Chemical Probes Targeting the Protein Arginine Deiminases

描述（由申请人提供）：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。一种同工酶，PAD 4，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形成。然而，PAD 4在这些过程中的每一个中的具体作用是不完全定义的。此外，它的调节模式及其对激酶信号传导的贡献才刚刚开始被理解和认识。其他人类PAD的生理作用（即，PAD 1、2、3和6）更不容易理解。基于先前描述PAD靶向不可逆抑制剂和基于活性的蛋白质组学探针（ABPP）的开发的工作，我们提出开发同工酶特异性PAD灭活剂。此外，我们将使用这些化合物来鉴定和表征调节PAD活性的因素，最初集中在PAD 4上。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之间的串扰。具体目标是：（1）本建议的第一个目标是集中 开发具有改进的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上，即带有卤代乙脒的化合物作为不可逆的PAD抑制剂。提出了两项战略：（i）用三唑和四唑文库替换Cl-脒（PI实验室开发的抑制剂）中的骨架酰胺;和（i）环状类肽文库方法。我们还将确定具有改变反应性的新型弹头，以克服氟乙脒和氯乙脒弹头的局限性。这些化合物最终将作为PAD功能的有用的化学探针，直接和转化为ABPP时发现调节特定同工酶活性的因子。(2)第二个目标集中于表征调节PAD 4活性的翻译后修饰（PTM）。这项工作建立在我们先前的工作基础上，表明PAD 4在体内被蛋白水解，乙酰化和泛素化，这些PTM与不同的活性状态相关。具体而言，我们描述了一个综合的化学生物学方法来检查这些PTM在体外和体内对PAD 4活性的影响。(3)第三个目标是研究瓜氨酸和丝氨酸磷酸化之间的串扰。我们专注于这些研究，因为我们假设存在这种串扰来“微调”细胞信号。具体来说，我们证明了串扰在调节ELK 1的磷酸化中起作用。此外，我们将采取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，而且将提供一套可用于研究瓜氨酸蛋白的化学探针。

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.