Haloacetamidine based inactivators of Protein Arginine Deiminase 4

精氨酸脱亚胺酶4(PAD4)在类风湿性关节炎(RA)中被广泛认为是 开发新型类风湿关节炎治疗药物的最佳新靶点。这种酶能将精氨酸残基降解成 形成瓜氨酸和氨水。该提案的第一个目标是在以下方面执行结构/活动关系 F-嘧啶，PI发现的一种基于氟乙酰胺的PAD4失活剂；从而鉴定PAD4 效力增强的灭活剂。第二个目标是采取两种方法来识别PAD4选择性 灭活剂。第一种方法将卤代乙酰胺弹头结合到PAD特异性多肽亚基中。 将使用ATES底物特异性研究来鉴定这些多肽。第二种方法使用 具有更高选择性的卤代乙酰胺化合物灭活剂的重点文库将被 通过筛选PAD4和PAD2文库进行鉴定，PAD2是表达最广泛的相关酶 PAD同工酶。这项提议的第三个目标是使用这些基于卤代乙酰胺的化合物作为 基于活性的蛋白质图谱(ABPP)试剂的合成基础。这些ABPP不仅将是 有助于表征PAD4灭活剂的体内选择性，但也将使分离和/或 从哺乳动物细胞系中富集内源性PAD4；从而有助于鉴定 这种蛋白质在体内被激活时发生的翻译后修饰。 一旦完成，拟议的研究将导致鉴定一种有效的和选择性的PAD4无活性。 Vator将代表一种用于治疗RA的先导化合物。此外，建议的化合物将 是重要的探针，将用于破译PAD4在人类细胞中尚未完全确定的角色 信号转导(如基因调控)以及这些通路的失调如何(或是否)导致类风湿关节炎。 Lay声明：该项目的目标是开发针对精氨酸脱亚胺酶4蛋白的抑制剂。 之所以追求这一目标，是因为这种酶的活性在失调时被认为有助于 类风湿性关节炎的发生和发展，这种疾病影响到大约1%的美国人口，并导致平均 预期寿命减少5到10年。本提案中描述的抑制剂不仅代表 治疗类风湿性关节炎的先导化合物，但也将有助于进一步定义这种酶如何 会导致疾病的发生。

Protein Arginine Deiminase 4 (PAD4) is widely regarded in the Rheumatoid Arthritis (RA) community as the best new target for the development of a novel RA therapeutic. This enzyme hydrolyses Arg residues to form Citrulline and ammonia. The first goal of this proposal is to perform structure/activity relationships on F-Amidine, a fluoroacetamidine-based PAD4 inactivator discovered by the PI; and thereby identify a PAD4 inactivator with improved potency. The second goal is to take two approaches to identify a PAD4-selective inactivator. The first approach incorporates the haloacetamidine warhead into PAD-specific peptide substr- ates - substrate specificity studies will be used to identify these peptides. The second approach uses a focused library of haloacetamidine-containing compounds - inactivators with improved selectivity willbe identified by screening the library with PAD4 and PAD2, a related enzyme that is the most widely expressed PAD isozyme. The third goal of this proposal is the use these haloacetamidine based compounds as the basis for synthesizing Activity Based Protein Profiling (ABPPs) Reagents. These ABPPs will not only be useful for characterizing the in vivo selectivity of PAD4 inactivators, but will also enable the isolation and/or enrichment of endogenous PAD4 from mammalian cell lines; and thereby facilitate the identification of the post-translational modifications that occur to this protein when it is activated in vivo. Once complete the proposed studies will lead to the identification of a potent and selective PAD4 inacti- vator that will represent a lead compound for the treatment of RA. In addition, the proposed compounds will be important probes that will be used to decipher the incompletely defined role of PAD4 in human cell signaling (e.g. gene regulation) and how (or if) dysregulation of these pathways contributes to RA. Lay Statement: The goal of this project is to develop inhibitors targeting Protein Arginine Deiminase 4. This goal is being pursued because the activity of this enzyme, when dysregulated, is believed to contribute to the onset and progression of RA, a disease that affects ~1% of the American population and causes a mean reduction in life expectancy of 5 to 10 years. The inhibitors described in this proposal not only represent lead compounds for the treatment of RA but will also be useful for further defining how this enzyme contributes to disease onset.