Haloacetamidine based inactivators of Protein Arginine Deiminase 4

描述（由申请人提供）：蛋白精氨酸脱亚胺酶4 （PAD4）在类风湿关节炎（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发现的基于氟乙脒的PAD4失活剂F-Amidine进行结构/活性关系；从而鉴定出具有改进效力的PAD4灭活剂。第二个目标是采用两种方法来识别pad4选择性失活剂。第一种方法将卤乙脒战斗部结合到pad特异性肽底物中-底物特异性研究将用于识别这些肽。第二种方法是使用含有卤乙脒的化合物的集中文库-选择性提高的失活剂将通过PAD4和PAD2筛选文库来鉴定，PAD4和PAD2是一种相关的酶，是最广泛表达的PAD同工酶。本提案的第三个目标是使用这些基于卤乙脒的化合物作为合成基于活性的蛋白质分析（ABPPs）试剂的基础。这些ABPPs不仅可用于表征PAD4失活剂的体内选择性，还可用于从哺乳动物细胞系中分离和/或富集内源性PAD4；从而促进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和选择性的PAD4灭活剂，这将代表治疗RA的先导化合物。此外，所提出的化合物将成为重要的探针，用于破译PAD4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声明：该项目的目标是开发针对蛋白精氨酸脱亚胺酶4的抑制剂。这一目标之所以被追求，是因为当这种酶的活性失调时，被认为与类风湿性关节炎的发病和进展有关。类风湿性关节炎影响约1%的美国人口，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类风湿性关节炎的先导化合物，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.