N-α-Benzoyl-N5-(2-Chloro-1-Iminoethyl)-l-Ornithine Amide, a Protein Arginine Deiminase Inhibitor, Reduces the Severity of Murine Collagen-Induced Arthritis

Abstract

Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0–35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine–treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus

“…Previous studies have shown that daily Cl-amidine treatment does not alter the frequency or absolute number of T cells (total and subsets), B cells, NK cells, or monocytes (32). We therefore predicted that Cl-amidine, in contrast to what we observed for neutrophils and NET formation, would not robustly inhibit lymphocyte phenotype and function in vitro.…”

“…Also, while Cl-amidine treatment robustly inhibited NET formation in vitro, it did not have any effect on T or B cell proliferation, apoptosis, or activation. Further, the work of others has shown that daily Cl-amidine treatment does not alter the frequency or absolute number of T cells (total and subsets), B cells, NK cells, or monocytes (32). Nevertheless, how PAD inhibition may directly affect other cells of the innate and adaptive immune systems relevant to SLE should be the focus of future investigation.…”

“…Indeed, data to date points toward ROS formation being upstream of PAD4 activation in neutrophils (69), supporting the idea that PAD inhibition is a more targeted strategy for inhibiting NET formation. Given the ability of Cl-amidine to also improve other inflammatory disease phenotypes (31,32), it will be of interest to revisit those inflammatory murine models in the context of what we now know about Cl-amidine's effectiveness in inhibiting NET formation in mice.…”

“…Indeed, N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-Lornithine amide, or Cl-amidine (30), has previously been shown to inhibit PADs in mice without significant toxicity and to improve disease phenotypes in animal models of inflammatory arthritis and inflammatory bowel disease (31,32). PADs are a family of 5 calcium-dependent enzymes (PAD1, -2, -3, -4, and -6) that catalyze the conversion of peptidyl-arginine residues to peptidyl-citrulline by a hydrolytic reaction, which is variably termed deimination or citrullination (33).…”

Citrullination: A Specific Target for the Autoimmune Response in Rheumatoid Arthritis

“…Thus, proinflammatory effects of citrullination that are independent of ACPAs could influence RA and many other diseases. Indeed, in the mouse RA model of collagen-induced arthritis, inhibition of PAD reduces disease severity even though collagen-induced arthritis is not dependent on ACPAs (19). Inhibition of PAD also reduces vascular damage in a murine model of systemic lupus (20).…”