Novel molecules as in vivo biological probes

项目概述：本申请的广泛的长期目标是开发一种 可逆地调节任何感兴趣的蛋白质的稳定性（并因此调节其功能）的一般方法， 合成的、生物可利用的小分子。本申请描述了基础生物医学研究，如果 如果成功，将使人类疾病的诊断和治疗取得广泛进展， 促进基础生物学研究以及人类疾病的新动物模型。 已知不稳定蛋白质结构域与第二蛋白质的融合产生嵌合蛋白质结构域。 在细胞中不稳定并被蛋白酶体降解的蛋白质。此应用程序的目标是 产生一个不稳定的蛋白质结构域，当与高亲和力配体结合时，该结构域是有条件稳定的。 为了在小鼠中使用，不稳定结构域将在敲除的情况下与感兴趣的蛋白质融合。 在小鼠中，稳定化配体将组成性地施用，直到它被释放时为止。 所期望的是询问感兴趣的蛋白质的作用。配体的退出会导致快速 目的蛋白质的降解。再次给予配体可以稳定嵌合蛋白 到正常水平。本申请的具体目标包括合理设计和多样性- 基于筛选技术来鉴定在哺乳动物中不稳定的FKBP蛋白突变体 细胞，但当与细胞可渗透的高亲和力配体结合时稳定。新的配体， 将开发出理想的药理学特性，并将使用 几种已知的科学和治疗重要性的蛋白质。 相关性：本研究的目标是开发一种通用的新方法， 在培养的人类细胞或小鼠中可逆地表达一种特定的蛋白质。这种方法 将能够开发许多新的人类疾病模型（例如，培养的细胞或 基因敲除小鼠），这些模型系统对正在进行的寻找新的和 改进药物来治疗人类疾病。

Project Summary: The broad, long-term objective of this application is the development of a general method to reversibly regulate the stability (and thus function) of any protein of interest using synthetic, bioavailable small molecules. This application describes basic biomedical research that, if successful, would enable widespread advances in the diagnosis and treatment of human disease by facilitating fundamental biological studies as well as new animal models of human disease. It is known that fusion of an unstable protein domain to a second protein creates a chimeric protein that is unstable in cells and degraded by the proteasome. The goal of this application is to create an unstable protein domain that is conditionally stabilized when bound to a high-affinity ligand. For use in mice, the unstable domain would be fused to a protein of interest in the context of a knock- in mouse, and the stabilizing ligand would be constitutively administered until such time as it was desired to interrogate the role of the protein of interest. Withdrawal of the ligand would cause rapid degradation of the protein of interest. Readministering the ligand would stabilize the chimeric protein to normal levels. The specific aims of this application incorporate both rational design and diversity- based screening techniques to identify mutants of the FKBP protein that are unstable in mammalian cells but stable when bound to a cell-permeable, high-affinity ligand. New ligands that possess desirable pharmacological properties will be developed, and this method will be validated using several proteins of known scientific and therapeutic importance. Relevance: The goal of this research is to develop a general new method to rapidly and reversibly inactivate a specific protein in either cultured human cells or in mice. This methodology would enable the development of many new models for human diseases (e.g., cultured cells or knock-out mice), and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases.