Development of a Selective Inhibitor of Protein Arginine Deiminase 2.

Protein arginine deiminase 2 (PAD2) plays a key role in the onset and progression of multiple sclerosis, rheumatoid arthritis, and breast cancer. To date, no PAD2-selective inhibitor has been developed. Such a compound will be critical for elucidating the biological roles of this isozyme and may ultimately be useful for treating specific diseases in which PAD2 activity is dysregulated. To achieve this goal, we synthesized a series of benzimidazole-based derivatives of Cl-amidine, hypothesizing that this scaffold would allow access to a series of PAD2-selective inhibitors with enhanced cellular efficacy. Herein, we demonstrate that substitutions at both the N-terminus and C-terminus of Cl-amidine result in >100-fold increases in PAD2 potency and selectivity (30a, 41a, and 49a) as well as cellular efficacy (30a). Notably, these compounds use the far less reactive fluoroacetamidine warhead. In total, we predict that 30a will be a critical tool for understanding cellular PAD2 function and sets the stage for treating diseases in which PAD2 activity is dysregulated.

[1]  G. Pruijn,et al.  Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen , 2014, Arthritis Research & Therapy.

[2]  N. L. La Thangue,et al.  Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses , 2016, Scientific Reports.

[3]  J. Hodgin,et al.  Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice , 2014, Annals of the rheumatic diseases.

[4]  M. Gross,et al.  Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design , 2015, ACS chemical biology.

[5]  Yuan Luo,et al.  A fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis, and in vitro and in vivo evaluation. , 2006, Journal of the American Chemical Society.

[6]  Graeme Winter,et al.  xia2: an expert system for macromolecular crystallography data reduction , 2010 .

[7]  P. Thompson,et al.  Chemical Proteomic Platform To Identify Citrullinated Proteins , 2015, ACS chemical biology.

[8]  P. Thompson,et al.  Peptidylarginine Deiminase Inhibition Reduces Vascular Damage and Modulates Innate Immune Responses in Murine Models of Atherosclerosis , 2014, Circulation research.

[9]  Xiangli Cui,et al.  D-amino acid based protein arginine deiminase inhibitors: Synthesis, pharmacokinetics, and in cellulo efficacy. , 2012, ACS medicinal chemistry letters.

[10]  Michael J. Bolt,et al.  Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor α target gene activation , 2012, Proceedings of the National Academy of Sciences.

[11]  P. Thompson,et al.  A FluoPol-ABPP PAD2 High-Throughput Screen Identifies the First Calcium Site Inhibitor Targeting the PADs , 2014, ACS chemical biology.

[12]  E. Girbal-Neuhauser,et al.  The Major Synovial Targets of the Rheumatoid Arthritis-Specific Antifilaggrin Autoantibodies Are Deiminated Forms of the α- and β-Chains of Fibrin1 , 2001, The Journal of Immunology.

[13]  Toshiyuki Shimizu,et al.  Structural basis for Ca2+-induced activation of human PAD4 , 2004, Nature Structural &Molecular Biology.

[14]  S. Chowdhury,et al.  Identification of multiple structurally distinct, nonpeptidic small molecule inhibitors of protein arginine deiminase 3 using a substrate-based fragment method. , 2015, Journal of the American Chemical Society.

[15]  D. Deforce,et al.  The relevance of citrullinated vimentin in the production of antibodies against citrullinated proteins and the pathogenesis of rheumatoid arthritis , 2011, Rheumatology.

[16]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[17]  M. Moscarello,et al.  Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain , 1993, Journal of neurochemistry.

[18]  Olof Svensson,et al.  Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF , 2013, Journal of applied crystallography.

[19]  Pojen P. Chen,et al.  NETs Are a Source of Citrullinated Autoantigens and Stimulate Inflammatory Responses in Rheumatoid Arthritis , 2013, Science Translational Medicine.

[20]  P. Thompson,et al.  Synthesis and screening of a haloacetamidine containing library to identify PAD4 selective inhibitors. , 2012, ACS chemical biology.

[21]  Reinout Raijmakers,et al.  Methylation of arginine residues interferes with citrullination by peptidylarginine deiminases in vitro. , 2007, Journal of molecular biology.

[22]  Paul R. Thompson,et al.  Chemical Biology of Protein Arginine Modifications in Epigenetic Regulation , 2015, Chemical reviews.

[23]  J. Ellman,et al.  Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors. , 2016, ACS medicinal chemistry letters.

