Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

Abstract

Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

Relative Binding Free Energy Calculations Applied to Protein Homology Models

“…Furthermore, a very potent inhibitor for the macrophage migration inhibitory factor (MIF or MMIF) was developed with the help of FEP calculations. 23 …”

A computational assessment of the predicted structures of Human Macrophage Migration Inhibitory Factor 1 orthologs in parasites and its affinity to human CD74 receptor

“…These residues establish interactions with the inhibitor in the crystal structure of the Hu‐MIF/inhibitor complex (PDB entry: 4WR8). In that complex, the polar residues Lys32 and Ser63 bind biaryltriazoles by hydrogen bonds, while Pro33, Tyr36, Trp108, and Phe113 keep the inhibitor bound by stacking interactions . Our structural alignments showed that all of these relevant residues (ie, Lys32, Pro33, Tyr36, Ser63, Trp108, and Phe113) were 100% conserved in Ts‐MIF with a very similar spatial orientation compared with Hu‐MIF (Figure 2B).…”

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor

“…26 First, a single point screening was done at a concentration of 25 μM and 50 μM and the compounds showing more than 50% inhibition of enzyme activity at 25 μM were tested for IC 50 values (Figs. S2 and S3).…”

“…The assay was done following the procedure of Dziedzic et al 26 4-hydrox-yphenyl pyruvate (4-HPP) was used as substrate to quantify tautomerase activity. Stock solutions of 10 mM 4-HPP were made in 50 mM ammonium acetate buffer pH 6.0, and incubated overnight at room temperature to allow equilibration between keto and enol form.…”

“…24–28 Using a structure-based virtual screening method, Orita-13 containing a chromen-4-one scaffold was identified as MIF inhibitor. 26,29 Additionally, covalent MIF inhibitors have been described, such as TP, as probes suitable for activity-based protein profiling. 30 Taken together, several small molecule binders of MIF have been developed (Fig.…”