PMC

Hit values are reported as percentage bound kinase.

(A) Aligned de-wetting patterns for C-Kit kinase (green) and JNK1 (blue) restricted to the C-Kit residues in contact with imatinib. (B) De-wetting hot spots arising as backbone hydrogen-bonded residues in C-Kit kinase (backbone in light blue; de-wetting hot spots in green), aligned with JNK (blue backbone; de-wetting hot spots in yellow). The JNK residues M111 and N114, aligned with the de-wetting hot spot C673-G676 in C-Kit, are not paired by a hydrogen bond. Yet M111 is a de-wetting hot spot for JNK (Figure E), hence a harnessing spot for the designed imatinib modification.

(A) Snapshot of the SBMD simulation for C-Kit kinase bound to imatinib at 1 ns. The main-chain hydrogen bond between C673 and G676 is competitively and irreversibly replaced by hydrogen bonding to a water molecule, revealing the instability of the intramolecular interaction. (B) Snapshot of the SBMD simulation of C-Kit kinase in complex with WBZ_4 at 1 ns. The main-chain hydrogen bond between C673 and G676 is stabilized by the water expulsion promoted by the added methyl on the inhibitor.

(A) Western blot of C-Kit inhibition. WBZ_4 inhibits phosphorylation of C-Kit kinase in GIST882 cells. Gel bands from the Western blot assays (see Methods) of C-Kit and its phosphorylated form in GIST cells treated with WBZ_4 and imatinib. The β-actin assay was adopted as control. (B) Western blot of Bcr-Abl inhibition. Phosphorylation of Bcr-Abl kinase is not significantly inhibited by WBZ_4 in CML K562 cells. Electrophoretic gel bands for Western blots for Bcr-Abl kinase and its phosphorylated form in CML cells treated with WBZ_4 and imatinib.

The kinases are inhibited by WBZ_4 (squares) and by the parental compound (triangles). Phosphorylation rate plots are given for Bcr-Abl (red) and C-Kit (blue). The open red symbols correspond to inhibition of unphosphorylated Bcr-Abl, while the filled red symbols correspond to the Tyr412-phosphorylated form. Error bars represent dispersion over 5 runs for each kinetic assay.

(A) Effect of WBZ_4 or imatinib therapy on in vivo GIST growth determined by longitudinal tumor volume measurements. Mice were randomized to treatment with either control (normal PBS and empty liposomes give indistinguishable results within experimental uncertainty), imatinib, or liposome-formulated WBZ_4. *P < 0.01. (B) Effect of WBZ_4 or imatinib therapy on in vivo GIST growth determined by weight measurements. Animals from all groups were sacrificed after 6 weeks of therapy, tumors were excised, and the weight was recorded. *P < 0.05. (C) Effect of WBZ_4 or imatinib therapy on in vivo CML growth induced through a xenograft of K562 tumor cells, determined by longitudinal tumor volume measurement. These results corroborate in vivo the in vitro finding of WBZ_4 selectivity.

(A) In vitro phosphorylation inhibition assay for Abl enzyme in the presence of WBZ_4 (pink) or imatinib (blue). Active recombinant Abl enzyme (1 μg/ml) and its substrate (Abl-tide; 1 μg/ml) were incubated for 1 hour at 37°C in the presence of various WBZ_4 or imatinib concentrations. ATP (100 nM) was added to the reaction mixture. Phosphorylation of Abl-tide peptide was detected by incubation in consecutive order with anti-rabbit phospho–Abl-tide antibody and anti-rabbit HRP antibody. TMB was added to initiate the chromophore reaction, and 2 minutes were allowed for color development. The reaction was terminated by the addition of 1 M H₂SO₄. Phosphorylation of the substrate was quantified as absorbance units (AU) by spectrophotometry at 450 nm. Values obtained with the enzyme without the inhibitors (WBZ_4 or imatinib) were assumed to represent 100% phosphorylation and were compared with the values obtained with the addition of the inhibitors. (B) In vitro phosphorylation inhibition assay for C-Kit in the presence of WBZ_4 (pink) or imatinib (blue). Active recombinant C-Kit kinase (25 ng/ml) and its substrate Poly(Glu4-Tyr) (150 nM) were incubated for 1 hour at 37°C in the presence of various WBZ_4 or imatinib concentrations. ATP (100 nM) was added to the reaction mixture. Phosphorylation of Poly(Glu4-Tyr) peptide was detected by incubation in consecutive order with anti-phosphotyrosine antibody and anti-rabbit HRP antibody.

