Replication Protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR 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 homogenous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of RPA70N. A 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 performed with a Z’ factor of ≥0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.
Stapled helix peptides can serve
as useful tools for inhibiting
protein–protein interactions but can be difficult to optimize
for affinity. Here we describe the discovery and optimization of a
stapled helix peptide that binds to the N-terminal domain of the 70
kDa subunit of replication protein A (RPA70N). In addition to applying
traditional optimization strategies, we employed a novel approach
for efficiently designing peptides containing unnatural amino acids.
We discovered hot spots in the target protein using a fragment-based
screen, identified the amino acid that binds to the hot spot, and
selected an unnatural amino acid to incorporate, based on the structure–activity
relationships of small molecules that bind to this site. The resulting
stapled helix peptide potently and selectively binds to RPA70N, does
not disrupt ssDNA binding, and penetrates cells. This peptide may
serve as a probe to explore the therapeutic potential of RPA70N inhibition
in cancer.
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