5 research outputs found
Discovery of Protein–Protein Interaction Inhibitors of Replication Protein A
Replication
protein A (RPA) is a ssDNA binding protein that is
essential for DNA replication and repair. The initiation of the DNA
damage response by RPA is mediated by protein–protein interactions
involving the N-terminal domain of the 70 kDa subunit with partner
proteins. Inhibition of these interactions increases sensitivity toward
DNA damage and replication stress and may therefore be a potential
strategy for cancer drug discovery. Toward this end, we have discovered
two lead series of compounds, derived from hits obtained from a fragment-based
screen, that bind to RPA70N with low micromolar affinity and inhibit
the binding of an ATRIP-derived peptide to RPA. These compounds may
offer a promising starting point for the discovery of clinically useful
RPA inhibitors
Discovery of a Potent Inhibitor of Replication Protein A Protein–Protein Interactions Using a Fragment-Linking Approach
Replication protein A (RPA), the
major eukaryotic single-stranded
DNA (ssDNA)-binding protein, is involved in nearly all cellular DNA
transactions. The RPA N-terminal domain (RPA70N) is a recruitment
site for proteins involved in DNA-damage response and repair. Selective
inhibition of these protein–protein interactions has the potential
to inhibit the DNA-damage response and to sensitize cancer cells to
DNA-damaging agents without affecting other functions of RPA. To discover
a potent, selective inhibitor of the RPA70N protein–protein
interactions to test this hypothesis, we used NMR spectroscopy to
identify fragment hits that bind to two adjacent sites in the basic
cleft of RPA70N. High-resolution X-ray crystal structures of RPA70N–ligand
complexes revealed how these fragments bind to RPA and guided the
design of linked compounds that simultaneously occupy both sites.
We have synthesized linked molecules that bind to RPA70N with submicromolar
affinity and minimal disruption of RPA’s interaction with ssDNA
Discovery of a Potent Stapled Helix Peptide That Binds to the 70N Domain of Replication Protein A
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
Diphenylpyrazoles as Replication Protein A Inhibitors
Replication Protein A is the primary
eukaryotic ssDNA binding protein
that has a central role in initiating the cellular response to DNA
damage. RPA recruits multiple proteins to sites of DNA damage via
the N-terminal domain of the 70 kDa subunit (RPA70N). Here we describe
the optimization of a diphenylpyrazole carboxylic acid series of inhibitors
of these RPA–protein interactions. We evaluated substituents
on the aromatic rings as well as the type and geometry of the linkers
used to combine fragments, ultimately leading to submicromolar inhibitors
of RPA70N protein–protein interactions
MOESM1 of Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery
Additional file 1. Six supplementary figures and three supplementary tables