An anticancer C-Kit kinase inhibitor is reengineered to make it more active and less cardiotoxic
Ariel Fernández, … , Anil K. Sood, Gabriel Lopez-Berestein
Ariel Fernández, … , Anil K. Sood, Gabriel Lopez-Berestein
Published December 3, 2007
Citation Information: J Clin Invest. 2007;117(12):4044-4054. https://doi.org/10.1172/JCI32373.
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Technical Advance

An anticancer C-Kit kinase inhibitor is reengineered to make it more active and less cardiotoxic

Abstract

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modification to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We established the molecular blueprint for target discrimination in vitro using spectrophotometric and colorimetric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibitory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up approach to control drug specificity.

Authors

Ariel Fernández, Angela Sanguino, Zhenghong Peng, Eylem Ozturk, Jianping Chen, Alejandro Crespo, Sarah Wulf, Aleksander Shavrin, Chaoping Qin, Jianpeng Ma, Jonathan Trent, Yvonne Lin, Hee-Dong Han, Lingegowda S. Mangala, James A. Bankson, Juri Gelovani, Allen Samarel, William Bornmann, Anil K. Sood, Gabriel Lopez-Berestein

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Figure 6

In vitro comparison of inhibitory impact of imatinib and WBZ_4 on Abl and C-Kit kinase.

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In vitro comparison of inhibitory impact of imatinib and WBZ_4 on Abl an...
(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.