Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3.
Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification, also termed deimination, is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e., F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated, the development of PAD-selective inhibitors is of paramount importance. To aid that goal, we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein, we report the results of these studies, which suggest that, like PAD4, PADs 1 and 3 employ a reverse protonation mechanism. Additionally, the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds, denoted F4- and Cl4-amidine, are the most potent PAD3 inhibitors ever described.
Knuckley
Bryan
B
Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, USA.
Causey
Corey P
CP
Jones
Justin E
JE
Bhatia
Monica
M
Dreyton
Christina J
CJ
Osborne
Tanesha C
TC
Takahara
Hidenari
H
Thompson
Paul R
PR
D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D002384:Catalysis; D002956:Citrulline / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D000080062:Protein-Arginine Deiminase Type 1; D000080063:Protein-Arginine Deiminase Type 3; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D002956:Citrulline; D000641:Ammonia; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080062:Protein-Arginine Deiminase Type 1; D000080063:Protein-Arginine Deiminase Type 3; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D002118:Calcium
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