Intracellular cyclic reagents for validating protein function

功能基因组分析提供了数据集，可以对蛋白质的治疗潜力进行假设。鉴于大量的蛋白质活性和相互作用被预测对任何特定疾病都很重要，很难确定药物开发的最佳靶点。针对所有这些目标启动药物开发项目也太耗时和昂贵。为了规避这些问题，需要成本效益高的方法来快速验证在疾病相关过程中重要的靶标，这些靶标容易受到小分子药物等反显性药物的抑制。本提案的目标是开发一个综合平台，用于分离环肽和抗体样单链可变区（scFvs），这些区域与蛋白靶点具有高亲和力和特异性相互作用。该建议建立在我们实验室先前开发的技术的基础上，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“幼虫”。lariat由环状肽或“套索”区域组成，其共价连接的“尾巴”包含转录激活域。套索区域随机化的lariats组合文库，可以使用酵母双杂交试验筛选与蛋白质靶标的相互作用。在本提案中，短期目标是开发提高lariat肽和scFvs的稳定性和溶解度的方法。这将提高我们针对蛋白质目标分离这些试剂的能力，并有效地将它们用作抑制剂来验证蛋白质的治疗潜力。本研究的长期目标是分离出抗慢性髓性白血病（CML）中参与调节自分泌IGF-1信号的蛋白靶点的lariats和scFvs，并利用这些试剂鉴定新的靶点，开发治疗CML的药物。

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.