Inhibition of macrophage migration inhibitory factor (MIF) tautomerase and biological activities by acetaminophen metabolites

Abstract

The cytokine macrophage migration inhibitory factor (MIF) has emerged to be an important regulator of the inflammatory response and is critically involved in the development of septic shock, arthritis, and glomerulonephritis. Although the biological activities of MIF are presumed to require a receptor-based mechanism of action, the protein is also a tautomerase and has a catalytically active N-terminal proline that is invariant in structurally homologous bacterial isomerases. This observation raises the possibility that MIF may exert its biological action via an enzymatic reaction. Physiologically relevant substrates for MIF have not been identified, nor have site-directed mutagenesis studies consistently supported the requirement for a functional catalytic site. Small molecule inhibitors of MIF's isomerase activity also have been developed, but none have been shown yet to inhibit MIF biological activity. We report herein that the iminoquinone metabolite of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI), inhibits both the isomerase and the biological activities of MIF. The reaction between NAPQI and MIF is covalent and produces a NAPQI-modified MIF species with diminished cell binding activity and decreased recognition by anti-MIF mAb. These data are consistent with a model by which the NAPQI reacts with the catalytic Pro-1 of MIF to disrupt the integrity of epitope(s) critical to MIF's biological activity and point to the importance of the catalytic domain, but not the catalytic activity per se, in MIF function. These results also point to a powerful approach for the design of small molecule inhibitors of MIF based on interaction with its catalytic site and constitute an example of a pharmacophore capable of irreversibly inhibiting the action of a proinflammatory cytokine.

ISO-1 Binding to the Tautomerase Active Site of MIF Inhibits Its Pro-inflammatory Activity and Increases Survival in Severe Sepsis

“…Unfortunately, L-Trp Schiff base compounds lack long term stability. We also have shown that a P450-dependent metabolite of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to MIF at its enzymatic site (23). The NAPQI adduct inactivates MIF cytokine activity in a number of in vitro bioassays, including interference with the anti-inflammatory effect of dexamethasone, After incubation with FL-ISO1, the cells were washed with PBS and fixed using 4% formaldehyde.…”

“…The medium was then replaced with ISO-1-free medium. The cells were then lysed and the tautomerase activity determined (17,23) (Fig. 1b).…”

The Tautomerase Active Site of Macrophage Migration Inhibitory Factor Is a Potential Target for Discovery of Novel Anti-inflammatory Agents

“…The involvement of the MIF catalytic site in biological activity has also been studied using small molecule inhibitors (37,38). We have previously studied a series of pharmacological inhibitors of MIF that were designed utilizing the known information about the MIF-binding site and the known chemical structures of substrates of MIF.…”

“…We have generated a series of pharmacological inhibitors of MIF based on structure-activity studies of other inhibitors and a non-physiological substrate. An acetaminophen metabolite, N-acetylbenzoquinone imine, forms a covalent bond with MIF and inhibits MIF catalytic activity (37). We recently used rational design and synthesis of p-hydroxyphenyl Schiff bases as inhibitors of MIF enzymatic activity (38).…”

Phenolic Hydrazones Are Potent Inhibitors of Macrophage Migration Inhibitory Factor Proinflammatory Activity and Survival Improving Agents in Sepsis

“…9 While the physiological role of the tautomerase activity is uncertain, we have previously found that compounds, which are structurally similar to D-and L-dopachrome, could bind to, and thereby block the MIF's tautomerase active site. 4,[10][11][12][13] We have previously shown that N-acetyl-p-benzoquinone imine (NAPQI) forms a covalent complex with MIF at its active site (Scheme 1) and is capable of irreversibly inhibiting the adverse biological effect of MIF. 13 However, the toxicity of NAPQI precluded its use as a viable clinical inhibitor of MIF and the development of non-toxic small molecule inhibitors of MIF tautomerase activity warranted further investigation.…”