4.5 Article

A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A

Journal

ANALYTICAL BIOCHEMISTRY
Volume 421, Issue 2, Pages 742-749

Publisher

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.11.025

Keywords

Cancer; Checkpoint pathways; RPA70N; ATRIP; Fluorescence polarization anisotropy assay; High-throughput screening

Funding

  1. U.S. National Institutes of Health (NIH) [5DP10D006933, 5RC2CA148375, R01GM065484, PO1CA092584, 5P0CA098131, 5T32ES7028-37]
  2. National Council for Scientific and Technological Development-CNPq
  3. Federal University of Minas Gerais/Brazil
  4. German Academic Exchange Service (DAAD)
  5. Cancer Research UK [11566] Funding Source: researchfish

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Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the AIR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with Z' factor of >= 0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface. (C) 2011 Elsevier Inc. All rights reserved.

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