Peptide Reporters for Estimation of BCR-Abl Kinase Activity in ALL Patient Cells

描述(申请人提供)：针对激酶的分子靶向治疗正在改变癌症治疗的面貌；然而，目前临床上还没有直接监测这些药物在患者细胞中的抑制情况的检测方法。此外，遗传和生化水平上的细胞异质性是抵抗这些疗法的基础，而大量的细胞分析不能准确地预测或监测治疗效果。建立了一种监测单细胞bcr-abl酶活性的生化分析方法。本试验定量检测了费城染色体阳性(Ph)急性淋巴细胞白血病(ALL)患者的bcr-Abl激酶活性。这笔赠款的目标是优化测试，以测试BCR-Abl的酶活性在Ph所有患者的单个细胞中表现出很大程度的变异性的假设，从而可以预测对靶向治疗的反应或抵抗。这项拟议的研究考虑了荧光标记多肽底物的设计、合成和评估，作为一种定量评估患者样本中bcr-Abl激酶活性的新方法。目的1采用标准的Fmoc固相肽合成方法合成报告分子，并用MALDI和高效液相色谱对其进行表征。选择了两种设计策略来产生具有更强的细胞内降解抗性的报告：1)通过连接物连接到底物N端的β-发夹多肽，以及2)在底物中战略性地掺入非天然氨基酸。在目标2中，在体外评估底物以确定动力学参数Km和Vmax，也在细胞裂解产物和完整的bcr-Abl细胞中评估磷酸化的定量和底物半衰期的测定。毛细管电泳仪用于细胞裂解物研究的自动化系统和定制的单细胞分析仪器的定量。最终，底物的选择将基于磷酸化和耐降解性。AIM 3将使用AIM 2中开发的铅底物在单细胞水平上对Ph所有患者样本中的bcr-Abl激酶活性进行量化。微注射用于将底物输送到细胞中，但将测试从电穿孔到肉豆蔻化的其他方法。这些数据将被用来更好地了解肿瘤细胞中的激酶磷酸化，预测药物靶向治疗对所有患者的疗效，并促进个性化治疗的发展。

DESCRIPTION (provided by applicant): Molecularly targeted therapies against kinases are changing the face of cancer treatment; however, there are no assays currently in clinical use that directly monitor the inhibition of these drugs in patient cells. Moreover, cellular heterogeneity both at the genetic and biochemical level underlies resistance to these therapies, while bulk cellular assays do not accurately predict or monitor therapeutic efficacy. A biochemical assay for monitoring BCR-Abl kinase activity in single cells has been developed. The assay quantitatively measures the enzymatic activity of BCR-Abl kinase in patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The goal of this grant is to optimize the assay to test the hypothesis that the enzymatic activity of BCR-Abl exhibits a great degree of variability amongst individual cells of Ph+ ALL patients allowing prediction of response or resistance to targeted therapies. The proposed research takes into consideration the design, synthesis and evaluation of fluorescently labeled peptide substrates as a novel method of quantitatively estimate BCR-Abl kinase activity in patient samples. Aim 1 is focused on reporter synthesis by standard FMOC solid phase peptide protocol and characterization by MALDI and HPLC. Two design strategies are selected to generate reporters with increased intracellular resistance to degradation: 1) a beta-hairpin peptide attached through a linker to N-terminus of the substrate, and 2) strategic incorporation of un-natural amino acids into the substrate. In Aim 2, the substrates are evaluated in vitro for determination of kinetic parameters KM and Vmax, and also in cell lysates and intact BCR-Abl+ cells for quantification of phosphorylation and determination of substrate half-life. Capillary electrophoreses is used for quantification with an automated system for cell lysate studies and a custom-built instrument for single cell analyses. Ultimately, the substrates will be selected based on phosphorylation and resistance to degradation. Aim 3 will quantify BCR-Abl kinase activity in Ph+ ALL patient samples at the single-cell level using the lead substrates developed in Aim 2. Microinjection is utilized to deliver the substrates into cells but other methods ranging from electroporation to myristoylation will be tested. These data will be used to gain a greater understanding on kinase phosphorylation in tumor cells, to predict the efficacy of drug-targeted therapy for ALL patients and to facilitate development of individualized therapies.