A synthetic biology approach to targeting erythropoietin-based therapeutics

描述（由申请人提供）：本提案的目的是测试从自然系统中推断的蛋白质相互作用的原理是否可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是贫血和自身免疫性疾病的有吸引力的治疗选择。然而，其临床应用受到其脱靶效应的抑制，包括 血栓形成和刺激肿瘤生长。为了满足对不同细胞类型具有更高特异性的EPO药物的需求，可以在细胞如何合成中找到合成生物学方法。 使用多组分受体复合物来特异性靶向细胞因子。例如，白细胞介素2受体由三个亚基组成，其中一个亚基作为其他两个亚基的高亲和力靶向结构域，以产生整体高亲和力受体[50]。我假设，这种精确蛋白质靶向的天然机制可以转化为基于EPO的合成治疗剂的工程化，其具有改善的红系祖细胞或巨噬细胞靶向，分别用于治疗贫血或自身免疫性疾病。我将构建嵌合蛋白质，它由突变形式的EPO组成，EPO对其受体的亲和力降低，一个与靶细胞强烈结合的靶向元件，以及一个允许同时结合的接头。我推测，通过结合动力学的定量信息与蛋白质配体和细胞表面受体的结构信息相结合，所得到的蛋白质（“嵌合激活剂”）应该只激活细胞中的信号转导与受体的两个元素。靶向元件应介导初始结合，并且突变的EPO应随后结合，这是由于其在细胞表面的高局部浓度，尽管其结合亲和力降低。这项工作将提供有关设计靶向蛋白质药物的定量方面的有价值的信息，EPO的多种生物学作用及其作为多效性治疗剂的价值，并且还可能实现工程细胞类型特异性疗法的新方法。总的来说，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产生影响。

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.