Although PAD4 has been most extensively studied as a potential therapeutic target in RA (mainly based on the availability of a crystal structure [51
]), PAD2 may also be important. It is proposed that selective inhibition of PAD would reduce the levels of citrullinated proteins and consequently suppress the humoral immune response directed to citrullinated antigens in RA. Because PAD4 has an important physiological role in regulating gene expression and PAD4 translocates into the nucleus from the cytosol, potential inhibitors may need to be selective for the extracellular compartment or other PAD isotypes to avoid unwanted effects on gene transcription. It is, however, not known whether intracellular or extracellular PAD is important in the pathophysiology of RA.
Paclitaxel is a chemotherapeutic agent that was initially derived from the bark of the Pacific yew tree. It inhibits angiogenesis by interfering with microtubule function in cell mitosis, migration, chemotaxis, and intracellular transport [55
]. In addition, in the millimolar range (half-maximal inhibitory concentration [IC50
] = approximately 5 mM), paclitaxel inhibits PAD isolated from bovine brain [56
]. It has been shown to prevent the induction of collagen-induced arthritis (CIA) and cause significant regression of existing CIA [57
]. An open-label multicenter phase II study of paclitaxel in patients with RA was completed in July 2008, although results of this are still pending [58
Other PAD inhibitors include F-amidine [N
-(2-fluoro-1-iminoethyl)-l-ornithine amide], Cl-amidine [N
-(2-chloro-1-iminoethyl)-l-ornithine amide], and 2-chloroacetamidine, of which Cl-amidine was reported to be the most potent (IC50
= 5.9 μM) [59
]. Ex vivo
studies with F-amidine and Cl-amidine, using a cell line and an assay measuring PAD4-mediated citrullination of a nuclear protein and the resulting enhancement in binding to another protein, indicated that these inhibitors are bioavailable [59
]. F-amidine irreversibly inhibits PAD4 via the specific modification of Cys 645, an active-site residue that is critical for enzyme catalysis. Cys 645 acts as a nucleophile to form a thiouronium intermediate that is hydrolyzed to form citrulline. Cl-amidine and 2-chloroacetamidine are thought to act via a similar mechanism [59
]. Inactivation by F-amidine and Cl-amidine is calcium-dependent [60
]. In vitro
studies with PAD4 have shown that calcium binding leads to a conformational change that moves Cys 645 and His 471 into positions that are competent for catalysis [51
] and presumably reactive with F-amidine and Cl-amidine. This is of therapeutic importance as these compounds would therefore be expected to inhibit PAD4 in its activated state only at sites of inflammatory activity such as the synovium and not the inactive enzyme at other sites in the body, limiting toxicity [59
]. Willis and colleagues [62
] recently showed that Cl-amidine treatment in CIA is able to inhibit clinical disease activity scores by 55%, 53%, and 42% in the 50, 10, and 1 mg/kg per day groups, respectively. Histological severity scores and complement C3 deposition scores paralleled the decreases in disease activity. In addition, mice receiving Cl-amidine showed reduced epitope spreading by peptide microarray, especially to citrullinated joint antigens. Interestingly, there were no changes in the percentages of T-cell, B-cell, or monocyte populations in treated mice compared with controls [62
]. These results suggest that Cl-amidine may represent a novel class of RA therapeutics that specifically target citrullination.
Bhattacharya and colleagues [63
] demonstrated that human astrocytes subject to pressure showed elevated PAD2 levels, increased intracellular calcium concentrations, and increased citrullination. Treatment with the cell-permeable calcium chelating agent BAPTA-AM (1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester) resulted in decreased intracellular calcium concentration and PAD2 levels. These results suggest that calcium modulation may be an alternative therapeutic strategy in modulating PAD activity and citrullination, although we would argue that this mechanism is too broad to be applicable in practice.
On the basis of the therapeutic use of tetracyclines in RA [23
], Knuckley and colleagues [28
] screened tetracycline derivatives (minocycline, doxycycline, tetracycline, and chlortetracycline) for their potential to inhibit PAD4 activity. Chlortetracycline was identified as the most potent inhibitor (IC50
= 100 μM) and was suggested to bind to a region distal from the active site [28
]. Streptomycin, an aminoglycoside antibiotic, was also tested because of its two guanidinium groups that could act as inhibitors of PAD4. Streptomycin was found to inhibit PAD4, though with a lower potency (IC50
= approximately 1.8 mM), and was suggested to bind within or in close proximity to the active site. The data suggest that these compounds could provide a valuable scaffold for engineering inhibitors with greater potency and selectivity.