Fig. 6. Chemical structure of prototype azaindole inhibitor 1 and cocrystal of 1 in complex with the T315I mutant of the Abl kinase domain. (A) Chemical structure azaindole inhibitor 1. (B) Cocrystal structure of azaindole inhibitor 1 displayed in stick format (yellow: carbon, blue: nitrogen, red: oxygen) bound to the T315I mutant of the Abl kinase domain (gray: carbon, cyan: nitrogen, pink: oxygen, yellow: sulfur), shown with a semitransparent molecular surface on the enzyme. A portion of the outer surface of the enzyme has been cut away to reveal the interior of the active site. The crystallographic R and Rf values are 21.6% and 24.2%, respectively, at 2.0Å resolution and with Mn(I)/sd = 1.5. Dashed yellow lines represent hydrogen bond interactions. (C) Cocrystal structure of azaindole inhibitor 1 (white, carbon; blue, nitrogen; red, oxygen) bound to the T315I mutant of the Abl kinase domain in an active conformation, shown as a ribbon diagram colored by sequence, beginning with blue at the amino terminus and progressing through the color spectrum to red at the carboxyl terminus. Crystallographic R and R_f values are 21.6% and 24.2%, respectively, at 2.0 Å resolution and with Mn(I)/sd = 1.5. (D) Cocrystal structure of dasatinib bound to the active conformation of native Abl kinase domain (PDB 2GQG), shown as a ribbon diagram colored by sequence (1). The color-coding is the same as in C.

Fig. 7. Comparison of effects of SGX393 and other Abl kinase inhibitors on enzymatic activity of GST-Abl and GST-Abl^T315I fusion proteins. (Left) Inhibition of in vitro autophosphorylation of purified GST-Abl and GST-Abl^T315I by imatinib, nilotinib, dasatinib, and SGX393. (Right) IC₅₀ values (nM) for inhibition of GST-Abl and GST-Abl^T315I autophosphorylation and peptide substrate phosphorylation by imatinib, nilotinib, dasatinib, and SGX393. For autophosphorylation experiments, GST-Abl and GST-Abl^T315I were purified, dephosphorylated, and incubated with the indicated inhibitors in the presence of [g-³²P]-ATP. GST-Abl and [g-³²P]-ATP were separated by gel electrophoresis and autophosphorylation was measured by autoradiography. Peptide substrate phosphorylation experiments were similar except that a biotinylated Abl-specific peptide substrate was included. At the end of the incubation period, the enzyme was quenched and the reaction mixture was transferred onto streptavidin-coated membranes. The membranes were washed, and scintillation counting was done to quantitate the level of phosphorylation of biotinylated substrate.

Fig. 8. Effects of SGX393 compared to other Abl kinase inhibitors on proliferation of Ba/F3 cells expressing native or kinase domain-mutant Bcr-Abl. (A) Ba/F3 cells expressing native Bcr-Abl (Upper) or Bcr-Abl^T315I (Lower) were incubated in the presence of graded concentrations of imatinib (green), nilotinib (blue), dasatinib (red), or SGX393 (orange). Cells were subjected to MTS assays to assess the extent of inhibition of cell proliferation. (B) Comparison of fold-change in IC₅₀ over native Bcr-Abl for imatinib, nilotinib, dasatinib, and SGX393. Each bar represents the fold-change in IC₅₀ value for inhibition of cellular proliferation of Ba/F3 cell lines expressing native Bcr-Abl, Bcr-Abl^Y253F, Bcr-Abl^Y253H, Bcr-Abl^E255K, Bcr-Abl^E255V, Bcr-Abl^T315I, or Bcr-Abl^F317L relative to the corresponding IC₅₀ value for native Bcr-Abl. The IC₅₀ value (nM) is displayed on the x axis. Broken bars indicate a fold change value >100. Data for imatinib, nilotinib, and dasatinib from ref. 2.

