Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain

Abstract

Many intracellular protein–protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein–protein interactions mediated by serine phosphorylation.

Conformational Restriction of Peptides Using Dithiol Bis-Alkylation

“…Thiol bis-alkylation has also been applied to mRNA display libraries (Schlippe, Hartman, Josephson, & Szostak, 2012). In one such selection, all the selected peptides were cyclic, even those containing a single cysteine; the mxy linker unexpectedly formed a small ring with the N-terminal methionine (White et al, 2015). The rapid cross-linking of cysteine and methionine using dibromoxylene linkers is an observation that we have confirmed in our lab (see later).…”

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display

“…The dibromoxylene cyclization chemistry has already been shown to be compatible with mRNA display. 56, 57, 58 However, others have shown that CuAAC reagents degrade nucleic acids over time and that RNA is particularly susceptible to oxidation. 59, 60 The use of polytriazole ligands, such as tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) or the water-soluble tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA), have been shown to protect Cu(I) from oxidation or disproportionation.…”

“…56, 57 We designed five libraries, each containing two cysteine codons (one fixed at the 3′ end of the random region and one that was varied), and one Phe codon that was varied in position between libraries (Figure 4a–c and Supporting Figure S6). The initiator Met codon fixed the position of the azide to the N-terminus.…”

Structure‐Guided Synthesis and Evaluation of Small‐Molecule Inhibitors Targeting Protein–Protein Interactions of BRCA1 tBRCT Domain

“…Natarajan and co‐workers later identified a difluorophosphonate‐containing compound with K d 0.71 μ m. White et al. developed a non‐phosphorylated peptide inhibitor Peptide 8.6 ( K d 3.6 μ m ) with glutamic acid as a pSer mimic, using mRNA display technology. The peptidic nature and/or charge of these inhibitors limits their further development for use in chemical biology or therapy.…”