Structure-based design of an osteoclast-selective, nonpeptide src homology 2 inhibitor with in vivo antiresorptive activity

Abstract

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine 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 demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3',4'-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC(50) = 0.30 microM for AP22408 and 5.5 microM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model.

Figure 1

Structure-based design of AP22408. (A) X-ray crystal structure of citrate complexed to the pTyr-binding pocket of Src SH2 depicting hydrogen bonding atoms. (B) Model of Dpp in the pTyr binding pocket of Src SH2, highlighting the hydrogen bonding network. (C) Model of 3 complexed with Lck SH2, illustrating the solvent accessible surface detailed in terms of both hydrophobic and hydrogen bonding interactions. Yellow colors indicate hydrophobic surfaces, blue colors indicate hydrogen bond accepting surfaces, and red colors indicate hydrogen bond donating surfaces. (D) Model of AP22408 in Lck SH2 highlighting the hydrophobic interactions of the bicyclic template.

Figure 2

Chemical structures of pTyr, citrate, and Dpp (2).

Figure 3

Chemical structures of 1, 3, AP22408, AP22409, and AP22650.

Figure 4

Chemical synthesis of amine intermediate 4 and AP22408. DMF, dimethylformamide; BH₃/THF, borane-tetrahydrofuran; Boc₂O di-tert-butyl dicarbonate; Boc, tert-butoxycarbonyl; NBS, N-bromosuccinimide; n-BuLi, n-butyllithium; THF, tetrahydrofuran; CO₂, carbon dioxide; HOBt, 1-hydroxybenzotriazole; EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; TFA, trifluoroacetic acid; Ac₂O, acetic anhydride; TMSI, iodotrimethylsilane; Me, methyl; Et, ethyl.

Figure 5

Chemical synthesis of Dpp intermediate 5. Tf, trifluoromethanesulfonyl; Boc, tert-butoxycarbonyl; NMM, 4-methylmorpholine; Me, methyl; Et, ethyl; Ph, phenyl.

Figure 6

Superposition of the model of AP22408 (white) and the x-ray (blue) crystal structure of the AP22408-Lck SH2 (S164C) complex. The water-mediated hydrogen bonds (determined crystallographically, blue) between the carboxamide of AP22408 and the protein were not predicted as no water molecules were included in our model.

Figure 7

In vivo antiresorptive activity of AP22408 in TPTX rats. Mixed model area under the curve analysis: three point moving average data (days 5–18).