Biochem. J. (2014) 462, 303–314 (Printed in Great Britain) doi:10.1042/BJ20140612 303 Potent inhibition of macrophage migration inhibitory factor (MIF) by myeloperoxidase-dependent oxidation of epicatechins Nina DICKERHOF* 1 , Nicholas J. MAGON*, Joel D. A. TYNDALL†, Anthony J. KETTLE* and Mark B. HAMPTON* 1 *Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, New Zealand †School of Pharmacy, University of Otago, Dunedin, New Zealand MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenance of the inflammatory response. It is implicated in a number of inflammatory diseases including sepsis, arthritis and colitis, and in diseases with an inflammatory component, such as atherosclerosis, diabetes and cancer. MIF has an unusual N-terminal proline with catalytic activity, and targeting of this residue by small-molecule inhibitors has been shown to interfere with the biological activity of MIF. The objective of the present study was to determine if MIF was susceptible to modification by epicatechins, a group of dietary flavonoids with known anti-inflammatory properties. Epicatechins are substrates for peroxidases including neutrophil- derived MPO (myeloperoxidase). In the present study we show that oxidation of the catechol moiety of epicatechins to an o-quinone by MPO generates potent MIF inhibitors. Near complete inhibition of MIF by the MPO/H 2 O 2 /epicatechin system was achieved at equimolar concentrations of epicatechin and MIF, even in the presence of other MPO substrates. We have characterized the modification introduced by oxidized (−)- epicatechin on MIF by LC-MS (liquid chromatography MS) and found it to occur at the N-terminal proline. We propose that MIF inhibition by oxidized epicatechins contributes to the anti- inflammatory activity of these compounds. Key words: dopachrome tautomerase, epicatechin, inflammation, macrophage migration inhibitory factor (MIF), myeloperoxidase (MPO). INTRODUCTION MIF (macrophage migration inhibitory factor) is emerging as an important therapeutic target for the treatment of multiple inflammatory and autoimmune diseases. Initially identified as a T-lymphocyte cytokine that inhibited the migration of macrophages during delayed hypersensitivity responses [1,2], MIF is now recognized to be released from a large number of cell types following exposure to infectious and inflammatory stimuli [3–7]. It acts by amplifying pro-inflammatory cytokine production [8], promoting cell survival [9,10] and counter-regulating the immunosuppressive action of glucocorticoids [11,12]. As such, MIF plays a key role in the pathogenesis of multiple diseases with direct or indirect inflammatory components. Elevated circulating levels of MIF have been observed in rheumatoid arthritis, atherosclerosis, asthma, lupus, diabetes, colitis, sepsis, cancer, and cardiovascular and infectious diseases [13–17]. Support for a significant role of MIF in the pathology of these diseases comes from the protection provided by gene targeting, anti-MIF antibodies and small-molecule inhibitors [13,18–22]. The details of MIF signalling pathways are yet to be resolved, but direct binding of MIF to the CD74–CD44 extracellular receptor complex accounts, in part, for its biological activity [23–25]. Intriguingly, MIF also has a keto-enol tautomerase activity, for which no physiological role has been attributed. This activity enables isomerization of substrates such as D-dopachrome and 4-hydroxyphenyl pyruvate [26,27] and is catalysed by the N-terminal proline residue. This conserved proline has an unusually low pK a value of 5.6 enabling it to act as a catalytic nucleophile at physiological pH [28]. Covalent modification of the proline inhibits tautomerase activity and impairs binding to the CD74 receptor [23,29–31]. Various small-molecule inhibitors have been developed to either bind the active site [32–34] or covalently modify the N-terminal proline residue [22,30]. These inhibitors display anti-inflammatory and anti-cancer activities in experimental models. Dietary isothiocyanates have been shown to inhibit MIF through covalent modification of the N-terminal proline residue [35]. We hypothesize that other electrophiles will be capable of reacting with MIF and modifying its biological activity. Epicatechins are the major constituents of green tea, but are also abundant in cocoa, black grapes and red wine [36,37]. They belong to the group of flavan-3-ols, with EC [(−)- epicatechin], ECG [(−)-epicatechin-3-gallate], EGC [(−)-epigal- locatechin] and EGCG [(−)-epigallocatechin-3-gallate] as the major epicatechins. They contain an o-diphenolic (catechol) moiety that can be oxidized to an o-quinone, thereby generating a potent electrophile (Figure 1A). Oxidation of o-diphenolic moieties to the o-quinone can be achieved chemically [38,39] or enzymatically by haem peroxidases [39–41]. EC is efficiently oxidized by the neutrophil-derived enzyme MPO (myeloperoxidase) [42], which is the predominant haem peroxidase present at sites of inflammation [43,44]. MPO oxidizes a number of physiological substrates via multiple mechanisms [45]. Hydrogen peroxide (H 2 O 2 ) converts ferric MPO into compound I. Chloride and other (pseudo)halides can directly reduce compound I back into the ferric enzyme via a two-electron reduction step, thereby being converted into hypochlorous and Abbreviations: AU, arbitrary units; AUC, area under the curve; CID, collision-induced dissociation; DCME, dopachrome methylester; L-Dopa-ME, L-3,4-dihydroxyphenylalanine methylester hydrochloride; EC, (−)-epicatechin; ECG, (−)-epicatechin-3-gallate; EGC, (−)-epigallocatechin; EGCG, (−)- epigallocatechin-3-gallate; HRP, horseradish peroxidase; HSA, human serum albumin; IAM, iodoacetamide; LC-MS, liquid chromatography MS; LPO, lactoperoxidase; MIF, macrophage migration inhibitory factor; MPO, myeloperoxidase; oxEC, oxidized EC; PPO, polyphenol oxidase; rhMIF recombinant human MIF. 1 Correspondence may be addressed to either of these authors (email nina.dickerhof@otago.ac.nz or mark.hampton@otago.ac.nz). c  The Authors Journal compilation c  2014 Biochemical Society Biochemical Journal www.biochemj.org