Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

Abstract

AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

Fig. 1.

Chemical structures and kinetic graphs of AV411, AV1013, and ISO-1. Each Lineweaver–Burk plot reveals the noncompetitive inhibition pattern of AV411 (A) and AV1013 (B), and the competitive inhibition pattern of ISO-1 (C). The Lineweaver–Burk of AV1013 demonstrates a small effect on K_m (B). The inserts in the Lineweaver–Burk plots show the intersections near the origin. Dissociation constants (K_ds) measured by Trp (AV411 and AV1013) and Tyr (ISO-1) fluorescence are presented below the chemical structures.

Fig. 2.

PBMC cell migration inhibition. PBMC migration dose response for (A) AV411 and (B) (R)-AV1013. For all panels, negative control (NEG) is without rhMIF. Positive control is with rhMIF but no drug added. P values represent significant difference relative to positive control. One, two, or three asterisks represent p < 0.05, 0.01, or 0.001, respectively. Inhibitors (INH) in (A) and (B) are AV411 and (R)-AV1013, respectively. (C) Inhibition of MIF-induced chemotaxis of PBMCs in the presence of individual or paired combinations of monoclonal antibodies (10 μg/mL each) directed against MIF receptors (CD74, CXCR2, and CXCR4). P values represent comparison to the MIF-mediated migration in the absence of the antibodies.

Fig. 3.

AV411 and AV1013 interactions with rhMIF in solution. (A) HSQC spectra of rhMIF with (red) or without (blue) AV411 are overlaid. (B) The surface of a rhMIF trimer is shown with the degree of chemical shift change, colored in red, upon binding of AV411 (Left). The highest intensity indicates 0.094 ppm of chemical shift change. On the right, is the surface of the trimer rotated by 90° along the trimer axis and clipped to show relative locations of the mapped residues. The residues are colored in the same manner as with the left. Only the residues revealing large chemical shifts are labeled.

Fig. 4.

rhMIF/(R)-AV1013 crystal structure. (A) Simulated annealing electron density calculated omitting (R)-AV1013 and contoured at 3.0σ, showing AV1013 (in magenta) between Tyr36 and Trp108. (B) Surface representation of AV1013-bound MIF in the same orientation displayed in Fig. 3 and Fig. S2. The inhibitor binds in a site predicted by the NMR results. (C) Superposition of the complex structure of rhMIF-(R)AV1013 (carbons in green for the allosteric site and yellow for the active site) and apo-rhMIF (carbons in white) showing the conformational shift of Tyr36 that allows AV1013 (all atoms in magenta) to bind. The superposition was performed by overlaying the positions of Cα atoms of residues 2–30 and 37–114 due to larger rms deviations for Pro1 and the loop including residues 31–36.

Fig. 5.

rhMIF-HPP-(R)AV1013 ternary complex. (A) An active site with only HPP bound. HPP can be modeled as the enol (carbon atoms in cyan) and keto (carbon atoms in pink) form within the same active site, suggesting that interconversion between these two forms occurs in the active site. The simulated annealing electron density displayed in A and B is 3.0σ F_o-F_c density calculated omitting all of substrate molecules as well as Pro1. (B) The ternary complex with HPP in the active site and (R)-AV1013 in the neighboring allosteric site. (C) Proximity of allosterically bound (R)-AV1013 to the active site bound HPP shown in stereo with carbon atoms of the protein shown in green for the allosteric site and yellow for the active site, those of the inhibitor shown in magenta, those of the enol form of HPP in cyan, and those of the Pro1 proximal HPP in gray. The conformational shift of Tyr36 observed in the binary structure (Fig. 4B) is also seen here in the ternary complex in the (R)-AV1013-bound subunit.