Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4
AuthorsMartin Monreal, Maria Teresa.
Rebak, Alexandra Stripp
Nielsen, Michael L.
Thompson, Paul R
Nielsen, Claus H.
Document TypeJournal Article
Keywordspeptidyl arginine deiminase (PAD)
small-molecule PAD inhibitors
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
MetadataShow full item record
AbstractCitrullination, 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.
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