Intracellular cyclic reagents for validating protein function

用于验证蛋白质功能的细胞内循环试剂

• 批准号: 249793-2008
• 负责人: Geyer, Clarence
• 金额: $ 1.82万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2008
• 资助国家: 加拿大
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=394500
• 关键词: Intracellular; cyclic; reagents; validating; protein

Functional genomic analyses are providing data sets that allow hypotheses to be formulated on the therapeutic potential of proteins. Given the large number of protein activities and interactions that are predicted to be important for any given disease, it is difficult to determine the best targets for drug development. It is also too time consuming and expensive to initiate drug development programs against all these targets. To circumvent these problems, cost-effective methods are needed to rapidly validate targets that are important in disease-associated processes and that are susceptible to inhibition by trans dominant agents such as small-molecule drugs. The goal of this proposal is to develop a comprehensive platform for isolating cyclic peptides and antibody-like single-chain variable regions (scFvs) that interact with protein targets with high affinity and specificity. The proposal builds on previous technology developed in our laboratory, whereby we designed a novel genetic selection for rapidly isolating "lariats" that function as inhibitors of protein function. A lariat consists of a cyclic peptide or "noose" region with a covalently attached "tail" that contains a transcription activation domain. Combinatorial libraries of lariats, where the noose region is randomized, can be screened for interactions with a protein target using the yeast two-hybrid assay. In this proposal, the short-term objective is to develop methods to increase the stability and solubility of lariat peptides and scFvs. This will improve our ability to isolate these reagents against protein targets and to use them effectively as inhibitors for validating the therapeutic potential of proteins. The long-term goal of this proposal is to isolate lariats and scFvs against protein targets involved in regulating autocrine IGF-1 signaling in chronic myeloid leukemia (CML) and to use these reagents to identify novel targets to develop drugs for treating CML.

功能基因组分析提供的数据集，使假设制定的治疗潜力的蛋白质。鉴于大量的蛋白质活性和相互作用被预测对任何给定的疾病都很重要，因此很难确定药物开发的最佳靶标。针对所有这些目标启动药物开发计划也太耗时和昂贵。为了避免这些问题，需要具有成本效益的方法来快速验证在疾病相关过程中重要的靶标，并且这些靶标容易受到反式显性药物如小分子药物的抑制。该提案的目标是开发一个综合平台，用于分离以高亲和力和特异性与蛋白质靶标相互作用的环肽和抗体样单链可变区（scFv）。该提案建立在我们实验室以前开发的技术基础上，我们设计了一种新的遗传选择，用于快速分离作为蛋白质功能抑制剂的“lariats”。lactone由环状肽或“套索”区与共价连接的“尾”组成，所述尾含有转录激活结构域。可以使用酵母双杂交测定筛选套索区域被随机化的lariats的组合文库与蛋白质靶标的相互作用。在该提案中，短期目标是开发增加乳肽和scFv的稳定性和溶解性的方法。这将提高我们针对蛋白质靶点分离这些试剂的能力，并将其有效地用作验证蛋白质治疗潜力的抑制剂。该提案的长期目标是分离针对参与调节慢性髓性白血病（CML）中自分泌IGF-1信号传导的蛋白质靶点的lariats和scFvs，并使用这些试剂来鉴定新靶点以开发用于治疗CML的药物。