Developing a strategy to identify & validate pharmacodynamic kinase inhibitor biomarkers

 描述（由申请人提供）：小分子激酶抑制剂是最有前途的肿瘤靶向治疗药物之一。在少数病例中已经取得了显著的临床成功，目前正在努力扩大这些成功。目前有100多种激酶抑制剂正在进行临床试验，另有数百种正在开发中。不幸的是，鉴于目前靶向肿瘤药物的成功率约为5%，这些药物中的绝大多数很可能会失败。这种不可接受的高失败率的主要促成因素是缺乏药效学生物标志物来评估药物开发每个阶段的药物疗效。激酶抑制剂生物标志物的缺乏是我们对大多数蛋白激酶的生理靶点缺乏了解的直接结果。在本申请中，我们建议开发和验证一种用于鉴定药效学激酶抑制剂生物标志物的创新新方法。当前行业的最新技术水平是采用抗磷酸酶抗体来半定量地测量当靶激酶被抑制时直接或甚至间接激酶底物中的磷酸化降低。适当生物标志物的选择通常由抗磷酸酶抗体的可用性驱动，而不是由响应底物的药效学特征驱动。在这里，我们打算审查一个新的范例，包括绝对定量的磷酸化和非磷酸化形式的激酶反应底物。我们打算以激酶特异性的方式描述蛋白磷酸化的化学计量，这是一个迄今为止尚未实现的目标。这将通过将稳定同位素标记纳入反向凝胶内激酶测定中，并使用高分辨率质谱法分析来自ERK 2转运蛋白处理的肿瘤的胰蛋白酶肽来实现。将使用AQUA肽作为内标物进行定量。通过在给药后的不同时间间隔定量底物反应，将生成并验证一组准确报告药物疗效的底物反应性底物。我们预计，生物标志物组将优于单一生物标志物，以确定有前途的新激酶抑制剂治疗的最佳剂量和时间表。这项工作的成功完成将满足开发新技术以降低激酶抑制剂失败率的迫切需要。

 DESCRIPTION (provided by applicant): Small molecule kinase inhibitors comprise one of the most promising classes of oncology-directed therapies. Phenomenal clinical successes have already been achieved in a few cases, and intense efforts to broaden these successes are currently underway. More than 100 kinase inhibitors are currently in clinical trials, and hundreds of others are in the development pipeline. Unfortunately, it is highly likely that the vast majorit of these will fail, given that the current success rate for targeted oncology drugs is ~5%. A primary contributing factor to this unacceptably high failure rate is the paucity of pharmacodynamic biomarkers to assess drug efficacy at each stage of drug development. The dearth of kinase inhibitor biomarkers is the direct result of our more general lack of knowledge regarding the physiological targets of most protein kinases. In this application, we propose to develop and validate an innovative new approach for the identification of pharmacodynamic kinase inhibitor biomarkers. The current state-of-the-art in the industry is to employ anti-phosphosite antibodies to semi-quantitatively measure phosphorylation decreases in direct, or even indirect kinase substrates when the target kinase is inhibited. The choice of an appropriate biomarker is most often driven by anti-phosphosite antibody availability, rather than by pharmacodynamic characteristics of the responsive substrate. Here, we intend to vet a new paradigm that incorporates absolute quantification of phosphorylated and non-phosphorylated forms of inhibitor-responsive kinase substrates. We intend to profile protein phosphorylation stoichiometry in a kinase-specific manner, a goal that has not been achieved to date. This will be accomplished by incorporating stable isotope labeling into a reverse in-gel kinase assay, and analyzing tryptic peptides from ERK2 inhibitor-treated tumors using high resolution mass spectrometry. Quantification will be achieved using AQUA peptides as internal standards. By quantifying substrate responses at various intervals after drug administration, a panel of inhibitor-responsive substrates that accurately report on drug efficacy will be generated and validated. We anticipate that panels of biomarkers will outperform single biomarkers to determine optimal dose and scheduling of promising new kinase inhibitor therapies. The successful completion of this work will fulfill a pressing need to develop new technologies to reduce the failure rate of kinase inhibitors.