Conformational Dynamics of Protein Tyrosine Kinases Src and Abl

描述(申请人提供)：蛋白激酶调节许多细胞信号事件，它们的严格控制对于调节从细胞分裂到能量代谢的基本过程是必不可少的。因此，蛋白激酶直接或间接参与许多疾病，并成为关键的药物靶点也就不足为奇了。例如，Src激酶是第一个被发现的原癌基因，而去调节Abl融合蛋白(bcr-abl)的形成是95%的慢性髓系白血病患者的致病原因。X-射线晶体结构表明，相同的激酶可以获得活性构象和各种非活性构象，这意味着激酶具有内在的灵活性。活性状态和非活性状态如何稳定以及状态如何相互转换是理解激酶调节的关键问题。由于X射线晶体结构只提供静态快照，因此我们将使用核磁共振实验来研究Abl和Src激活域结构转换的时间尺度和幅度。 Bcr-Abl是临床上非常成功的药物伊马替尼(Gleevec(R)，Novartis)治疗慢性粒细胞白血病(CML)的靶点。为什么伊马替尼结合并抑制c-Abl，而不是结构上密切相关的c-Src激酶？尽管伊马替尼对Src的亲和力比Abl低一个数量级，但与伊马替尼形成的络合物中Src的晶体结构表现出与Abl相似的蛋白质-药物相互作用。由于伊马替尼只与非活性构象的激酶结合，药物结合与活性和非活性状态之间的相互转换密切相关。这项研究的目的是检验这种相互转化中的差异是否是伊马替尼的差异敏感性的基础。因此，我们将通过核磁共振实验来比较在伊马替尼存在的情况下，Src和Abl激酶之间骨架运动的时间尺度和幅度。为了准备这些动力学实验，我们已经建立了表达系统和核磁共振条件，接下来将进行Src和Abl核磁共振谱的指定。 像伊马替尼这样的激酶抑制药物有很大的治疗潜力，因为蛋白激酶介导了许多信号事件。然而，这些药物必须对其目标激酶具有特殊的特异性，耐药突变可能会使这些药物无效，就像在接受伊马替尼治疗的白血病患者中所看到的那样。拟议的实验将阐明像伊马替尼这样的抑制剂如何利用激酶蛋白的特征运动，而不仅仅是它们的结构，来实现特异性。此外，这些结果将对理解激酶调节的基本机制和已知在接受激酶抑制治疗的癌症患者中出现的耐药突变产生更广泛的影响。

DESCRIPTION (provided by applicant): Protein kinases mediate many cell signaling events, and their tight control is essential for regulating essential processes ranging from cell division to energy metabolism. Thus, it is not surprising that protein kinases are directly or indirectly involved in many diseases and that kinases are key drug targets. For example, Src kinase was the first identified proto-oncogene and the formation of a de-regulated Abl fusion protein (BCR-Abl) is the cause of disease in 95% of patients with chronic myeloid leukemia. X-ray crystal structures have shown that the same kinases can obtain an active and various inactive conformations, implying that kinases are inherently flexible. How the active and inactive states are stabilized and how the states interconvert are key questions in understanding kinase regulation. Because X-ray crystal structures provide only static snapshots, we will use nuclear magnetic resonance (NMR) experiments to study the time scales and amplitudes of structural interconversions in Abl and Src kinase domains. BCR-Abl is the target of the clinically highly successful drug imatinib (Gleevec(r), Novartis) in the treatment of chronic myelogenous leukemia (CML). Why does imatinib bind and inhibit c-Abl but not the structurally closely related c-Src kinase? The crystal structure of Src in complex with imatinib shows protein-drug interactions similar to that of Abl, even though the affinity of imatinib for Src is orders of magnitude lower than for Abl. Because imatinib binds only to the inactive conformation of the kinase, drug binding is intimately related to the interconversion between active and inactive states. The goal of this study is to examine whether differences in this interconversion underlie the differential sensitivities for imatinib. Therefore, we will compare the time scales and amplitudes of backbone motions between Src and Abl kinases in the presence of imatinib by NMR experiments. In preparation for these dynamics experiments, we have established expression systems and NMR conditions and will next pursue the assignment of the Src and Abl NMR spectra. Kinase inhibitory drugs such as imatinib have a great therapeutic potential because of the many signaling events that protein kinases mediate. However, these drugs have to be exceptionally specific for their target kinase and resistance mutations can render these drugs ineffective as seen in leukemia patients under imatinib treatment. The proposed experiments will clarify how inhibitors such as imatinib exploit the characteristic movements of kinase proteins, rather than just their structures, to achieve specificity. Furthermore, the results will have broader impact on the understanding of the fundamental mechanisms of kinase regulation and of drug resistance mutations that are known to arise in cancer patients undergoing kinase inhibitory treatment.