Structural and kinetic analyses of macrophage migration inhibitory factor active site interactions.

Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

Macrophage migration inhibitory factor promotes intestinal tumorigenesis.

BACKGROUND & AIMS: The cytokine macrophage migration inhibitory factor (MIF) is expressed throughout the human gastrointestinal tract. Recently, protumorigenic activity of MIF has been described in several cancer models. Therefore, we investigated the expression and function of MIF during the early stages of intestinal tumorigenesis. METHODS: MIF messenger RNA, protein, and tautomerase activity were measured in normal intestinal mucosa and adenomas from patients with sporadic colorectal adenomas and in the adenomatous polyposis coli (Apc)Min/+ mouse model of intestinal tumorigenesis. MIF function was investigated by using VACO-235 human colorectal adenoma cells in vitro and by testing the effect of genetic deletion of Mif on ApcMin/+ mouse intestinal tumorigenesis. RESULTS: MIF expression and tautomerase activity were increased in human and ApcMin/+ mouse intestinal adenomas compared with adjacent normal mucosa. Up-regulation of MIF occurred mainly in epithelial cells (associated with an increasing grade of dysplasia), but also in stromal plasma cells. Exogenous MIF inhibited apoptosis and promoted anchorage-independent growth of VACO-235 cells (maximal at 100 ng/mL). Homozygous deletion of Mif was associated with a reduction in the number and size of ApcMin/+ mouse adenomas (P = .025 for the difference in large [>7-mm] tumors) and decreased angiogenesis (43% decrease in mean tumor microvessel density), but there was no alteration in epithelial cell apoptosis or proliferation. CONCLUSIONS: MIF expression is increased in sporadic human colorectal adenomas, and exogenous MIF drives tumorigenic behavior of epithelial cells in vitro. Mif also promotes intestinal tumorigenesis (predominantly via angiogenesis) in the ApcMin/+ mouse. Therefore, MIF is a potential colorectal cancer chemoprevention target.

Inhibition of MIF bioactivity by rational design of pharmacological inhibitors of MIF tautomerase activity.

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.

The tautomerase active site of macrophage migration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents.

Macrophage migration inhibitory factor (MIF) is an immunoregulatory protein that is a potential therapeutic target for a number of inflammatory diseases. Evidence exists that an unexpected catalytic active site of MIF may have a biological function. To gain further insight into the role of the catalytic active site, a series of mutational, structural, and biological activity studies were performed. The insertion of an alanine between Pro-1 and Met-2 (PAM) abolishes a non-physiological catalytic activity, and this mutant is defective in the in vitro glucocorticoid counter-regulatory activity of MIF. The crystal structure of MIF complexed to (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF d-dopachrome tautomerase activity, reveals that ISO-1 binds to the same position of the active site as p-hydroxyphenylpyruvic acid, a substrate of MIF. ISO-1 inhibits several MIF biological activities, further establishing a role for the catalytic active site of MIF.