Chemical Probes Targeting the Protein Arginine Deiminases

描述（由申请人提供）：蛋白精氨酸脱亚胺酶（PAD）活性在多种人类疾病中异常上调，包括类风湿关节炎、结肠炎和癌症。因此，这些酶是潜在的治疗靶点。一种同工酶PAD4有助于控制许多生理过程，包括基因转录、细胞凋亡、细胞生长和中性粒细胞胞外陷阱的形成。然而，PAD4在这些过程中的具体作用还没有完全定义。此外，它的调节模式和它对激酶信号的贡献才刚刚开始被理解和欣赏。其他人类pad（即pad 1、2、3和6）的生理作用甚至不太清楚。在先前研究PAD靶向不可逆抑制剂和基于活性的蛋白质组学探针（ABPPs）的基础上，我们建议开发针对同工酶的PAD失活剂。此外，我们将使用这些化合物来识别和表征调节PAD活性的因素，最初的重点是PAD4。此外，我们将在分子和细胞生物学水平上研究丝氨酸磷酸化和精氨酸瓜氨酸化之间的串扰。具体目标是：(1)本提案的第一目标是集中的

DESCRIPTION (provided by applicant): Protein Arginine Deiminase (PAD) activity is aberrantly upregulated in multiple human diseases, including rheumatoid arthritis, colitis, and cancer. Thus, these enzymes are potential therapeutic targets. One isozyme, PAD4, helps control a number of physiological processes, including gene transcription, apoptosis, cell growth, and neutrophil extracellular trap formation. However, the specific roles of PAD4 in each of these processes are incompletely defined. Additionally, its mode of regulation and its contribution kinase signaling are only beginning to be understood and appreciated. The physiological roles of the other human PADs (i.e., PADs 1, 2, 3, and 6) are even less well understood. Building on previous work that described the development of PAD- targeted irreversible inhibitors and activity based proteomic probes (ABPPs), we propose to develop isozyme specific PAD inactivators. Additionally, we will use these compounds to identify and characterize the factors that regulate PAD activity, focusing initially on PAD4. Additionally, we will examine crosstalk between serine phosphorylation and arginine citrullination at both the molecular and cell biology levels. Specific aims are: (1) The first aim of this proposal is focused on developing isozyme specific PAD inhibitors with improved potency and bioavailability. This aim builds on our exciting discovery that haloacetamidine bearing compounds act as irreversible PAD inhibitors. Two strategies are proposed: (i) the replacement of the backbone amides in Cl-amidine, an inhibitor developed by the PI's lab, with libraries of triazoles and tetrazoles; and (i) a cyclic peptoid library approach. We will also identify novel warheads with altered reactivity tha overcome the limitations of the fluoro- and chloroacetamidine warheads. These compounds will ultimately serve as useful chemical probes of PAD function both directly and when converted into ABPPs to discover the factors that regulate the activity of a particular isozyme. (2) The second aim focuses on characterizing the post-translational modifications (PTMs) that regulate PAD4 activity. This work builds on our prior efforts showing that PAD4 is proteolyzed, acetylated, and ubiquitinated in vivo and these PTMs correlate with different activity states. Specifically, we describe an integrated chemical biology approach to examine the effects of these PTMs on PAD4 activity both in vitro and in vivo. (3) The third aim will study crosstalk between citrullination and serine phosphorylation. We are focused on these studies because we hypothesize that such crosstalk exists to 'fine-tune' cell signaling. Specifically, we demonstrate that crosstalk plays a role in regulating the phosphorylation of ELK1. Additionally, we will take candidate and proteomic approaches to determine the scope of crosstalk between these two PTMs. Once complete, the proposed studies will not only increase our understanding of PAD biology but will provide a suite of chemical probes that can be used to study protein citrullination.