Abstract

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.

Original languageEnglish (US)
Pages (from-to)46-52
Number of pages7
JournalAnalytical Biochemistry
Volume569
DOIs
StatePublished - Mar 15 2019

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Assays
Proteins
Fluorescence
Replication Protein A
Protein Transport
Screening
Small Molecule Libraries
Throughput
Pathologic Processes

Keywords

  • Fluorescent assay
  • High throughput screening
  • Protein-protein interaction
  • Small molecule inhibitor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A simple fluorescent assay for the discovery of protein-protein interaction inhibitors. / Al-Mugotir, Mona; Kolar, Carol; Vance, Krysten; Kelly, David L.; Natarajan, Amarnath; Borgstahl, Gloria E.O.

In: Analytical Biochemistry, Vol. 569, 15.03.2019, p. 46-52.

Research output: Contribution to journalArticle

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AU - Vance, Krysten

AU - Kelly, David L.

AU - Natarajan, Amarnath

AU - Borgstahl, Gloria E.O.

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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.

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