Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast -mediated resorption, We have investigated whether compounds that bind to t he Src SH2 domain inhibit Src activity in cells and decrease osteoclast-med iated resorption, Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in ce lls, demonstrate blockade of Src signaling, and lack cellular toxicity, Cel l-based assays included: (1) a mammalian two-hybrid assay; (2) morphologica l reversion and growth inhibition of cSrcY527F-transformed cells; and (3) i nhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assay s, The compounds described were synthesized to contain nonhydrolyzable phos photyrosine mimics that bind to bone. These compounds were further tested a nd found to inhibit rabbit osteoclast-mediated resorption of dentine, These results indicate that compounds that bind to the Src SH2 domain can inhibi t Src activity in cells and inhibit osteoclast-mediated resorption, (C) 200 1 by Elsevier Science Inc. All rights reserved.