title: Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain.
abstract: The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.
authors.affiliation: Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 5C02, Bethesda, Maryland, 20892, USA.
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Long
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Yao
authors.forename: J H
authors.initials: JH
authors.lastname: Voigt
authors.forename: F D
authors.initials: FD
authors.lastname: Lung
authors.forename: J H
authors.initials: JH
authors.lastname: Luo
authors.forename: T R
authors.initials: TR
authors.lastname: Burke
authors.forename: C R
authors.initials: CR
authors.lastname: King
authors.forename: D
authors.initials: D
authors.lastname: Yang
authors.forename: P P
authors.initials: PP
authors.lastname: Roller
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D051380:GRB2 Adaptor Protein; D006801:Humans; D008024:Ligands; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D008024:Ligands; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D014443:Tyrosine; C495948:GRB2 protein, human