Solid-state analysis of kinase activity in cell extracts

描述(由申请人提供)：本提案中描述的工作的目标是构建一种定量的和临床有用的分析方法来测量慢性粒细胞白血病(CML)和急性髓细胞白血病(AML)患者细胞提取物中多种酪氨酸激酶的活性。大多数CML病例与染色体易位相关，导致BCR-ABL酪氨酸激酶的持续活性，而大量的AML患者则过度表达或存在Flt3酪氨酸激酶的激活突变。甲磺酸伊马替尼是一种bcr-abl的抑制剂，在体内灭活bcr-abl并实现CML患者的缓解方面显示出巨大的疗效。然而，某些bcr-abl突变是伊马替尼耐药的。因此，体外测量评估bcr-abl活性水平及其在伊马替尼治疗后的剂量依赖性抑制将为定制CML患者的个体化治疗方案提供有价值的信息。同样，Flt3激酶抑制剂正在进行临床试验，预计同样会存在耐药突变。将合成BCR-ABL和Flt3底物，并用丙烯酸部分标记。然后将这些底物与丙烯酰胺和交联剂在乳液聚合中进行共聚，通过两相微流体流形成微球。微球将与表达bcr-abl和flt3的细胞系的提取物以及CML患者的白细胞提取物接触。底物磷酸化的定量水平将通过抗磷酸酪氨酸抗体标记和流式细胞术来检测。此外，将开发一种使用硫代磷酸化和随后标记硫代磷酸盐的方法作为抗体标记的替代方法。通过抗磷酸酪氨酸抗体和硫代磷酸化检测的敏感性和特异性将进行比较。在敏感性和特异性方面，这项研究中开发的方法将与标准的体外激酶分析以及目前用于诊断CML的方法进行比较。在这项研究中，我们的具体目标是：1.开发用于测量细胞提取物中bcr-abl活性的丙烯酰胺-多肽共聚微球2.定量甲磺酸伊马替尼对慢性粒细胞白血病患者细胞提取物和组织样本中bcr-abl的抑制作用3.使用丙烯酰胺-多肽共聚微球检测多种激酶的活性相关性：这项建议中描述的工作将开发一种直接诊断与慢性髓系白血病相关的蛋白酪氨酸激酶活性升高的分析方法，并预测单个患者对药物治疗的反应。这种检测方法可能会比目前的方法更快速、更便宜，并具有预测药物治疗反应的优势。

DESCRIPTION (provided by applicant): The goal of the work described in this proposal is to construct a quantitative and clinically useful assay to measure activity of multiple tyrosine kinases from cellular extracts of chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML) patients. Most CML cases are associated with a chromosomal translocation resulting in the consitutively active BCR-ABL tyrosine kinase while a large number of AML patients overexpress or have activating mutations in the FLT3 tyrosine kinase. Imatinib mesylate, an inhibitor of BCR-ABL, demonstrates tremendous efficacy in inactivating BCR-ABL in vivo and achieving remission in CML patients. However, certain BCR-ABL mutations are imatinib-resistant. Therefore, an in vitro measurement to assess the level of BCR-ABL activity and its dose-dependent inhibition upon treatment with imatinib would provide valuable information for tailoring individual CML patient treatment programs. Similarly, FLT3 kinase inhibitors are in clinical trials and resistant mutants are likewise expected to exist. BCR-ABL and FLT3 substrates will be synthesized and labeled with an acrylic moiety. Then these substrates will be copolymerized with acrylamide and crosslinker in an emulsion polymerization to generate microspheres via two-phase microfluidic flow. The microspheres will be exposed to extracts from cell lines expressing BCR-ABL and FLT3 and to white blood cell extracts derived from CML patients. Quantitative levels of substrate phosphorylation will be detected by anti-phosphotyrosine antibody labeling and flow cytometry. In addition, a method using thiophosphorylation and subsequent labeling of thiophosphates will be developed as an alternative to antibody labeling. Sensitivity and specificity of detection via antiphosphotyrosine antibodies and thiophosphorylation will be compared. The assay developed in this study will be compared to standard in vitro kinase assays as well as to methods currently used to diagnose CML, with respect to sensitivity and specificity. In this study our specific aims are: 1. Develop acrylamide-peptide copolymer microspheres for measuring Bcr-Abl activity in cell extracts 2. Quantify Bcr-Abl inhibition by imatinib mesylate in cell extracts and tissue samples from CML patients 3. Assess activity of multiple kinases using an acrylamide-peptide copolymer microsphere assay Relevance: The work described in this proposal will develop an assay to directly diagnose elevated protein tyrosine kinase activity related to chronic myeloid leukemia and predict how an individual patient will respond to drug therapy. This assay will likely be more rapid and less expensive than current methods, and have the advantage of predicting response to drug treatment.