mTOR Substrate Phosphorylation: A New Bioassay for Therapeutics

点击翻译按钮获取中文摘要

 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.