A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction.
The GoLoco motif is a short Galpha-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 Galpha (i1). 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 microL 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 LOPAC(1280) 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.
Kimple
Adam J
AJ
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA. asimeono@mail.nih.gov
Yasgar
Adam
A
Hughes
Mark
M
Jadhav
Ajit
A
Willard
Francis S
FS
Muller
Robin E
RE
Austin
Christopher P
CP
Inglese
James
J
Ibeanu
Gordon C
GC
Siderovski
David P
DP
Simeonov
Anton
A
D020816:Amino Acid Motifs; D001681:Biological Assay / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D020549:GTP Phosphohydrolase Activators / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020710:RGS Proteins / Q000378:metabolism; D013194:Staining and Labeling
D020549:GTP Phosphohydrolase Activators; D020710:RGS Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go