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

描述（由申请人提供）：针对激酶的分子靶向治疗正在改变癌症治疗的面貌；然而，目前还没有临床使用的检测方法直接监测这些药物在患者细胞中的抑制作用。此外，在遗传和生化水平上的细胞异质性是对这些疗法产生耐药性的基础，而大量细胞试验并不能准确预测或监测治疗效果。建立了一种监测单细胞BCR-Abl激酶活性的生化检测方法。该方法定量测定费城染色体阳性（Ph+）急性淋巴细胞白血病（ALL）患者BCR-Abl激酶酶活性。该资助的目的是优化该检测方法，以验证以下假设：BCR-Abl的酶活性在Ph+ ALL患者的单个细胞中表现出很大程度的差异，从而预测对靶向治疗的反应或耐药性。提出的研究考虑了设计、合成和评价荧光标记肽底物作为定量估计患者样品中BCR-Abl激酶活性的新方法。目的1是通过标准的FMOC固相肽方案合成报告基因，并通过MALDI和HPLC进行表征。研究人员选择了两种设计策略来产生细胞内降解抗性增强的报告蛋白：1)β -发夹肽通过连接物连接到底物的n端，以及2)将非天然氨基酸战略性地结合到底物中。在Aim 2中，对底物进行体外评估，以确定动力学参数KM和Vmax，并在细胞裂解物和完整的BCR-Abl+细胞中进行磷酸化定量和底物半衰期测定。毛细管电泳用于定量与细胞裂解研究的自动化系统和单细胞分析的定制仪器。最终，底物将根据磷酸化和抗降解性来选择。Aim 3将使用Aim 2中开发的先导底物，在单细胞水平上定量Ph+ ALL患者样品中的BCR-Abl激酶活性。显微注射用于将底物递送到细胞中，但将测试其他方法，从电穿孔到肉豆蔻酰化。这些数据将用于更好地了解肿瘤细胞中的激酶磷酸化，预测ALL患者药物靶向治疗的疗效，并促进个体化治疗的发展。

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.