(A) De-wetting propensities of C-Kit residues in contact with imatinib (PDB.1T46; green bars) and of aligned residues in Bcr-Abl kinase (PDB.1FPU; red bars) and Lck (PDB.3LCK; black bars). Residue i is in contact with the ligand if an atom of the latter lies within its domain, D(i) (see Methods). The de-wetting propensity is quantified by the mean residence time of solvating water molecules. Error bars denote Gaussian dispersion over 5 molecular dynamics runs. (B) Pattern of de-wetting hot spots that arise as backbone hydrogen-bonded residues in C-Kit kinase (ribbon backbone representation in light blue; de-wetting hot spots in green), aligned with Bcr-Abl kinase (magenta backbone; de-wetting hot spots in red). The imatinib methylation site leading to the expulsion of water from C-Kit C673-G676 de-wetting hot spot is highlighted (yellow rectangle). Imatinib is thus modified to favorably expel interfacial water molecules from the C-Kit microenvironment, a feature not conserved in Bcr-Abl. Hydrogen bonds are represented as segments joining α-carbons of the paired residues, and those lacking the propensity for dehydration are shown as light gray segments. Residues from the C-Kit chain are labeled in green, and those from Bcr-Abl are labeled in white. (C) Differences in de-wetting hot spots upon alignment of C-Kit (light blue backbone; hot spots in green) and Lck kinase (gold backbone; hot spots in black). Labels for Lck residues are shown in white. (D) Prototype molecule WBZ_4 (N-{5-[4-(4-methyl piperazine methyl)-benzoylamido]-2-methylphenyl}-4-[3-(4-methyl)-pyridyl]-2-pyrimidine amine). The added methyl group is indicated in red.

(A) Cell proliferation assay for GIST882 cells. WBZ_4 inhibits cell proliferation of C-Kit–positive ST882 cells. GIST cancer cells ST882 (GIST882) were seeded in 96-well plates at a density of 8 × 10³ cells per well. The cells were treated with various concentrations of WBZ_4 (red) and imatinib (light blue) for an additional 48 hours. Cell proliferation was determined by Alamar blue assay (see Methods). Cell proliferation is expressed as the percentage of proliferating cells relative to untreated cells. The WBZ_4 compound was incorporated into liposomes (see Methods) to facilitate cellular delivery. (B) Cell proliferation assay for K562 cells. WBZ_4 does not significantly inhibit cell proliferation of Bcr-Abl–expressing K562 cells. K562 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well in 50 μl of medium. Two hours later, 50 μl medium containing different concentrations (0.01, 0.1, 1 μM) of liposome-encapsulated WBZ_4 (red) or soluble imatinib (light blue) were added to the wells to reach a final volume of 100 μl per well. Following 48 hours of exposure, the Alamar blue assay was performed (see Methods). Plates were read at dual wavelength (570 and 595 nm) in an ELISA plate reader.

(A) Western blot of JNK inhibition in cardiomyocytes. Equal amounts of extracted cellular protein (50 μg per lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Western blots were then probed with primary antibodies specific to the phosphorylated forms of ERKs, JNKs, and p38^MAPK. To ensure equal loading, blots were also probed with an antibody specific to GAPDH. The position of molecular weight standards is indicated to the left of each blot. (B) Effect of WBZ_4 or imatinib therapy on mouse heart BNP. The mRNA levels of BNP (a sensitive marker of myocardial hypertrophy and cardiac impairment) were examined in the left ventricle of mice from the groups in Figure A. The BNP mRNA levels were about 58% higher in the ventricles from imatinib-treated animals (*P = 0.02), but no significant difference was noted in the WBZ_4-treated animals. (C) Comparison of left-ventricular EF after 6 weeks of control (groups treated with either PBS or empty liposomes), imatinib, or WBZ_4 therapy in mice (doses are described in Methods). *P = 0.02 compared with PBS or empty liposomes; ^†P < 0.01 compared with WBZ_4.