Discovery of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP - Primary and Confirmatory Screens
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: 2057
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
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.
The assay buffer was composed of 15 mM HEPES (pH 7.4), 20 mM NaCl, 1 mM EGTA, 0.1 mg/ml BSA, 0.150 mM GTP, 0.02% Tween-20, and 2% DMSO. The following reagents were added using a BioRaptr to a Corning 3891 black, tissue-cultured treated 1536-well plate. For the substrate mix, 0.3 mM of GTP (Sigma) was added to each well in 1.25 uL of assay buffer. For the enzyme mix, 1.2 uM of GST- Rac1F28L and 8 nM of MgcRacGAP was added in 1.25 uL of assay buffer. The background was determined using assay buffer alone in control wells. The plates are incubated for 1 hour at room temperature to allow for GAP-induced GTP hydrolysis. The ADP hunter kit was equilibrated to room temperature and thoroughly mixed. Addition of the ADP Hunter reagents and incubation occurred with minimal light to prevent the signal from decreasing. 1.25 uL of ADP Hunter Plus solution A was added to every well in the plate and the plates were allowed to incubate briefly before the addition of 2.5 uL of ADP Hunter Plus solution B. After a 45 minute incubation with the end point detection, the plates were read on an Envision using a bottom read fluorescence 530/590 detection.
Data Analysis: Data was analyzed using ActivityBase software (IDBS, Inc, Guilford, UK). The mean Z prime for the campaign was >/= 0.8. Results are reported as percent (%) inhibition and were calculated using the following formula: % inhibition = 100*(Test Cmpd - Med Pos Ctrl)/(Med Neg Ctrl - Med Pos Ctrl).
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Using the criteria of 3 standard deviations greater than the mean, a hit cutoff of 31.89% Inhibition was used to define activity in the primary screen. Of the compounds available for testing in the confirmatory screen, those that showed at least 30% inhibition at any tested dose were considered active. Compounds that exceeded the activity cutoff in the primary screen, but were available for confirmatory testing were designated with the outcome of 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 inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the IC50 result while compounds that did not confirm as actives were given the score of 0.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: confirmatory
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: assay design: enzyme reporter: coupled enzyme: protease coupled enzyme
BAO: detection technology: fluorescence: fluorescence intensity
* Activity Concentration. ** Test Concentration.
Data Table (Concise)