Novel molecules as in vivo biological probes

项目摘要：此应用程序的广泛、长期目标是开发 用来可逆地调节任何感兴趣蛋白质的稳定性(和功能)的一般方法 人工合成的生物可利用的小分子。该应用程序描述了基础生物医学研究，如果 成功，将使人类疾病的诊断和治疗通过以下方式取得广泛进展 促进基础生物学研究以及人类疾病的新动物模型。 众所周知，不稳定的蛋白质结构域与第二种蛋白质的融合会产生嵌合体 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 创建一个不稳定的蛋白质结构域，当与高亲和力配体结合时，该结构域有条件地稳定。 为了在小鼠身上使用，不稳定的结构域将在敲击的背景下与感兴趣的蛋白质融合- 在小鼠体内，稳定配体将被结构性地给予，直到它被 想要询问感兴趣的蛋白质的作用。配体的退出将导致快速 目标蛋白质的降解。重新注射配体将稳定嵌合蛋白 恢复到正常水平。本申请的具体目标既包括合理的设计，也包括多样性- 基于筛选技术鉴定哺乳动物中不稳定的FKBP蛋白突变体 细胞，但结合在细胞渗透性、高亲和力的配体上时稳定。新的配体拥有 将开发理想的药理特性，并将使用以下方法验证该方法 几种已知具有科学和治疗重要性的蛋白质。 相关性：这项研究的目标是开发一种通用的新方法来快速和 在培养的人类细胞或小鼠中，可逆地使特定蛋白质失活。这种方法论 将使许多新的人类疾病模型的开发成为可能(例如，培养细胞或 基因敲除小鼠)，而这些模型系统对正在进行的寻找新的和 改进了治疗人类疾病的药物。

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.