Novel molecules as in vivo biological probes

描述(由申请人提供)：项目摘要：本申请的广泛、长期目标是开发一种通用方法，使用合成的生物可用小分子可逆地调节任何感兴趣的蛋白质的稳定性(和功能)。这项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和人类疾病的新动物模型，在人类疾病的诊断和治疗方面取得广泛进展。众所周知，不稳定的蛋白结构域与第二个蛋白融合会产生一个在细胞中不稳定的嵌合蛋白，并被蛋白酶体降解。这项应用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，该结构域是有条件的稳定的。为了在小鼠身上使用，不稳定的结构域将在敲入小鼠的背景下与感兴趣的蛋白质融合，稳定配体将被结构性地给予，直到需要询问感兴趣的蛋白质的作用的时候。配体的退出会导致目标蛋白质的快速降解。重新注射配体将使嵌合蛋白稳定到正常水平。这项应用的具体目的是结合合理的设计和基于多样性的筛选技术来鉴定FKBP蛋白的突变体，这些突变体在哺乳动物细胞中不稳定，但当与细胞渗透的高亲和力配体结合时稳定。将开发具有理想药理特性的新配体，并将使用几种已知的科学和治疗重要性的蛋白质来验证该方法。相关性：这项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快速和可逆地灭活特定的蛋白质。这种方法将使许多新的人类疾病模型(例如培养细胞或基因敲除小鼠)的发展成为可能，这些模型系统对正在进行的治疗人类疾病的新的和改进的药物的寻找有极大的帮助。

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.