Abstract
The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cellsurface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fullyautomated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
Keywords: Fluorescence anisotropy, fluorescence polarization, GoLoco motif, heterotrimeric G-proteins, high throughput screening
Combinatorial Chemistry & High Throughput Screening
Title: A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction
Volume: 11 Issue: 5
Author(s): Adam J. Kimple, Adam Yasgar, Mark Hughes, Ajit Jadhav, Francis S. Willard, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. Ibeanu, David P. Siderovski and Anton Simeonov
Affiliation:
Keywords: Fluorescence anisotropy, fluorescence polarization, GoLoco motif, heterotrimeric G-proteins, high throughput screening
Abstract: The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cellsurface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fullyautomated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
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Kimple J. Adam, Yasgar Adam, Hughes Mark, Jadhav Ajit, Willard S. Francis, Muller E. Robin, Austin P. Christopher, Inglese James, Ibeanu C. Gordon, Siderovski P. David and Simeonov Anton, A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction, Combinatorial Chemistry & High Throughput Screening 2008; 11 (5) . https://dx.doi.org/10.2174/138620708784534770
DOI https://dx.doi.org/10.2174/138620708784534770 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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