Novel molecules as in vivo biological probes

项目概述：本申请的广泛、长期目标是开发一种通用方法，利用合成的、生物可利用的小分子来可逆地调节任何感兴趣的蛋白质的稳定性（和功能）。本申请描述了基础生物医学研究，如果成功，将通过促进基础生物学研究以及人类疾病的新动物模型，使人类疾病的诊断和治疗取得广泛进展。众所周知，将一个不稳定的蛋白质结构域与另一个蛋白质融合会产生一种嵌合蛋白，这种嵌合蛋白在细胞中是不稳定的，并被蛋白酶体降解。本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。对于小鼠来说，在敲入小鼠的情况下，不稳定结构域将被融合到感兴趣的蛋白质上，稳定配体将被组成性地给予，直到需要询问感兴趣的蛋白质的作用。退出配体会导致目标蛋白的快速降解。重新注射配体可以将嵌合蛋白稳定在正常水平。本应用的具体目的包括合理设计和基于多样性的筛选技术，以鉴定在哺乳动物细胞中不稳定但与细胞渗透性高亲和力配体结合时稳定的FKBP蛋白突变体。具有理想药理特性的新配体将被开发出来，并且该方法将使用几种已知的科学和治疗重要性的蛋白质进行验证。相关性：本研究的目标是开发一种通用的新方法来快速和可逆地灭活培养的人类细胞或小鼠中的特定蛋白质。这种方法将能够开发许多新的人类疾病模型（例如，培养细胞或敲除小鼠），这些模型系统对正在进行的寻找新的和改进的药物来治疗人类疾病有很大的帮助。

DESCRIPTION (provided by applicant): 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.