Novel molecules as in vivo biological probes

作为体内生物探针的新型分子

• 批准号: 7164408
• 负责人: THOMAS James WANDLESS
• 金额: $ 25.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7164408
• 关键词: Adoption; Affinity; Animal Model; Animals; Appendix; Binding; Bioavailable; Biological; Biological Assay; Biological Models; Biomedical Research; Cells; Chimeric Proteins; Communities; Complementary DNA; Cultured Cells; Development; Diagnosis; Embryo; Financial compensation; Gene Silencing; Genes; Goals; Human; Knock-in Mouse; Knock-out; Knockout Mice; Life; Ligands; Mammalian Cell; Methodology; Methods; Modeling; Mus; Pharmaceutical Preparations; Phenotype; Physiology; Property; Proteins; Research; Research Personnel; Role; Screening procedure; System; Tacrolimus Binding Proteins; Techniques; Technology; Tertiary Protein Structure; Therapeutic; Time; Transgenic Organisms; Withdrawal; base; design; desire; functional restoration; homologous recombination; human disease; improved; in vivo; interest; mouse model; multicatalytic endopeptidase complex; mutant; novel; null mutation; protein degradation; protein function; small molecule; tool

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.

描述（由申请人提供）：项目摘要：该应用程序的广泛，长期目标是开发一种通用方法，该方法是使用合成的，可生物利用的小分子可逆地调节任何感兴趣蛋白质的稳定性（以及功能）。该应用描述了基本的生物医学研究，如果成功，该研究将通过促进基本生物学研究以及新的人类疾病动物模型来实现人类疾病诊断和治疗的广泛进步。众所周知，不稳定蛋白结构域与第二蛋白的融合会产生一种嵌合蛋白，该蛋白质在细胞中不稳定并被蛋白酶体降解。该应用的目的是创建一个不稳定的蛋白质结构域，该蛋白质结构域在绑定到高亲和力配体时有条件地稳定。为了在小鼠中使用，不稳定的结构域将在小鼠中的敲门型中融合到感兴趣的蛋白质中，并且稳定的配体将被组成式施用，直到需要询问感兴趣蛋白质的作用为止。配体的提取会导致感兴趣的蛋白质迅速降解。调整配体将使嵌合蛋白稳定在正常水平上。该应用的具体目的既包含了理性设计和基于多样性的筛选技术，又鉴定了在哺乳动物细胞中不稳定的FKBP蛋白突变体，但在与细胞可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，该方法将使用已知的科学和治疗意义的几种蛋白质进行验证。相关性：这项研究的目的是开发一种一般的新方法，以快速而可逆地使培养的人类细胞或小鼠中的特定蛋白质失活。这种方法将使许多新的人类疾病模型（例如培养细胞或淘汰小鼠）的发展发展，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非常有帮助。