Fig. 9. Efficacy of SGX393 in murine bone marrow colony-forming assays. Murine bone marrow mononuclear cells were transduced with retrovirus expressing p210 native Bcr-Abl, Bcr-Abl ^Y253H, Bcr-Abl ^E255K, Bcr-Abl ^T315I, Bcr-Abl ^M351T, or Bcr-Abl ^H396P. Transduced cells were plated in methylcellulose without cytokines in the presence of imatinib, dasatinib, nilotinib, or SGX393 at the indicated doses or in the absence of drug (NT). Colonies were counted 7 days after plating and all results are expressed as percentage of NT colony number. Values represent mean ±SEM.

Fig. 10. Efficacy of SGX393 against native Bcr-Abl in xenograft models. (A) Ba/F3 cells expressing native Bcr-Abl were s.c. implanted in nude mice at 2 ´ 10⁶ cells per mouse. Tumor volume was allowed to reach 100 mm³, and treatment by IP injection was initiated with vehicle (black; 50% PEG400, 50% saline), imatinib (green), or SGX393 (orange) at 50 mg/kg every other day for 12-13 days. At the conclusion of the study, plasma levels of SGX393 were measured and CrkL phosphorylation in tumor tissue was determined by immunoblot analysis. (B) K562 cells were used in a parallel xenograft experiment: vehicle (black; 50% PEG400, 50% saline) or SGX393 (orange).

Fig. 11. Inhibition of cellular tyrosine phosphorylation by SGX393 in primary Bcr-Abl^T315I cells. (A) (Top) Bcr-Abl^T315I CML lymphoid blast crisis (CML L-BC) mononuclear cells incubated in the presence of SGX393, imatinib, dasatinib, or nilotinib overnight and subjected to immunoblot analysis for total CrkL. (Middle) The relative proportion of phosphorylated CrkL to non-phosphorylated CrkL was determined by densitometry. (Bottom) In a parallel experiment, cells were stained with anti-phosphotyrosine-FITC antibody and analyzed by FACS for total cellular phosphorylation. Results are shown as mean fluorescence intensity relative to unstained controls ± SEM. (B) Results for Bcr-Abl^T315I mononuclear cells from a patient with Ph-positive B-ALL (Ph+ ALL).

Fig. 12. SGX393 does not exhibit toxicity toward normal bone marrow progenitors. Normal bone marrow mononuclear cells from three individuals were cultured in duplicate in the presence of SGX393 (0, 500, 1000, 2000, and 5000 nM) in media optimized for assessing either granulocyte/macrophage (CFU-GM) colony formation (green bars) or erythroid (BFU-E) colony formation (purple bars). Results are displayed as the percentage of colony formation compared with untreated control; error bars represent the SEM.

Fig. 13. Simultaneous exposure of Ba/F3 cells expressing Bcr-Abl to SGX393 in dual combination with imatinib, dasatinib, or nilotinib results in additive to synergistic cytotoxicity. Viability of Ba/F3 cells expressing Bcr-Abl was measured after a 3-day incubation in escalating concentrations of SGX393 alone (0-70 nM), imatinib alone (0-1000 nM), dasatinib alone (0-5 nM), nilotinib alone (0-40 nM), or dual combinations of SGX393 (0-70 nM) with imatinib (0-1000 nM), dasatinib (0-5 nM), or nilotinib (0-40 nM). As described in ref. 3, this range of concentrations is suitable for evaluating the combination index (CI), using CalcuSyn dose effect analysis software (Biosoft). Synergy level classifications were adapted from the CalcuSyn manual. Additivity corresponds to C.I. = 1; values of >1, antagonism; values of >3, strong antagonism; values of <1, synergy; values of <0.3, strong synergy. ED is the effective dose for each combination; for example, ED₅₀ corresponds to a combination that results in 50% inhibition of proliferation. Two independent experiments were conducted. Results from a representative experiment are displayed.

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