Structure-based design of an osteoclast-selective, nonpeptide Src homology2 inhibitor with in vivo antiresorptive activity

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosin e kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that d emonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citr ate [in the phosphotyrosine (pTyr) binding pocket], we designed 3',4'-dipho sphonophenytalanine (Dpp) as a pTyr mimic. In addition to its design to bin d Src SH2, the Dpp moiety exhibits bone-targeting properties that confer os teoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 al so illustrates a bicyclic template to replace the post-pTyr sequence of cog nate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Clu-lle (I). An x-ray stru cture of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interac tions of both the Dpp and bicyclic template of AP22408 as predicted from mo lecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC50 = 0.30 mu M for AP22 408 and 5.5 mu M for 1). Furthermore, AP22408 inhibits rabbit osteoclast-me diated resorption of dentine in a cellular assay, exhibits bone-targeting p roperties based on a hydroxyapatite adsorption assay, and demonstrates in v ivo antiresorptive activity in a parathyroid hormone-induced rat model.