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A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
Published on: December 7, 2014
Characterization of compound 584, an Abl kinase inhibitor with lasting effects
Miriam Puttini1, Sara Redaelli, Loris Moretti
1School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, 1211 Genève 4, Switzerland.
Background:
Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584).
Design And Methods:
To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor.
Results:
cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib.
Conclusions:
The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.
Insights
A new imatinib analog, compound 584 (cmp-584), demonstrates superior potency against Bcr-Abl kinase in leukemia cells. Its slower dissociation rate from the target kinase explains its enhanced cellular activity compared to imatinib.
Area of Science:
- Oncology
- Pharmacology
- Biochemistry
Background:
- Imatinib resistance is a significant challenge in treating Philadelphia chromosome-positive leukemias.
- New drugs like dasatinib, bosutinib, and nilotinib are being developed to overcome imatinib resistance.
- Compound 584 (cmp-584) is a novel imatinib analog designed to enhance potency.
Purpose of the Study:
- To design, synthesize, and evaluate the biological properties of cmp-584, an imatinib analog.
- To assess the in vitro and in vivo efficacy of cmp-584 against Bcr-Abl kinase and relevant cell lines.
- To investigate the binding kinetics of cmp-584 compared to imatinib.
Main Methods:
- Rational design of cmp-584 for enhanced shape complementarity with the Abl kinase domain.
- In vitro kinase assays against a panel of 67 kinases and cell-based assays using Bcr-Abl-positive cell lines.
- In vivo studies using a murine xenotransplanted model and wash-out experiments to assess binding kinetics.
Main Results:
- Cmp-584 exhibited potent anti-Abl activity (IC50: 8 nM for recombinant protein, 0.1-10 nM in cells).
- It maintained activity against PDGF receptors and c-KIT, and Lyn kinase (IC50: 301 nM), with no inhibition of other kinases at nanomolar doses.
- Cmp-584 was 20- to 300-fold more potent than imatinib in cell-based assays, with sustained effects observed in vivo.
Conclusions:
- Cmp-584 displays significantly enhanced cellular activity compared to imatinib.
- A slower off-rate (dissociation rate) of cmp-584 from Bcr-Abl is proposed as the mechanism for its increased efficacy.
- These findings suggest cmp-584 as a promising candidate for overcoming imatinib resistance in leukemia treatment.
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