title: 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.
authors: †Department of Chemistry, Virginia Commonwealth University (VCU), 1001 West Main Street, P.O. Box 842006, Richmond, Virginia 23284, United States.
authors: E Railey
authors: ER
authors: White
authors: ⊥Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: Luxin
authors: L
authors: Sun
authors: Zhong
authors: Z
authors: Ma
authors: Jason M
authors: JM
authors: Beckta
authors: †Department of Chemistry, Virginia Commonwealth University (VCU), 1001 West Main Street, P.O. Box 842006, Richmond, Virginia 23284, United States.
authors: Brittany A
authors: BA
authors: Danzig
authors: †Department of Chemistry, Virginia Commonwealth University (VCU), 1001 West Main Street, P.O. Box 842006, Richmond, Virginia 23284, United States.
authors: David E
authors: DE
authors: Hacker
authors: Melissa
authors: M
authors: Huie
authors: David C
authors: DC
authors: Williams
authors: ⊥Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: Ross A
authors: RA
authors: Edwards
authors: Kristoffer
authors: K
authors: Valerie
authors: ⊥Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors: J N Mark
authors: JN
authors: Glover
authors: †Department of Chemistry, Virginia Commonwealth University (VCU), 1001 West Main Street, P.O. Box 842006, Richmond, Virginia 23284, United States.
authors: Matthew C T
authors: MC
authors: Hartman
mesh_terms: D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004249:DNA Damage; D006801:Humans; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D019151:Peptide Library; D012694:Serine
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