ 1.56 uM Rep 3 (1.56μM**)Float%
44% Viability @ 0.78 uM Rep 3 (0.78μM**)Float%
45% Viability @ 0.39 uM Rep 3 (0.39μM**)Float%
46% Viability @ 0.19 uM Rep 3 (0.19μM**)Float%
47% Viability @ 0.09 uM Rep 3 (0.09μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084103-01

Data Table (Concise)
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