A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF)

Abstract

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

FIGURE 1.

High throughput screening for rhMIF HPP tautomerase inhibitors. A, keto-enol tautomerization of HPP catalyzed by rhMIF. B, spectrophotometric detection of rhMIF-catalyzed HPP tautomerase reaction in a 384-well plate format. C, scatter plot of HTS outcome for one of the two replicates of 230,000 compounds. Compounds giving >50% inhibition above the line were classified as hits. D, distribution of the 2,430 hits, categorized into five groups with respect to percent inhibition.

FIGURE 2.

Tautomerase inhibitors block rhMIF binding to CD74-receptor (A) and the structure of p425 (B). A, inhibition of rhMIF-CD74 interaction by four tautomerase inhibitors (p425, 1D2, 1H2, and 2F10) identified from HTS. Biotinylated rhMIF binding to soluble CD74 was detected colorimetrically (A₄₀₅) with a streptavidin-alkaline phosphatase conjugate and p-nitrophenyl phosphate substrate. The most potent confirmed screening hit was p425 with an IC₅₀ of 0.81 μm, ∼10-fold higher than that of anti-MIF antibody (anti-MIF). B, chemical structure of p425 also known as Chicago Sky Blue 6B.

FIGURE 3.

p425 impairs MIF-mediated production of IL-6, IL-8, and MMP-3 and inhibition of p53-dependent apoptosis. A, inhibition of rhMIF-induced IL-6 (left panel) and IL-8 (right panel) production in human foreskin fibroblast cells in the presence of p425 (white), ISO-1 (stipple), and anti-MIF antibody (black). IL-6 and IL-8 levels in the culture medium were determined by ELISA, and the percent inhibition was calculated with respect to a no-inhibitor control. Dose-dependent inhibition of rhMIF-induced IL-6 (B) and IL-8 (C) production in the presence of p425 and ISO-1. D, inhibition of MMP-3 production in the presence of p425 and ISO-1 in primary human synovial fibroblast cells. MMP-3 levels in the culture medium were determined by ELISA, and the percent inhibition was calculated with respect to a no-inhibitor control. E, p425 impairs the ability of rhMIF to inhibit p53-dependent apoptosis. HeLa cells were exposed to camptothecin (CAM) and then treated with rhMIF or rhMIF plus p425. Apoptosis was measured by ELISA. The plot shows the level of apoptosis in untreated (CAM), treated with rhMIF alone (CAM+MIF) and rhMIF in the presence of 2, 10, and 50 μm p425. F, p425 does not cause significant cytotoxicity in human foreskin fibroblast cells.

FIGURE 4.

Crystallographic analysis of rhMIF-p425 complex. A, ribbon diagram depicting the unique binding of the p425 at the interface of two of the tightest packed rhMIF crystallographic trimers. The ligand makes contacts with monomer A (blue) and monomer C (cyan) of one trimer and with monomer B (magenta) of an adjacent trimer. B, ligplot representation of rhMIF-p425 interaction reveals hydrogen bonding (dashed lines) with four residues, Lys³² and Asn¹¹⁰ from one monomer, Glu⁵⁴ from a second monomer, and Ser⁵³ from a monomer of an adjacent trimer. C and D, superimposition of p425-rhMIF complex with HPP-rhMIF and coumarin derivative-rhMIF, respectively. Arrowheads indicate the respective ligands in the active site.