Haloacetamidine based inactivators of Protein Arginine Deiminase 4

描述（由申请人提供）：蛋白质精氨酸脱亚胺酶4（PAD 4）在风湿性关节炎（RA）领域被广泛认为是开发新型RA治疗药物的最佳新靶点。这种酶水解精氨酸残基形成瓜氨酸和氨。本提案的第一个目标是对PI发现的一种基于氟乙酰胺的PAD 4灭活剂F-脒进行结构/活性关系分析;从而鉴定出具有改善效力的PAD 4灭活剂。第二个目标是采用两种方法来鉴定PAD 4选择性灭活剂。第一种方法将卤代乙脒弹头结合到PAD特异性肽底物中-底物特异性研究将用于鉴定这些肽。第二种方法使用含卤代乙脒化合物的集中文库-通过用PAD 4和PAD 2（一种相关酶，其是最广泛表达的PAD同工酶）筛选文库来鉴定具有改进选择性的灭活剂。该提议的第三个目标是使用这些基于卤代乙脒的化合物作为合成基于活性的蛋白质谱（ABPP）试剂的基础。这些ABPP不仅可用于表征PAD 4灭活剂的体内选择性，而且还能够从哺乳动物细胞系中分离和/或富集内源性PAD 4;从而有助于鉴定该蛋白在体内活化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PAD 4灭活剂，这将代表治疗RA的先导化合物。此外，提出的化合物将是重要的探针，将用于破译PAD 4在人类细胞信号传导（例如基因调控）中的不完全定义的作用，以及这些途径的失调如何（或是否）导致RA。Lay Statement：本项目的目标是开发针对蛋白质精氨酸脱亚胺酶4的抑制剂。这一目标正在追求，因为这种酶的活性，当失调时，被认为有助于RA的发作和进展，RA是一种影响约1%的美国人口并导致平均预期寿命减少5至10年的疾病。本提案中描述的抑制剂不仅代表了治疗RA的先导化合物，而且还可用于进一步确定这种酶如何促进疾病发作。

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.