TY - JOUR
T1 - A simple fluorescent assay for the discovery of protein-protein interaction inhibitors
AU - Al-Mugotir, Mona
AU - Kolar, Carol
AU - Vance, Krysten
AU - Kelly, David L.
AU - Natarajan, Amarnath
AU - Borgstahl, Gloria E.O.
N1 - Funding Information:
We thank William Lutz, Jacob Remsza, Admir Kellezi, Jeff Lovelace and Amy Wells for technical assistance. We also thank UNMC Fred & Pamela Cancer Center Molecular Biology/HTS Core Facility. The research was supported by the Fred & Pamela Buffett Cancer Center Support Grant ( P30CA036727 ) pilot project funding, the Nebraska Research Initiative and the Nebraska Department of Health and Human Services ( 2018-11 and 2019-08 ). MAM also acknowledges the U.S. Department of Education GAANN ( P200A120231 ) and Nebraska NASA EPSCoR Space Grant for student fellowships.
Publisher Copyright:
© 2019 The Authors
PY - 2019/3/15
Y1 - 2019/3/15
N2 - Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors, called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example, and the FluorIA protocol design, optimization and successful application to HTS of large chemical libraries are described. Here enhanced green fluorescent protein (EGFP)-tagged RAD52 detected the PPI using full-length RPA heterotrimer coated, black microtiter plates and loss in fluorescence intensity identified small molecule inhibitors (SMIs) that displaced the EGFP-tagged RAD52. The FluorIA design and protocol can be adapted and applied to detect PPIs for other protein systems. This should push forward efforts to develop targeted therapeutics against protein complexes in pathological processes.
AB - Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors, called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example, and the FluorIA protocol design, optimization and successful application to HTS of large chemical libraries are described. Here enhanced green fluorescent protein (EGFP)-tagged RAD52 detected the PPI using full-length RPA heterotrimer coated, black microtiter plates and loss in fluorescence intensity identified small molecule inhibitors (SMIs) that displaced the EGFP-tagged RAD52. The FluorIA design and protocol can be adapted and applied to detect PPIs for other protein systems. This should push forward efforts to develop targeted therapeutics against protein complexes in pathological processes.
KW - Fluorescent assay
KW - High throughput screening
KW - Protein-protein interaction
KW - Small molecule inhibitor
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U2 - 10.1016/j.ab.2019.01.010
DO - 10.1016/j.ab.2019.01.010
M3 - Article
C2 - 30707898
AN - SCOPUS:85060987662
SN - 0003-2697
VL - 569
SP - 46
EP - 52
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -