A synthetic biology approach to targeting erythropoietin-based therapeutics

描述(申请人提供)：这项提案的目标是测试从自然系统中推断出的蛋白质相互作用原理是否可以用于蛋白质工程，以设计有用的基于促红细胞生成素(EPO)的分子。由于其刺激红细胞生成和抗炎途径，EPO是一种有吸引力的贫血和自身免疫性疾病的治疗选择。然而，它的临床应用受到它的非靶向影响的限制，包括 血栓形成和刺激肿瘤生长。为了解决基于EPO的药物对不同细胞类型更具特异性的需求，可以在如何在细胞中找到合成生物学方法 使用多组分的受体复合体来特异性地靶向细胞因子。例如，白介素2受体由三个亚基组成，其中一个亚基作为另两个亚基的高亲和力靶向结构域，从而创建一个整体的高亲和力受体[50]。我推测，这种精确蛋白质靶向的自然机制可以转化为以合成EPO为基础的疗法的工程，改进了对红系祖细胞或巨噬细胞的靶向，分别用于治疗贫血或自身免疫性疾病。我将构建由EPO突变形式与其受体亲和力降低的嵌合蛋白，与靶细胞强烈结合的靶向元件，以及允许同时结合的连接子。我假设，通过将关于结合动力学的定量信息与关于蛋白质配体和细胞表面受体的结构信息相结合，所产生的蛋白质(嵌合激活物)应该只在具有这两种元素的受体的细胞中激活信号转导。靶向元件应介导初始结合，突变的EPO由于其在细胞表面的高局部浓度而随后应结合，尽管其结合亲和力降低。这项工作将提供关于设计基于蛋白质的靶向药物的定量方面的有价值的信息，EPO的不同生物学作用及其作为多效性治疗的价值，也可能使设计细胞类型特异性治疗的新方法成为可能。总体而言，这些发现将对改善贫血或自身免疫性疾病患者的生活质量和疾病预后具有重要意义。

DESCRIPTION (provided by applicant):The goal of this proposal is to test whether principles of protein interaction that have been inferred from natural systems can be used in protein engineering to design useful erythropoietin (EPO)-based molecules. Due to its stimulation of erythropoiesis and anti-inflammatory pathways, EPO is an attractive treatment option for anemia and autoimmune disease. However, its clinical use is inhibited by its off-target affects, including thrombosis and stimulation of tumor growth. To address the need for EPO-based drugs with greater specificity for distinct cell types, a synthetic biology approach can be found in how cells use multi-component receptor complexes to specifically target cytokines. For example, the interleukin 2 receptor is composed of three subunits, one of which acts as a high-affinity targeting domain for the other two to create an overall high-affinity receptor [50]. I hypothesize that this natural mechanism for precise protein targeting can be translated to the engineering of synthetic EPO-based therapeutics with improved targeting of erythroid progenitors or macrophages, for the treatment of anemia or autoimmune disease, respectively. I will build chimeric proteins composed of a mutated form of EPO with reduced affinity for its receptor, a targeting element that binds strongly to the target cell, and a linker that allows simultaneous binding. I hypothesize that by combining quantitative information about binding kinetics with structural information about protein ligands and cell surface receptors, the resulting proteins ("chimeric activators") should activate signal transduction only in cells with receptors for both elements. The targeting element should mediate initial binding, and the mutated EPO should subsequently bind due to its high local concentration at the cell surface, despite its decreased binding affinity. This work will provide valuable information about the quantitative aspects of designing targeted protein-based drugs, the diverse biological roles of EPO and its value as a pleiotropic therapeutic, and may also enable new methods for engineering cell type-specific therapies. Overall, the findings will have implications for improving the quality of life and diseae prognosis in patients suffering from anemia or autoimmune disease.