Growth factor receptor-bound protein 2 (Grb2) links tyrosine-phosphorylated proteins to a guanine nucleotide releasing factor of the son of sevenless (Sos) class by attaching to the former by its Src homology 2 (SH2) moiety and to the latter by its SH3 domains. An isoform of grb2 complementary DNA (cDNA) was cloned that has a deletion in the SH2 domain. The protein encoded by this cDNA, Grb3-3, did not bind to phosphorylated epidermal growth factor receptor (EGFR) but retained functional SH3 domains and inhibited EGF-induced transactivation of a Ras-responsive element. The messenger RNA encoding Grb3-3 was expressed in high amounts in the thymus of rats at an age when massive negative selection of thymocytes occurs. Microinjection of Grb3-3 into Swiss 3T3 fibroblasts induced apoptosis. These findings indicate that Grb3-3, by acting as a dominant negative protein over Grb2 and by suppressing proliferative signals, may trigger active programmed cell death.
The synthesis and structure-activity relationships of a series of [(hydroxybenzylidene)amino]salicylates and a series of [(hydroxybenzyl)amino]salicylates as inhibitors of EGF receptor-associated tyrosine kinase activity are described. Their inhibitory potency was evaluated in vitro using ER 22 cell membranes (CCL 39 cells transfected with EGF receptor) as an enzyme source and the tridecapeptide RRSrc (RRLIEDAEYAARG) as substrate. Their cellular activity was measured by inhibition of the EGF-stimulated DNA synthesis of ER 22 cells. Chemical modifications were made to analyze the role of the different substituents. The amino series was found to be more active than the imino series. The hydroquinone moiety appears to be essential for tyrosine kinase inhibitory activity in the series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Comparison of the imino and amino series by molecular modeling techniques provides further evidence in support of the hypothesis that the important reduced linking chain, CH2NH, allows the correct positioning of the 2,5-dihydroxybenzyl ring, possibly in a cis-like conformational arrangement.
Potent inhibitors of EGF-dependent protein tyrosine kinase (PTK) activity were synthesized in a series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Several of these compounds inhibited EGF-dependent DNA synthesis in ER 22 cells with IC50 < 1 microM. In this series of PTK inhibitors, the role of the salicylate moiety as a potential divalent ion chelator was tested and found to be nonessential in all cases. The length and ramification of the substituting carboxyl group were investigated to improve cellular bioavailability, and this analysis provided compounds with increased inhibitory effect on EGF-induced DNA synthesis. Salicylates esterified with long hydrophobic chains were shown to be noncompetitive inhibitors of ATP, in contrast to the free acid and methyl salicylate. Moreover, all the tested inhibitors were shown to be noncompetitive inhibitors of the peptide substrate. Structure-activity relationships allowed us to suspect a hydrophobic pocket in the tyrosine kinase domain, preferentially interacting with aromatic rings. Finally, the selectivity of the best inhibitors was tested against other kinases, and they were found to be selective for tyrosine kinase. They were also shown to be good inhibitors of EGF-receptor autophosphorylation.
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