TY - JOUR
T1 - A High-Throughput Time-Resolved Fluorescence Energy Transfer Assay to Screen for Modulators of RGS7/Gβ5/R7BP Complex
AU - Muntean, Brian S.
AU - Patil, Dipak N.
AU - Madoux, Franck
AU - Fossetta, James
AU - Scampavia, Louis
AU - Spicer, Timothy P.
AU - Martemyanov, Kirill A.
N1 - Funding Information:
B.S.M. designed experiments, performed the experiments, organized the data, and wrote the article. D.N.P. performed purification of 6xHis-RGS7/Gb5 and GST-R7BP, and untagged-R7BP. F.M. designed experiments and organized data. J.F. designed experiments and organized data. L.S. designed experiments and organized data. T.P.S. designed experiments and organized data. K.A.M. designed the study, analyzed data, and wrote the article. We would like to thank Smitha Kota and Joseph Kissil (The Scripps Research Institute) for providing the MicroSource Spectrum compound library. This work was supported by National Institutes of Health Grants DA041207 (to B.S.M.), DA042746 (to K.A.M. and L.S.), and DA026405 (to K.A.M.).
Publisher Copyright:
© 2018, Mary Ann Liebert, Inc. 2018.
PY - 2018/4
Y1 - 2018/4
N2 - G protein-coupled receptors (GPCRs) are excellent drug targets exploited by majority of the Food and Drug Administration-approved medications, but when modulated, are often accompanied by significant adverse effects. Targeting of other elements in GPCR pathways for improved safety and efficacy is thus an unmet need. The strength of GPCR signaling is tightly regulated by regulators of G protein signaling (RGS) proteins, making them attractive drug targets. We focused on a prominent RGS complex in the brain consisting of RGS7 and its binding partners Gβ5 and R7BP. These complexes play critical roles in regulating multiple GPCRs and essential physiological processes, yet no small molecule modulators are currently available to modify its function. In this study, we report a novel high-throughput approach to screen for small molecule modulators of the intramolecular transitions in the RGS7/Gβ5/R7BP complex known to be involved in its allosteric regulation. We developed a time-resolved fluorescence energy transfer-based in vitro assay that utilizes full-length recombinant proteins and shows consistency, excellent assay statistics, and high level of sensitivity. We demonstrated the potential of this approach by screening two compound libraries (LOPAC 1280 and MicroSource Spectrum). This study confirms the feasibility of the chosen strategy for identifying small molecule modulators of RGS7/Gβ5/R7BP complex for impacting signaling downstream of the GPCRs.
AB - G protein-coupled receptors (GPCRs) are excellent drug targets exploited by majority of the Food and Drug Administration-approved medications, but when modulated, are often accompanied by significant adverse effects. Targeting of other elements in GPCR pathways for improved safety and efficacy is thus an unmet need. The strength of GPCR signaling is tightly regulated by regulators of G protein signaling (RGS) proteins, making them attractive drug targets. We focused on a prominent RGS complex in the brain consisting of RGS7 and its binding partners Gβ5 and R7BP. These complexes play critical roles in regulating multiple GPCRs and essential physiological processes, yet no small molecule modulators are currently available to modify its function. In this study, we report a novel high-throughput approach to screen for small molecule modulators of the intramolecular transitions in the RGS7/Gβ5/R7BP complex known to be involved in its allosteric regulation. We developed a time-resolved fluorescence energy transfer-based in vitro assay that utilizes full-length recombinant proteins and shows consistency, excellent assay statistics, and high level of sensitivity. We demonstrated the potential of this approach by screening two compound libraries (LOPAC 1280 and MicroSource Spectrum). This study confirms the feasibility of the chosen strategy for identifying small molecule modulators of RGS7/Gβ5/R7BP complex for impacting signaling downstream of the GPCRs.
KW - R7 binding protein
KW - allosteric modulators
KW - high-throughput screen
KW - regulator of protein signaling 7
KW - time-resolved FRET
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U2 - 10.1089/adt.2017.839
DO - 10.1089/adt.2017.839
M3 - Article
C2 - 29658790
AN - SCOPUS:85045553798
SN - 1540-658X
VL - 16
SP - 150
EP - 161
JO - Assay and Drug Development Technologies
JF - Assay and Drug Development Technologies
IS - 3
ER -