Substrate Specificity and Kinetic Studies of PADs 1, 3, and 4 Identify Potent and Selective Inhibitors of Protein Arginine Deiminase 3

摘要

Protein citrullination has been shown to regulate numerous physiological pathways (e.g., theninnate immune response and gene transcription) and is, when dysregulated, known to be associated withnnumerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification,nalso termed deimination, is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). Innmammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specificnexpression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 wasnrecently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e.,nF- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidinenalso exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor.nGiven the increasing number of diseases in which dysregulated PAD activity has been implicated, thendevelopment of PAD-selective inhibitors is of paramount importance. To aid that goal, we characterized thencatalytic mechanism and substrate specificity of PADs 1 and 3. Herein, we report the results of these studies,nwhich suggest that, like PAD4, PADs 1 and 3 employ a reverse protonation mechanism. Additionally, thensubstrate specificity studies provided critical information that aided the identification of PAD3-selectiveninhibitors. These compounds, denoted F4- and Cl4-amidine, are the most potent PAD3 inhibitors everndescribed.