A novel inhibitor of the tyrosine kinase Src suppresses phosphorylation ofits major cellular substrates and reduces bone resorption in vitro and in rodent models in vivo

The tyrosine kinase Src has been implicated in the process of osteoclast-me diated bone resorption, Here, we describe a novel class of Src inhibitors, substituted 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines, and characterize one of them, CGP77675, in vitro and in models of bone resorption in vivo. In vitr o, CGP77675 inhibited phosphorylation of peptide substrates and autophospho rylation of purified Src (concentration producing half-maximal inhibition [ IC50] values 5-20 and 40 nmol/L, respectively), The compound was selective toward other protein kinases: the Src IC50 value was lower than those for C dc2 (>500-fold), epidermal growth factor (EGF) receptor (7.5-fold), and vas cular endothelial growth factor receptor (>50-fold), and for v-Abl (15-fold ) and focal adhesion kinase (Fak) (>25-fold), The Src kinase family members Lck and Yes were inhibited with IC50 values 20-fold higher than or equal t o Src, To measure the inhibition of cellular Src activity, we identified th e major tyrosine-phosphorylated proteins in an Src-overexpressing cell line IC8.1 as Src, Fak, and paxillin, CGP77675 potently inhibited tyrosine phos phorylation of the Src substrates Fak and paxillin, but had much less effec t on Src (IC50 values 0.3, 0.5, and 5.7 mu mol/L). The phosphorylation of S rc in IC8.1 cells reflected phosphorylation of the negative regulatory tyro sine 527 (Y527); thus, the inhibitor was selective against the Y527 C-termi nal Src kinase Csk, In osteoblastic :MC3T3-E1 cells, CGP77675 inhibited sig naling induced by :PDGF at the receptor level, but not signaling by EGF, ba sic fibroblast growth factor, insulin-like growth factor-1, and phorbol 12- myristate 13-acetate, The effect of CGP77675 on bone resorption was evaluat ed in vitro and in vivo. The parathyroid hormone-induced bone resorption in rat fetal long bone cultures was inhibited with an IC50 of 0.8 mu mol/L. C GP77675 dose-dependently reduced the hypercalcemia induced in mice by inter leukin-1 beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone w as exerted via the inhibition of bone resorption. Thus, specific Src family kinase inhibitors may be useful for the treatment of diseases associated w ith elevated bone loss. (C) 1999 by Elsevier Science Inc, All rights reserv ed.