mTOR Substrate Phosphorylation: A New Bioassay for Therapeutics

 描述(申请人提供)：有越来越多的人类疾病与哺乳动物雷帕霉素靶标(MTOR)信号级联异常增强有关，mTOR信号级联是控制真核蛋白质合成的中心途径。这些疾病包括与癫痫、自闭症和智力残疾相关的神经发育障碍，以及癌症、肥胖症和糖尿病。FDA最近批准了部分mTOR抑制剂(MTORi)，如针对常染色体显性遗传性神经发育障碍结节性硬化症(TSC)的埃博利莫斯，这为测试mTORi埃博利莫斯对影响大脑、肾脏、肺和皮肤的多系统疾病的疗效提供了一个临床平台。埃博利莫斯也已被批准用于乳腺癌和肾癌。在这一应用中，我们将开发一种简单的血液检测方法来确定接受mTORi治疗的TSC患者淋巴细胞中的mTOR信号，以此作为提高mTORi治疗的临床疗效的策略。目前，mTORi有效性的评估是基于血清mTORi水平和一个标准范围。目前还没有一个指标来实际评估mTORi剂量或血液水平对mTORi信号的影响程度，mTORi信号是mTORi的核心作用机制。因此，我们将在mTORi治疗开始前和治疗4周后使用中尺度Discovery Sector 2400多重ELISA系统评估TSC患者淋巴细胞中4种mTOR底物(p70S6激酶、核糖体S6蛋白、4E-BP-1和Akt)的磷酸化情况。较低的磷酸化水平可作为较高的mTOR抑制和较低的mTOR信号转导的指标，所有这些都是mTORi疗效的间接衡量标准。我们将把这些数据与血清药物水平和TSC缩小脑和肾病变的临床效果联系起来。在PI实验室，我们将评估白细胞蛋白质合成的变化，作为mTORi的功能性测试，并将这些变化与底物磷酸化水平相关联。血样将在天普大学TSC诊所常规门诊期间抽取。这些样品中的蛋白质裂解产物将在天普大学医学院的克里诺博士的实验室和由SBIR资助的初创公司Cogizance Biomarkers LLC(CB)进行检测。长期计划是将这种检测方法商业化，使其成为接受mTORi治疗的患者的一种容易获得、负担得起的筛查治疗性血液测试。基于与mTOR信号异常相关的疾病的快速演变，我们预计我们的测试将在广泛的疾病和临床环境中具有非常广泛的适用性。

 DESCRIPTION (provided by applicant): There are a growing number of human diseases that have been linked to abnormally enhanced activation of the mammalian target of rapamycin (mTOR) signaling cascade, a central pathway governing eukaryotic protein synthesis. These include neurodevelopmental disorders associated with epilepsy, autism, and intellectual disability, as well as cancer, obesity, and diabetes. Recent FDA approval for select mTOR inhibitors (mTORi) such as everolimus for the autosomal dominant neurodevelopmental disorder tuberous sclerosis complex (TSC) has provided a clinical platform to test the efficacy of mTORi everolimus in a multisystem disorder affecting the brain, kidney, lung, and skin. Everolimus has also been approved for use in breast cancer and renal cancer. In this application, we will develop a simple blood assay to define mTOR signaling in lymphocytes from TSC patients treated with mTORi as a strategy to improve the clinical efficacy of mTORi treatment. Currently, the assessment of mTORi effectiveness is predicated on serum mTORi levels and a normative range. There is no metric to actually assess the degree to which any mTORi dose or blood level impacts mTOR signaling, the central mechanism of action of mTORi. Thus, we will assess the phosphorylation of 4 mTOR substrates (p70S6kinase, ribosomal S6 protein, 4E-BP-1 and Akt) in lymphocytes from TSC patients prior to the onset of mTORi treatment and then after 4 weeks of treatment using a MesoScale Discovery Sector 2400 multiplex ELISA system. Lower levels of phosphorylation serve as a metric for higher mTOR inhibition and lower mTOR signaling, all indirect measures of mTORi efficacy. We will correlate these data with serum drug levels and clinical effect on reduction in size of brain and renal lesions in TSC. In the PI laboratory, we will assess changes in leukocyte protein synthesis as a functional assay of mTORi and correlate these changes with levels of substrate phosphorylation. Blood samples will be drawn at the Temple University TSC Clinic during routine outpatient visits. Protein lysates from these samples will be assayed at Temple University School of Medicine in Dr. Crino's laboratory and at Cognizance Biomarkers LLC, (CB), an SBIR funded startup company. The long-term plan will be to commercialize this assay as a readily available, and affordable screening therapeutic blood test for patients being treated with mTORi. Based on the rapid evolution of disorders being associated with aberrant mTOR signaling, we anticipate that our test will have very wide applicability in a broad range of disorders and clinical settings.