Inhibition of binding of phospholipase Cγ1 SH2 domains to phosphorylated epidermal growth factor receptor by phosphorylated peptides

A series of tyrosine-containing peptides 1–12: Asp-Ala-Asp-Glu-Tyr⁹⁹²(PO₃H₂)-Leu-Ile-Pro-Gln-Gln-Gly-OH (1) Asp-Ala-Asp-Glu-Tyr⁹⁹² -Leu-Ile-Pro-Gln-Gln-Gly-OH (2) Phe-Leu-Pro-Val-Pro-Glu-Tyr¹⁰⁶⁸(PO₃H₂)-Ile-Asn-Gln-Ser-Val-OH (3) Phe-Leu-Pro-Val-Pro-Glu-Tyr¹⁰⁶⁸ -Ile-Asn-Gln-Ser-Val-OH (4) Asp-Asn-Pro-Asp-Tyr¹¹⁴⁸JR(PO₃H₂)-Gln-Gln-Asp-Phe-Phe-OH (5) Asp-Asn-Pro-Asp-Tyr¹¹⁴⁸ -Gln-Gln-Asp-Phe-Phe-OH (6) Ala-Glu-Tyr¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (7) Ala-Glu-Tyr¹¹⁷³ -Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (8) Ala-Glu-Tyr¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-OH (9) Ala-Glu-Tyr¹¹⁷³ -Leu-Arg-Val-Ala-OH (10) Tyrl¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (11) Tyr¹¹⁷³ -Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (12) (six pairs with and without the tyrosine phosphorylated) has been synthesized. The peptides were derived from tyrosine autophosphorylation sites in the epidermal growth factor receptor (EGFR): Tyr 992, 1068, 1148 and 1173. Peptide 1, derived from the Tyr 992 site, inhibited binding of a ³⁵S-labelled fusion protein containing both of the SH2 domains from PLCγ1 to the phosphorylated EGFR with an IC₅₀ of 8 μM. All of the phosphorylated peptides except 11 (1, 3, 5, 7 and 9) inhibited this binding to some degree (20–55%) at 10 p μM. The nonphosphorylated peptides were inactive in this assay. The nonphosphorylated peptides 2, 4, 6, 8, 10 and 12 were obtained by standard solid-phase synthetic methodologies using both Boc/benzyl and Fmoc/tert-butyl strategies. The phosphorylated peptides 1, 3, 5, 7, 9 and 11 were similarly obtained using a Fmoc/tert-butyl strategy incorporating unprotected N^x-Fmoc-Tyr, followed by phosphitylation and oxidation of the tyrosine in the resin-bound peptide. In addition, Asp-Ala-Asp-Glu-Phe⁹⁹²(4-CH₂PO₃H₂)-Leu-Ile-Pro-Gln-Gln-Gly-OH (15), an analog of 1 incorporating an enzymatically stable phosphotyrosine mimic, 4-phosphonomethyl-l -phenylalanine, was synthesized and found to be inactive.