[24]  Philip R. Evans,et al.  How good are my data and what is the resolution? , 2013, Acta crystallographica. Section D, Biological crystallography.

[25]  F. Mastronardi,et al.  Peptidylarginine deiminase: a candidate factor in demyelinating disease , 2002, Journal of neurochemistry.

[26]  Graeme Winter,et al.  Decision making in xia2 , 2013, Acta crystallographica. Section D, Biological crystallography.

[27]  P. Thompson,et al.  Mechanistic Studies of Protein Arginine Deiminase 2: Evidence for a Substrate-Assisted Mechanism , 2014, Biochemistry.

[28]  P. Thompson,et al.  Kinetic characterization of protein arginine deiminase 4: a transcriptional corepressor implicated in the onset and progression of rheumatoid arthritis. , 2005, Biochemistry.

[29]  P. Evans,et al.  Scaling and assessment of data quality. , 2006, Acta crystallographica. Section D, Biological crystallography.

[30]  N. Greene,et al.  Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability. , 2011, Developmental biology.

[31]  S. Knapp,et al.  Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response , 2016, Science Advances.

[32]  N. Shimizu,et al.  Monomeric Form of Peptidylarginine Deiminase Type I Revealed by X-ray Crystallography and Small-Angle X-ray Scattering. , 2016, Journal of molecular biology.

[33]  Paul Tempst,et al.  Histone Deimination Antagonizes Arginine Methylation , 2004, Cell.

[34]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[35]  E. Vossenaar,et al.  PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[36]  O. Griffith,et al.  Identification of PADI2 as a potential breast cancer biomarker and therapeutic target , 2012, BMC Cancer.

[37]  P. Thompson,et al.  Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3. , 2010, Biochemistry.

[38]  M. Knipp,et al.  A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline. , 2000, Analytical biochemistry.

[39]  Mariana J. Kaplan,et al.  Neutrophils in the pathogenesis and manifestations of SLE , 2011, Nature Reviews Rheumatology.

[40]  P. Thompson,et al.  Haloacetamidine‐Based Inactivators of Protein Arginine Deiminase 4 (PAD4): Evidence that General Acid Catalysis Promotes Efficient Inactivation , 2010, Chembiochem : a European journal of chemical biology.

[41]  P. Thompson,et al.  Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic potential. , 2009, Current opinion in drug discovery & development.

[42]  P. Thompson,et al.  Design, Synthesis, and Biological Evaluation of Tetrazole Analogs of Cl-Amidine as Protein Arginine Deiminase Inhibitors , 2015, Journal of medicinal chemistry.

[43]  Remco Loos,et al.  Citrullination regulates pluripotency and histone H1 binding to chromatin , 2014, Nature.

[44]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[45]  W. Robinson,et al.  N-α-Benzoyl-N5-(2-Chloro-1-Iminoethyl)-l-Ornithine Amide, a Protein Arginine Deiminase Inhibitor, Reduces the Severity of Murine Collagen-Induced Arthritis , 2011, The Journal of Immunology.

[46]  Yuan Luo,et al.  Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization. , 2006, Biochemistry.

[47]  P. Thompson,et al.  Potential Role of Peptidylarginine Deiminase Enzymes and Protein Citrullination in Cancer Pathogenesis , 2012, Biochemistry research international.

[48]  Yuan Luo,et al.  The development of N-α-(2-carboxyl)benzoyl-N(5)-(2-fluoro-1-iminoethyl)-l-ornithine amide (o-F-amidine) and N-α-(2-carboxyl)benzoyl-N(5)-(2-chloro-1-iminoethyl)-l-ornithine amide (o-Cl-amidine) as second generation protein arginine deiminase (PAD) inhibitors. , 2011, Journal of medicinal chemistry.

[49]  C. Allis,et al.  Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation , 2009, The Journal of cell biology.

[50]  Steven Clarke,et al.  Human PAD4 Regulates Histone Arginine Methylation Levels via Demethylimination , 2004, Science.

[51]  A. Nicholls,et al.  Automated ligand placement and refinement with a combined force field and shape potential. , 2006, Acta crystallographica. Section D, Biological crystallography.

[52]  P. Emery,et al.  Autoantibodies to Posttranslational Modifications in Rheumatoid Arthritis , 2014, Mediators of inflammation.

[53]  Deepak Poudyal,et al.  Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor. , 2011, American journal of physiology. Gastrointestinal and liver physiology.