Discovery of small molecule inhibitors of the oncogenic and cytokinetic protein MgcRacGAP - Counter Screen Coupled Enzyme
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: 1364
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. In order to delineate between compounds that targeted the G-protein involved in MgcRacGAP interaction and compounds that inhibited the end-point Hunter ADP enzyme reaction, a counter screen was employed to filter out the compounds that were not target specific.
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 GDP, 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, 0.03 mM of GDP (Sigma G7127) was added to each well in 1.25 uL of assay buffer. 2.5 uL of assay buffer was added in lieu of the MgcRacGAP enzymes. The background was determined using assay buffer alone in control wells. The plates were incubated for 1 hour at room temperature. 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 the 45 minute incubation with the end point detection, the plates were read on an Envision using a bottom read fluorescence 530/590 detection.
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.
Compounds showing 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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: enzyme reporter: coupled enzyme: protease coupled enzyme
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
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: bioassay specification: assay stage: confirmatory
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: version: 1.4b1090
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)