A Homogeneous Platform to Discover Inhibitors of the GoLoco Motif/G-alpha Interaction
Pp. 187-218 (32)
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
The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found
in proteins that regulate cell-surface 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 (Zfactor)
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 fully-automated
run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library
compound being tested over a range of concentrations following the quantitative highthroughput
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.
Fluorescence anisotropy, fluorescence polarization, GoLoco motif,
heterotrimeric G-proteins, high-throughput screening.
NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892-3370 USA.