Puttini Miriam, Redaelli Sara, Moretti Loris, Brussolo Stefania, Gunby Rosalind H, Mologni Luca, Marchesi Edoardo, Cleris Loredana, Donella-Deana Arianna, Drueckes Peter, Sala Elisa, Lucchini Vittorio, Kubbutat Michael, Formelli Franca, Zambon Alfonso, Scapozza Leonardo, Gambacorti-Passerini Carlo
School of Pharmaceutical Sciences, University of Geneva, Quai Ernest-Ansermet 30, 1211 Genève 4, Switzerland.
Haematologica. 2008 May;93(5):653-61. doi: 10.3324/haematol.12212. Epub 2008 Mar 26.
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).
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.
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.
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.
对伊马替尼耐药是费城染色体阳性白血病治疗中的一个重要临床问题，目前正在通过研发新的、更有效的药物来解决，如双重Src/Abl酪氨酸激酶抑制剂达沙替尼和博舒替尼以及伊马替尼类似物尼罗替尼。在本研究中，我们描述了一种具有氯取代苯甲酰胺的伊马替尼类似物即化合物584（cmp - 584）的设计、合成及生物学特性。
为提高效力，我们合理设计了cmp - 584，一种与Abl激酶结构域具有增强形状互补性的化合物。使用放射性和酶联免疫吸附激酶测定法在体外合成并对一组67种丝氨酸/苏氨酸和酪氨酸激酶对cmp - 584进行了表征。我们使用Bcr/Abl阳性人细胞系、增殖实验中的小鼠转染细胞以及小鼠异种移植模型研究了抑制细胞活性。还对分离的Bcr/Abl蛋白进行了激酶测定。最后，我们在全细胞上采用洗脱方法研究抑制剂的结合动力学。
cmp - 584在重组蛋白上（IC(50)：8 nM）和基于细胞的测定中（IC(50)：0.1 - 10 nM）均显示出强效的抗Abl活性。该药物对血小板衍生生长因子受体和c - KIT保持抑制活性，并且对Lyn也有活性（IC(50)：301 nM）。该组中的其他激酶在纳摩尔剂量下均未被抑制。cmp - 584在细胞中的活性比伊马替尼高20至300倍。这种优越的活性在完整细胞中很明显，完整细胞中存在全长Bcr - Abl。体内实验证实了cmp - 584的活性。洗脱实验表明，与伊马替尼相比，短时间暴露于该药物会在更长时间内损害细胞增殖和Bcr - Abl磷酸化。
目前的结果表明，与伊马替尼相比，cmp - 584的解离速率（off - rate）较慢，这可以解释前者细胞活性增加的原因。
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