TY - JOUR

T1 - Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4

AU - Martín Monreal, María Teresa

AU - Rebak, Alexandra Stripp

AU - Massarenti, Laura

AU - Mondal, Santanu

AU - Šenolt, Ladislav

AU - Ødum, Niels

AU - Nielsen, Michael L.

AU - Thompson, Paul R.

AU - Nielsen, Claus H.

AU - Damgaard, Dres

N1 - Publisher Copyright: © Copyright © 2021 Martín Monreal, Rebak, Massarenti, Mondal, Šenolt, Ødum, Nielsen, Thompson, Nielsen and Damgaard.

PY - 2021

Y1 - 2021

N2 - 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 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, 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.

AB - 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 μM, while BB-Cl-amidine was cytotoxic at concentrations above 1 μM, 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.

KW - cell viability

KW - citrullination

KW - peptidyl arginine deiminase (PAD)

KW - rheumatoid arthritis

KW - small-molecule PAD inhibitors

U2 - 10.3389/fimmu.2021.716250

DO - 10.3389/fimmu.2021.716250

M3 - Journal article

C2 - 34737738

AN - SCOPUS:85118584824

VL - 12

JO - Frontiers in Immunology

JF - Frontiers in Immunology

SN - 1664-3224

M1 - 716250

ER -