MIF is a proinflammatory cytokine that has been implicated in the pathogenesis of sepsis, arthritis, and other inflammatory diseases. Antibodies against MIF are effective in experimental models of inflammation, and there is interest in strategies to inhibit its deleterious cytokine activities. Here we identify a mechanism of inhibiting MIF pro-inflammatory activities by targeting MIF tautomerase activity. We designed small molecules to inhibit this tautomerase activity; a lead molecule, "ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)," significantly inhibits the cytokine activity in vitro. Moreover, ISO-1 inhibits tumor necrosis factor release from macrophages isolated from LPStreated wild type mice but has no effect on cytokine release from MIFdeficient macrophages. The therapeutic importance of the MIF inhibition by ISO-1 is demonstrated by the significant protection from sepsis, induced by cecal ligation and puncture in a clinically relevant time frame. These results identify ISO-1 as the first small molecule inhibitor of MIF proinflammatory activities with therapeutic implications and indicate the potential of the MIF active site as a novel target for therapeutic interventions in human sepsis.MIF is an important pro-inflammatory cytokine that has been implicated in the pathogenesis of inflammatory disorders (1-6). Administration of neutralizing anti-MIF antibodies has proven therapeutically effective in numerous animal models of systemic inflammation, including Gram-negative, Grampositive, and polymicrobial sepsis, arthritis, and autoimmune diabetes (1-4, 7, 8). Circulating MIF levels are elevated in animals with sepsis and in patients with severe sepsis and septic shock (1). These and other results indicate that inhibiting MIF is a promising approach to develop new anti-inflammatory agents.Three-dimensional x-ray crystallography of MIF shows that the molecule exists as a homotrimer (9 -11). This trimer possesses the ability to catalyze the tautomerization of the non-physiological substrates DL-dopachrome methyl esters (supplemental Fig. 1) into their corresponding indole derivatives (11,12). Crystallographic analysis of MIF complexed with p-hydroxyphenylpyruvic acid, a known MIF substrate (13), has revealed an active site which lies in a hydrophobic cavity formed between two adjacent subunits of the homotrimer (14). Tautomerase activity is an evolutionarily ancient phenomenon, which early life forms presumably utilized for synthesis, but there is no evidence that modern species use this in synthetic pathways. We reasoned that molecules that bind this site could be useful to target MIF function, because the tautomerase activity is expendable. We have designed a molecule to fit into the catalytic site and shown that (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) 2 is a potent inhibitor of MIF tautomerase activity (15). The crystal structure of MIF complexed to ISO-1 reveals that ISO-1 binds to the enzymatic active site. MATERIALS AND MET...

Macrophage migration inhibitory factor (MIF) is an immunoregulatory protein that is

The pro-inflammatory mediator macrophage migration inhibitory factor (MIF) is produced by immune and endocrine cells and inhibits the antiinflammatory activities of glucocorticoids. MIF also catalyzes the tautomerization of the non-naturally occurring D-isomer of dopachrome, phenylpyruvate, and certain catecholamines, suggesting that MIF might exert its biological effects via enzymatic action on a substrate. However, no physiologically relevant substrate for MIF has been identified. Site-directed mutagenesis studies have not consistently supported a requirement for an intact, functional catalytic site as a prerequisite for MIF bioactivity. We hypothesized that the catalytically active site, but not the enzymatic activity per se, nevertheless plays a critical role in MIF pro-inflammatory activity. Accordingly, we designed small druglike molecules that bind at the catalytically active tautomerase site of MIF and tested the complex for MIF bioactivity. We describe herein the rational design and synthesis of a class of imine conjugates produced by coupling amino acids to a range of benzaldehyde derivatives that inhibit MIF tautomerase and biological activities. We found that aromatic amino acid Schiff bases were better inhibitors of MIF enzymatic and bioactivities compared to the aliphatic ones. For instance, the IC(50) inhibition of MIF tautomerase activity by aromatic amino acid Schiff base methyl esters was achieved at a concentration between 1.65 and 50 microM, suggesting a critical role for the additional binding of the aromatic residues within the vicinity of the active site. The most potent inhibitor of MIF tautomerase activity was 2-[(4-hydroxybenzylidene)amino]-3-(1H-indol-3-yl)propionic acid methyl ester (8), with an IC(50) of 1.65 microM. We found that compound 8 binding to MIF active site resulted in the inhibition of MIF bioactivity in three established bioassays: ERK-1/2 MAP kinase activation, p53-dependent apoptosis, and proliferation of serum-starved cells. Compound 8 inhibited MIF interaction with its as yet unidentified cognate cell surface receptor as shown by flow cytometry, concluding a critical role for the tautomerase active site in receptor binding. Thus the inhibitory effect of compound 8 on MIF bioactivities strongly correlated with the inhibition of MIF tautomerase activity, a connection not made previously through use of small-molecule MIF inhibitors. The inhibitory activity of amino acid-benzaldehyde Schiff base-type MIF antagonists is the first step toward a meaningful structure/function analysis of inhibitors of MIF cellular bioactivities.

Previous studies have shown that amyloid β protein (Aβ), the essential molecule for the formation of toxic oligomers and, subsequently, Alzheimer plaques, has been associated in vivo with the immune modulator, macrophage migration inhibitory factor (MIF) (17). To further investigate this association in vivo we used the APP transgenic mouse model. Serial brain sections of transgenic APP mice were stained for Aβ plaques and MIF and we observed MIF immunolabeling in microglial cells in association with Aβ plaques in the transgenic mouse brain sections. In addition, functional studies in murine and human neuronal cell lines revealed that Aβ-induced toxicity could be reversed significantly by a small molecule inhibitor of MIF (ISO-1). Finally, to elucidate the role of MIF in Alzheimer's Disease (AD) we measured MIF levels in the brain cytosol and cerebrospinal fluid (CSF) of AD patients and age-matched controls. Our results demonstrate a marked increase of MIF levels within the CSF of AD patients compared with controls. Combined, our results indicate a strong role for MIF in the pathogenesis of AD and furthermore suggest that inhibition of MIF may provide a valuable avenue of investigation for the prevention of disease onset, progression and/or severity.