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BioAssay: AID 624300

Discovery of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP - HeLa Cytotoxicity

The Ras superfamily of small G-protein comprises more than 150 proteins (Wennerberg et al., 2005) and in their function as molecular switches they regulate steps in almost all aspects of cellular signaling, including both physiological and pathological processes. The discovery that the Ras proteins are frequently mutated in human cancers and that the oncogenic mutations are causing increased more ..
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 Tested Compounds
 Tested Compounds
All(2360)
 
 
Active(356)
 
 
Inactive(2004)
 
 
 Tested Substances
 Tested Substances
All(2362)
 
 
Active(356)
 
 
Inactive(2006)
 
 
 Related BioAssays
 Related BioAssays
AID: 624300
Data Source: Southern Research Specialized Biocontainment Screening Center (MgcRacGap_HeLaTox01)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-06-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 356
Related Experiments
AIDNameTypeComment
624334of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP - SummarySummarydepositor-specified cross reference
624351Discovery of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP - Counter Screen Coupled EnzymeConfirmatorydepositor-specified cross reference
652142Biochemical MgcRacGAP assay detecting GTPase activation of the target GTPase Rac1 through the measurement of inorganic phosphate.Otherdepositor-specified cross reference
652148Biochemical RhoGAP selectivity assay - p50RhoGAPConfirmatorydepositor-specified cross reference
652149Biochemical RhoGAP selectivity assay - BCR GAPConfirmatorydepositor-specified cross reference
652153Biochemical RhoGAP selectivity assay - MgcRacGAP inhibitionConfirmatorydepositor-specified cross reference
624330Discovery of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP - Primary and Confirmatory ScreensConfirmatorysame project related to Summary assay
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Krister Wennerberg, FIMM & Southern Research Institute
Award: 1 R03 MH096578-01

The Ras superfamily of small G-protein comprises more than 150 proteins (Wennerberg et al., 2005) and in their function as molecular switches they regulate steps in almost all aspects of cellular signaling, including both physiological and pathological processes. The discovery that the Ras proteins are frequently mutated in human cancers and that the oncogenic mutations are causing increased level of GTP loading, the hope of them as druggable proteins have existed. Their binding to a small molecule, a guanine nucleotide, raises the thought that they would be good target for small molecule inhibition. However, the Ras superfamily G-proteins generally have very high affinities for their nucleotide compared to druggable ATP hydrolyzing proteins. However, no specific GTP-competitive Ras inhibitors have ever been described.
Together, it makes this class of proteins a potentially exciting target for modulation by chemical probes and from a drug discovery perspective. Beyond these MLP probes, there are very few small G-protein signaling modulating small molecules available to the scientific community. Therefore developing novel, specific and well-characterized small G-protein inhibitors would be of great innovation value and they could serve as important biological tools.
Protocol
Cell Culture: HeLa cells obtained from ATCC (CCL-2) were cultured and maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS (Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen 10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged 1:4 every 3-4 days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media. Cells were passaged no more than ten times after being thawed.

Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 5X 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 9000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 24h 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: Thirty two control wells containing untreated were included on each assay plate and used to normalize the data on a per plate basis. The normalized % cell viability was plotted against the tested compound concentrations and CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0 for active compounds (those showing a decrease in cell viability less than 70% of the median cell control value).
Comment
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.

Outcome: Compounds that showed cell viability decrease below 70% for at least one concentration were defined as Active (showing toxicity). If viability remained >70% at all doses, the compound was defined as Inactive.

Score: Because of the inherent error in all high throughput screens including the fallacy of over-interpreting single dose data, the following tiered scoring system has been implemented at SRSBSC. Active compounds in the confirmatory screen are scored based on CC50 results on a tier of 40-80 with compounds failing confirmation scoring 0. Synthesized/Analog active compounds are also scored based on CC50 results and fall into the most reliable tier where actives will be scored from 80-100. Inactive compounds show a score of 0.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: viability reporter:atp content
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: BSL2
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay stage: Secondary:counterscreening
BAO: detection technology: luminescence: chemiluminescence
BAO: format detail: reagent: inducer: none
BAO: meta target detail: binding reporter specification: interaction: protein:small molecule
BAO: meta target: biological process target: cell death
BAO: meta target: molecular target: protein target: enzyme: transferase: kinase
BAO: version: 1.4b1090
Screening Concentration Range Max: 100
Screening Concentration Range Min: 0.2
From PubChem:
Assay Type: Toxicity
Assay Cell Type: HeLa
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50 *FloatμM
3CC50 Std Dev ModifierString
4CC50 Std DevFloat
5CC50 Hill SlopeFloat
6CC50 Normaized Chi2Float
7% Cell Viability @ 100 uM (100μM**)Float%
8% Cell Viability @ 50 uM (50μM**)Float%
9% Cell Viability @ 25 uM (25μM**)Float%
10% Cell Viability @ 12.5 uM (12.5μM**)Float%
11% Cell Viability @ 6.25 uM (6.25μM**)Float%
12% Cell Viability @ 3.13 uM (3.13μM**)Float%
13% Cell Viability @ 1.56 uM (1.56μM**)Float%
14% Cell Viability @ 0.78 uM (0.78μM**)Float%
15% Cell Viability @ 0.39 uM (0.39μM**)Float%
16% Cell Viability @ 0.2 uM (0.2μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH096578-01

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