Chemical Probes Targeting the Protein Arginine Deiminases

描述(申请人提供)：蛋白质精氨酸脱亚胺酶(PAD)活性在多种人类疾病中异常上调，包括类风湿性关节炎、结肠炎和癌症。因此，这些酶是潜在的治疗靶点。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确定。此外，它的调节模式和它对激酶信号的作用才刚刚开始被理解和欣赏。其他人体垫(即垫1、2、3和6)的生理作用更不清楚。在前人工作的基础上，描述了PAD靶向不可逆抑制剂和基于活性的蛋白质组探针(ABPP)的发展，我们建议开发同工酶特异的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：(一)聚焦本提案的第一个目标 关于开发具有更高效力和生物利用度的同工酶特异性PAD抑制剂。这一目标建立在我们令人兴奋的发现之上，即含卤代乙酰胺的化合物作为不可逆垫片抑制剂发挥作用。提出了两种策略：(I)用三氮唑和四氮唑的文库取代PI实验室开发的抑制剂氯-酰胺中的主链酰胺；(I)环肽文库方法。我们还将确定具有改变反应性的新型弹头，以克服氟化和氯乙烷化弹头的局限性。这些化合物最终将直接用作PAD功能的有用化学探针，并在转化为ABPP时用于发现调节特定同工酶活性的因素。(2)第二个目标是研究调节PAD4活性的翻译后修饰(PTM)。这项工作建立在我们之前的工作基础上，表明PAD4在体内是蛋白质降解、乙酰化和泛素化的，这些PTM与不同的活性状态相关。具体地说，我们描述了一种综合的化学生物学方法来检测这些PTM在体外和体内对PAD4活性的影响。(3)第三个目标将研究瓜氨酸化和丝氨酸磷酸化之间的串扰。我们之所以关注这些研究，是因为我们假设这种串扰的存在是为了“微调”细胞信号。具体地说，我们证明了串扰在调节ELK1的磷酸化方面发挥了作用。此外，我们将采用候选和蛋白质组学方法来确定这两个PTM之间的串扰范围。一旦完成，拟议的研究不仅将增加我们对PAD生物学的理解，还将提供一套可用于研究蛋白质瓜氨酸化的化学探针。

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.