Discovery Of Novel Inhibitors Targeting Macrophage Migration Inhibitory Factor (MIF) As Anti-Inflammatory Agents

Macrophage migration inhibitory factor(MIF), a multifunctional protein, is involved in regulating innate and adaptive immune response. As a proinflammation cytokine, MIF can promote other inflammatory cytokines and override the immunosuppressive effect of glucocorticoids(GCs). As such MIF has been regarded as an attractive anti-inflammatory pharmacological target. In addition, MIF has enzymatic activity. Blocking of endogenous MIF by tautomarese activity inhibitorhas shown to be therapeutically beneficial in a variety of animal models of inflammatory diseases. Therefore, development of novel small-molecule inhibitors of MIF are relentlessly sought as potential anti-inflammatory agents for treatming various MIF-related inflammatory diseases.In this study, molecular docking-based virtual screening and in vitro bioassays were utilized to identify novel small-molecule inhibitors of MIF. The in vitro enzyme-based assay identified that ten chemically diverse compounds exhibited potent inhibitory activity against MIF in the micromolar regime, including three compounds with IC50 values below 10 ?M and one below 1 ?M(0.55 ?M), the later is 26-fold more potent than that of the reference compound ISO-1. Further in vitro cell-based glucocorticoid overriding, chemotaxis and western bloting assays revealed that the three compounds can effectively inhibit the biological functions of MIF in vitro, suggesting that these compounds could be potential agents for treating inflammatory diseases.As the role of MIF inhibitors in neuroinflmmation has not been explored. Then we studied the anti-inflammatory effects of compound 1(Z-590). Our results demonstrated that MIF inhibitor Z-590 significantly decreased the production of nitric oxide(NO), tumor necrosis factor-?(TNF-?), interleukin-6(IL-6), interleukin-1?(IL-1?), cyclooxygenase-2(COX-2), inducible nitric oxide synthase(iNOS) as well as reactive oxygen species(ROS) in lipopolysaccharide(LPS)-activated BV-2 cells. We also found that Z-590 inhibited mitogen-activated protein kinase(MAPKs) signal pathway which may contribute to the drug-mediated inhibition on LPS-induced microglia activation. Furthermore, we found that Z-590 reduced cytotoxicity of activated microglia toward HT22 hippocampal cells in a microglia-conditional medium system.To further study the anti-inflammation of MIF tautomerase inhibitor, a series of analogue of compound 1(Z-590) derived from similarity search and chemical synthesis were evaluated for MIF tautomerase activities, and their structure-activity relationships were then analyzed. The most potent inhibitor(compound 5) with an IC50 of 370 nM strongly suppressed LPS-induced production of TNF-? and IL-6 in a dose-dependent manner and significantly enhanced the survival rate of mice with LPS-induced endotoxic shock from 0 to 35% at 0.5 mg/kg and to 45% at 1 mg/kg, highlighting the therapeutic potential of the MIF tautomerase inhibition in inflammatory diseases.Taken together, we discovered a series of novel MIF inhibitors and found they were not only inhibited the MIF induced biological activity, but also had protective effect on LPS-induced endotoxic shock. In addition, we provided the first evidence depiciting that MIF tautomerse inhibitor elicited strong anti-inflammatory effect in activated microglia, implying that MIF tautomerse inhibitors may provide a novel approach for drug discovery of inflammatory diseases including neuro-immunological diseases.