Haloacetamidine based inactivators of Protein Arginine Deiminase 4

描述(申请人提供)：蛋白精氨酸脱亚胺酶4(PAD4)在类风湿性关节炎(RA)社区被广泛认为是开发新的RA治疗方法的最佳新靶点。这种酶能分解精氨酸残基，生成瓜氨酸和氨。这项建议的第一个目标是对PI发现的一种基于氟乙酰胺的PAD4失活剂F-嘧啶进行结构/活性关系研究，从而确定具有更高效力的PAD4失活剂。第二个目标是采取两种方法来鉴定PAD4选择性失活剂。第一种方法将卤代乙酰胺弹头结合到PAD特异性多肽底物中-底物特异性研究将用于鉴定这些多肽。第二种方法使用一个含卤代乙酰胺化合物的集中文库-通过用PAD4和PAD2筛选文库来鉴定具有更高选择性的灭活剂，PAD4和PAD2是一种表达最广泛的PAD同工酶。这项建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合成基于活性的蛋白质图谱(ABPP)试剂的基础。这些ABPP不仅有助于表征PAD4失活剂的体内选择性，而且还将使从哺乳动物细胞系中分离和/或浓缩内源性PAD4成为可能，从而有助于识别该蛋白在体内激活时发生的翻译后修饰。一旦完成，建议的研究将导致鉴定一种有效的和选择性的PAD4失活剂，它将代表用于治疗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在人类细胞信号转导中的未完全确定的作用(例如，基因调节)以及这些途径的失调如何(或是否)导致类风湿性关节炎。Lay声明：该项目的目标是开发针对精氨酸脱亚氨酶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是RA的发生和发展的原因，RA是一种疾病，影响约1%的美国人口，并导致平均预期寿命缩短5至10年。本提案中描述的抑制剂不仅代表了治疗RA的先导化合物，而且还将有助于进一步确定这种酶如何促进疾病的发生。

DESCRIPTION (provided by applicant): 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 substrates - 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 will be 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 inactivator 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.