UMass Chan Medical School Faculty Publications

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Department of Biochemistry and Pharmacology; Thompson Lab

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Amino Acids, Peptides, and Proteins | Biochemical Phenomena, Metabolism, and Nutrition | Biochemistry, Biophysics, and Structural Biology | Enzymes and Coenzymes | Immune System Diseases | Immunology and Infectious Disease | Musculoskeletal Diseases


Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4(+) T cells, CD8(+) T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 muM, while BB-Cl-amidine was cytotoxic at concentrations above 1 muM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.


peptidyl arginine deiminase (PAD), citrullination, small-molecule PAD inhibitors, cell viability, rheumatoid arthritis

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Copyright © 2021 Martín Monreal, Rebak, Massarenti, Mondal, Šenolt, Ødum, Nielsen, Thompson, Nielsen and Damgaard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

DOI of Published Version



Martín Monreal MT, Rebak AS, Massarenti L, Mondal S, Šenolt L, Ødum N, Nielsen ML, Thompson PR, Nielsen CH, Damgaard D. Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4. Front Immunol. 2021 Oct 19;12:716250. doi: 10.3389/fimmu.2021.716250. PMID: 34737738; PMCID: PMC8560728. Link to article on publisher's site

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Journal/Book/Conference Title

Frontiers in immunology

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Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.