Discovery of a Potent Stapled Helix Peptide That Binds to the 70N Domain of Replication Protein A

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.

Stapled peptide design: principles and roles of computation

“…The most important rule-of-thumb in the placement of staples is not to replace critical interacting residues, so that the entropic penalty for binding is decreased without compromising the enthalpy of binding. This core principle has guided the design of the majority of stapled peptides reported in the literature, such as those targeting BCL-2 [43], HIV integrase [27], and Rep Protein A [53]. In the last case, comprehensive alanine scanning [54] was carried out to identify important binding residues.…”

“…Negative charges can be neutralised by mutating Asp and Glu to their respective uncharged analogues, Asn and Gln [39]. Lastly, negatively charged residues can be replaced by a strategically placed hydrocarbon staple [53]. However, charge alteration should be practised with caution because peptides with high positive charges tend to cause cell membrane disruption, thus reducing target specificity [72].…”

Macrocyclic α-Helical Peptide Drug Discovery

“…[19][20][21][22][23][24][27][28][29] and 62 for reviews) for the modulation a plethora of intracellular targets (i.e. Bcl2-family, 19,[22][23][24]43,47,48,58,[67][68][69][70][71]76,77,83,92,93,98 p53:MDM2/X, 42,57,65,66,72,79,81,82,87,89,94,95 MAML:Notch, 49 eIF4E, 158 ERa/ERb, 50 IRS1, 84 HIF-1:p300, 56,90 RAS:SOS, 59 Rab-GTPase, 96 b-catenin, 64,78,80 protein kinase-A, 91 RPA, 97 HIV-1 integrase, …”

Tightening up the structure, lighting up the pathway: application of molecular constraints and light to manipulate protein folding, self-assembly and function

“…Therefore, various sidechain-to-sidechain cross-linking strategies have been put forth to reduce the conformational entropy of the unfolded state, thus forcing short peptides to fold into α-helices or β-sheets (Figure 1). Such examples include disulfide bond formation [4-10], ring-closing metathesis [11-14], lactam bridge formation [15-19], hydrocarbon bridges [20-23], and hydrazone [24] and oxime linkages [25-27]. In particular, due to the ease of incorporation and natural abundance of cysteines in biological systems, cysteine alkylation [28-30] has become a popular method for incorporating cross-linkers that stabilize α-helical conformations [31, 32].…”