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BioAssay: AID 602228

Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M1 Aspartyl Aminopeptidase (PFM1AAP) (2)

This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the PFM1AAP screen. ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(3)
 
 
Inactive(2)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(3)
 
 
Inactive(2)
 
 
Inconclusive(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 602228
Data Source: Southern Research Specialized Biocontainment Screening Center (PfM1_Tox_02)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-01-26
Hold-until Date: 2012-12-30
Modify Date: 2013-01-03

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 3
Related Experiments
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AIDNameTypeComment
434966Summary of the probe development effort to identify inhibitors of the Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)Summarydepositor-specified cross reference
588682Counterscreen for inhibitors of PFM1AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP)Confirmatorydepositor-specified cross reference: PFM17LAP Counter Screen for M1
588686QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PFM1AAP)Confirmatorydepositor-specified cross reference: PFM1AAP Main Screen
588687Counterscreen for inhibitors of PFM1 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP)Confirmatorydepositor-specified cross reference: PFM18AAP Counter Screen for M1
588717Vero Cytoxicity Assay: A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Plasmodium falciparum M1 Aspartyl Aminopeptidase (PFM1AAP)Confirmatorydepositor-specified cross reference
1445Inhibitors of Plasmodium falciparum M1- Family Alanyl Aminopeptidase (M1AAP)Confirmatorysame project related to Summary assay
2214Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Cathepsin L proteinase (CTSL1).Confirmatorysame project related to Summary assay
2215Counterscreen for inhibitors of M1 and M17 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP).Confirmatorysame project related to Summary assay
463209Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (M1AAP): radiolabel-based cell-based assay to identify compounds that inhibit P. falciparum growth in RBCsOthersame project related to Summary assay
492976Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PfM1AAP): fluorescence-based biochemical assay to identify inhibitors of malaria cell lysateScreeningsame project related to Summary assay
492978Late stage assay provider results from the probe development effort to identify inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PfM1AAP): fluorescence-based biochemical assay to identify inhibitors of rPfM1AAPScreeningsame project related to Summary assay
602209Counterscreen for inhibitors of PFM1AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (2)Confirmatorysame project related to Summary assay
602212Counterscreen for inhibitors of PFM1 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (2)Confirmatorysame project related to Summary assay
602213QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PFM1AAP) (2)Confirmatorysame project related to Summary assay
624163QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M1 Alanyl Aminopeptidase (PFM1AAP) (3)Confirmatorysame project related to Summary assay
624164Counterscreen for inhibitors of PFM1AAP: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M17 Leucine Aminopeptidase (PFM17LAP) (3)Confirmatorysame project related to Summary assay
624165Counterscreen for inhibitors of PFM1 aminopeptidases: QFRET-based biochemical high throughput dose response assay for inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PFM18AAP) (3)Confirmatorysame project related to Summary assay
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 PFM1AAP screen.

In this assay, we treated Vero E6 cells with compounds selected as hits in the PFM1AAP assay for 72 hours over a 10 point 2-fold dilution series, ranging from 100 uM to 0.19 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". If the % viability at all doses was >80%, the compound was defined as "Inactive". 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 - additional comments and annotations
From BioAssay Depositor:
Assay Detection: Bio-luminescence
Assay Format: Cell-based
Assay Method: End-point
Assay Type: Viability/Toxicity
BSL: BSL1
Multiplexing: No
Phenotypic Screen: Yes
Screening Concentration:
Screening Concentration Range Max: 50
Screening Concentration Range Min: 0.098
Secondary Assay Sub-type: Counter-screen Assay
Used during SAR?: Yes
Used for Hit Validation?: Yes
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: VERO 76
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 MH082342-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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