A Chemical-Genetic Platform for Interrogating Specific mTOR Functions

用于询问特定 mTOR 功能的化学遗传学平台

• 批准号: 10200712
• 负责人: Douglas Ryan Wassarman
• 金额: $ 3.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200712
• 关键词: Active Sites; Address; Apoptosis; Apoptotic; Area; Autophagocytosis; Binding; Biological Assay; Biology; CCI-779; Cancerous; Cell Proliferation; Cell Survival; Cell model; Cell physiology; Cells; Cellular Stress; Chemicals; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Development; Drug Targeting; Engineering; Equilibrium; FRAP1 gene; Feedback; Genes; Genetic; Genetic Screening; Genetic study; Goals; Growth; Growth Factor; Human; Hyperactivity; In Vitro; Individual; Knock-out; Knowledge; Light; Malignant Neoplasms; Mammalian Cell; Metabolism; Methods; Mutation; Natural Products; Nutrient; Oncogenic; Pharmacology; Phosphorylation; Population; Problem Solving; Process; Protein Biosynthesis; Protein Kinase; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Sirolimus; Site; Specificity; System; Tacrolimus Binding Protein 1A; Techniques; Testing; Therapeutic; Tissues; X-Ray Crystallography; Yeasts; analog; arm; base; cancer cell; cell growth; cell motility; cell type; chemical genetics; gain of function; genetic approach; genetic predictors; inhibitor/antagonist; interest; knock-down; mTOR inhibition; mutant; novel; novel therapeutic intervention; novel therapeutics; population based; protein complex; rapid technique; recruit; small molecule; small molecule inhibitor; targeted treatment; therapeutic target; tool; tumor growth; tumorigenesis; yeast genetics

Project Summary / Abstract Mechanistic Target of Rapamycin (mTOR) is a protein kinase that acts through two distinct protein complexes to control critical cellular processes including protein synthesis, autophagy, and cell motility. Hyperactivation of mTOR drives oncogenesis through increases in cell growth and survival. In recent years, new pharmacological tools have helped make sense of the intricate mTOR signaling network, revealing the necessity of certain downstream mTOR effectors in cancerous tissue and the importance of mTOR complex 2 (mTORC2) as a therapeutic target. The goal of this proposal is to develop chemical-genetic tools for studying specific aspects of mTOR signaling. In particular, we propose to develop a method by which we can specifically inhibit mTORC2 to study its role in cancer. Currently there are no pharmacological inhibitors specific to mTORC2. Genetic methods for inhibiting mTORC2 require knockout or knockdown of genes encoding proteins in mTORC2. This approach disrupts the balance between the two mTOR complexes and suffers from slow onset, leading to the activation of complex feedback networks. In Aim 1, we propose to develop a small molecule that inhibits mTOR if and only if an auxiliary protein is present. This system would allow us to express the auxiliary protein in a specific cell type or even subcellular compartment, thereby sensitizing mTOR to small molecule inhibition in only those cells or compartments. This approach has several advantages; namely, it functions through a gain-of-function mechanism where an inert auxiliary protein is introduced, leaving the endogenous system intact and unchanged until the small molecule inhibitor is introduced. Preliminary data show that we have successfully identified a small molecule that selectively inhibits mTOR in yeast or mammalian cells exogenously expressing an auxiliary protein. In Aim 2, we propose to apply this tool to specifically inhibit mTORC2 and study how it contributes to cell growth and survival in cancer. We hypothesize that phosphorylation of particular mTORC2 substrates is critical for tumor growth and survival, and these substrates can be revealed using specific mTORC2 inhibitors. Completion of this proposal will provide a new chemical-genetic tool to address a previously unmet need in the area of mTOR biology: the ability to selectively inhibit subpopulations of mTOR. This will shed light on the critical roles of mTOR dysregulation in cancer and provide novel therapeutic strategies.

项目总结/摘要 雷帕霉素机制靶点（mTOR）是一种蛋白激酶，通过两种不同的途径起作用。 蛋白质复合物来控制关键的细胞过程，包括蛋白质合成，自噬， 和细胞运动性。mTOR的过度激活通过增加细胞生长驱动肿瘤发生 和生存近年来，新的药理学工具有助于理解 复杂的mTOR信号网络，揭示了某些下游mTOR的必要性 癌组织中的mTOR复合物2（mTORC 2）的重要性， 治疗靶点这项提案的目标是开发化学遗传学工具， mTOR信号传导的特定方面。特别是，我们建议开发一种方法， 可以特异性抑制mTORC 2，研究其在癌症中的作用。目前没有 mTORC 2特异性药理学抑制剂。用于抑制mTORC 2的遗传方法需要 敲除或敲低mTORC 2中编码蛋白质的基因。这种方法破坏了 两个mTOR复合物之间的平衡，并遭受缓慢的发病，导致 激活复杂的反馈网络。在目标1中，我们提出开发一种小分子， 当且仅当辅助蛋白存在时抑制mTOR。这个系统可以让我们 在特定细胞类型或甚至亚细胞区室中表达辅助蛋白，从而 使mTOR仅对那些细胞或区室中的小分子抑制敏感。这 这种方法有几个优点，即它通过功能增益机制发挥作用 其中引入惰性辅助蛋白，使内源系统保持完整， 在引入小分子抑制剂之前保持不变。初步数据显示， 在酵母或哺乳动物中成功鉴定了选择性抑制mTOR的小分子 外源表达辅助蛋白的细胞。在目标2中，我们建议将此工具应用于 特异性抑制mTORC 2，并研究它如何有助于癌症细胞的生长和存活。 我们假设特定mTORC 2底物的磷酸化对于肿瘤的发生是至关重要的。 生长和存活，并且这些底物可以使用特异性mTORC 2抑制剂来揭示。 该提案的完成将提供一种新的化学遗传工具，以解决以前的 mTOR生物学领域未满足的需求：选择性抑制肿瘤细胞亚群的能力 mTOR。这将揭示mTOR失调在癌症中的关键作用，并为癌症的治疗提供新的思路。 新的治疗策略。