title: Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Knuckley
authors.forename: Corey P
authors.initials: CP
authors.lastname: Causey
authors.forename: Justin E
authors.initials: JE
authors.lastname: Jones
authors.forename: Monica
authors.initials: M
authors.lastname: Bhatia
authors.forename: Christina J
authors.initials: CJ
authors.lastname: Dreyton
authors.forename: Tanesha C
authors.initials: TC
authors.lastname: Osborne
authors.forename: Hidenari
authors.initials: H
authors.lastname: Takahara
authors.forename: Paul R
authors.initials: PR
authors.lastname: Thompson
mesh_terms: 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
chemical_list: 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
references.citation: Biochem J. 2003 Feb 15;370(Pt 1):167-74
references.pmid: 12416996
references.citation: Biochemistry. 2005 Aug 9;44(31):10570-82
references.pmid: 16060666
references.citation: Nat Genet. 2003 Aug;34(4):395-402
references.pmid: 12833157
references.citation: Biochemistry. 2006 Jan 31;45(4):1162-72
references.pmid: 16430212
references.citation: J Neurosci Res. 2005 Apr 1;80(1):120-8
references.pmid: 15704193
references.citation: Curr Biol. 2001 Jun 26;11(12):996-1000
references.pmid: 11448779
references.citation: ACS Chem Biol. 2006 Aug 22;1(7):433-41
references.pmid: 17168521
references.citation: Bioessays. 2003 Nov;25(11):1106-18
references.pmid: 14579251
references.citation: J Clin Invest. 1998 Jan 1;101(1):273-81
references.pmid: 9421490
references.citation: J Leukoc Biol. 2001 Jul;70(1):46-51
references.pmid: 11435484
references.citation: Biochemistry. 2006 May 2;45(17):5618-30
references.pmid: 16634643
references.citation: Biochemistry. 2005 Aug 23;44(33):11188-200
references.pmid: 16101303
references.citation: Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5291-6
references.pmid: 16567635
references.citation: J Immunol. 2008 Feb 1;180(3):1895-902
references.pmid: 18209087
references.citation: Cell. 2004 Sep 3;118(5):545-53
references.pmid: 15339660
references.citation: Biochemistry. 2007 Nov 20;46(46):13370-81
references.pmid: 17960915
references.citation: J Immunol. 2003 Jul 15;171(2):538-41
references.pmid: 12847215
references.citation: Methods Enzymol. 1982;87:390-405
references.pmid: 7176923
references.citation: Anal Biochem. 2000 Nov 15;286(2):257-64
references.pmid: 11067748
references.citation: Clin Immunol. 2008 Feb;126(2):165-71
references.pmid: 17980669
references.citation: J Invest Dermatol. 2005 Jul;125(1):34-41
references.pmid: 15982300
references.citation: J Appl Physiol (1985). 2009 Oct;107(4):1213-22
references.pmid: 19644031
references.citation: Mol Carcinog. 2006 Mar;45(3):183-96
references.pmid: 16355400
references.citation: Biochem Biophys Res Commun. 1998 Feb 24;243(3):641-6
references.pmid: 9500980
references.citation: J Invest Dermatol. 2000 Apr;114(4):701-5
references.pmid: 10733676
references.citation: Tetrahedron Lett. 2008 Jul 7;49(28):4383-4385
references.pmid: 19587776
references.citation: Biochemistry. 2007 Jun 5;46(22):6578-87
references.pmid: 17497940
references.citation: Biochem Cell Biol. 2008 Oct;86(5):437-47
references.pmid: 18923545
references.citation: Chembiochem. 2010 Jan 25;11(2):161-5
references.pmid: 20014086
references.citation: J Invest Dermatol. 2005 Feb;124(2):384-93
references.pmid: 15675958
references.citation: J Biol Chem. 1996 Nov 29;271(48):30709-16
references.pmid: 8940048
references.citation: J Biochem. 1981 Mar;89(3):771-4
references.pmid: 7287639
references.citation: Biochemistry. 2005 Apr 19;44(15):5827-34
references.pmid: 15823041
references.citation: Gene. 2004 Apr 14;330:19-27
references.pmid: 15087120
references.citation: Biochem Biophys Res Commun. 2005 Feb 4;327(1):192-200
references.pmid: 15629448
references.citation: J Biol Chem. 2005 Dec 16;280(50):41360-5
references.pmid: 16223729
references.citation: Cell Mol Life Sci. 2005 Sep;62(17):1984-95
references.pmid: 16091842
references.citation: J Histochem Cytochem. 1998 Nov;46(11):1303-9
references.pmid: 9774629
references.citation: Anal Biochem. 2007 Oct 15;369(2):232-40
references.pmid: 17716614
references.citation: J Biol Chem. 2002 Dec 20;277(51):49562-8
references.pmid: 12393868
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 8;102(10):3611-6
references.pmid: 15731352
references.citation: J Cell Biol. 2009 Jan 26;184(2):205-13
references.pmid: 19153223
references.citation: Arthritis Rheum. 2008 Aug;58(8):2287-95
references.pmid: 18668562
references.citation: Int J Cosmet Sci. 2007 Jun;29(3):147-68
references.pmid: 18489346
references.citation: BMC Cancer. 2009 Jan 30;9:40
references.pmid: 19183436
references.citation: Curr Opin Drug Discov Devel. 2009 Sep;12(5):616-27
references.pmid: 19736621
references.citation: Science. 2004 Oct 8;306(5694):279-83
references.pmid: 15345777
references.citation: Eur J Biochem. 1999 Feb;259(3):660-9
references.pmid: 10092850
references.citation: Biochemistry. 2008 Jun 10;47(23):6242-50
references.pmid: 18470998
references.citation: Nat Struct Mol Biol. 2004 Aug;11(8):777-83
references.pmid: 15247907
references.citation: Neurochem Res. 2007 Feb;32(2):251-6
references.pmid: 17031564
references.citation: J Invest Dermatol. 2005 Apr;124(4):778-85
references.pmid: 15816836
references.citation: Biochemistry. 2006 Oct 3;45(39):11727-36
references.pmid: 17002273
references.citation: J Am Chem Soc. 2006 Feb 1;128(4):1092-3
references.pmid: 16433522
references.citation: J Am Chem Soc. 2008 Feb 13;130(6):1918-31
references.pmid: 18205354