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. 2014 Mar 27;57(6):2455-61.
doi: 10.1021/jm401730y. Epub 2014 Feb 19.

Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A

Andreas O Frank  1 , Bhavatarini Vangamudi, Michael D Feldkamp, Elaine M Souza-Fagundes, Jessica W Luzwick, David Cortez, Edward T Olejniczak, Alex G Waterson, Olivia W Rossanese, Walter J Chazin, Stephen W Fesik
Affiliations

Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A

Andreas O Frank et al. J Med Chem. .

Abstract

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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Figures

Figure 1
Figure 1
X-ray structures of fragments and peptides in complex with RPA70N reveal a preferred binding site within the basic cleft. (A) Overlay of cocrystal structures of fragments bound to RPA70N. (B) Superpositioned structures of RPA70N bound to fragments and the p53 peptide (red). (C) The dichlorophenyl ring of an elaborated compound (yellow) binds in the same hydrophobic pocket as F54 of the p53 peptide (red). (D) Superposition of RPA70N/p53 peptide (red) and RPA70N/peptide-33 (blue).
Figure 2
Figure 2
Peptide-39 is selective for binding to RPA70N. Peptide-37 and peptide-39 were incubated with increasing concentrations of the indicated RPA70 constructs. Because of the different sizes of the protein constructs used, data are normalized to the maximal anisotropy for each construct.
Figure 3
Figure 3
Peptide-39 does not displace RPA from ssDNA. (A) RPA and ssDNA were incubated with increasing concentrations of peptide-39 prior to separation on a polyacrylamide gel. (B) Quantification of three replicates.
Figure 4
Figure 4
FITC-labeled stapled helix peptides are visible within U2OS cells.
See this image and copyright information in PMC

References

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