L-O-(2-MALONYL)TYROSINE - A NEW PHOSPHOTYROSYL MIMETIC FOR THE PREPARATION OF SRC-HOMOLOGY-2 DOMAIN INHIBITORY PEPTIDES

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent s ignaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pT yr-containing protein ligands. Such pTyr-containing peptides are limit ed in their utility as SH2 domain inhibitors in vivo due to their hydr olytic lability to protein-tyrosine phosphatases (PTPs) and the poor c ellular penetration of the ionized phosphate moiety. An important aspe ct of SH2 domain inhibitor design is the creation of pTyr mimetics whi ch are stable to PTPs and have reasonable bioavailability. To date, mo st PTP-resistant pTyr mimetics which bind to SH2 domains are phosphona tes such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosp hono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)-methyl]-ph enylalanine (F(2)Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT , 5), into SH2 domain inhibitory peptides using the protected analogue L-N-alpha-Fmoc-O'-(O '', O ''-di-tert-butyl-2-malonyl)tyrosine (6) an d solid-phase peptide synthesis techniques. Five OMT-containing peptid es were prepared against the following SH2 domains: the PI-3 kinase C- terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 m u M), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 mu M, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 mu M), the Grb 2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 mu M), and th e N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 mu M). These results show that peptides 10, 11, 13, and 1 4 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTPB SH2 domain-directed peptide 13 being equiva lent to that previously observed for the corresponding F(2)Pmp-contain ing peptide. OMT may afford a new structural starting point for the de velopment of novel and useful SH2 domain inhibitors.