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 ..
BioActive Compounds: 356
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.
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).
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
Assay Type: Toxicity
Assay Cell Type: HeLa
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)