A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors

Abstract

Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders.

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor

“…37 The activity of Orita-13 has been reported to be similar to ISO-1. 27 The most potent chromene compounds were active at lower concentrations compared to the reference compound ISO-1. Therefore, compounds 10 and 17 were taken for further investigation.…”

Optimization of Pyrazoles as Phenol Surrogates to Yield Potent Inhibitors of Macrophage Migration Inhibitory Factor

“…The results were confirmed by measurement of Kd values in a fluorescence polarization assay. [12] Exemplary potent compounds are 1 (NVS-2[10]) and 2[11] with Ki values of ca. 0.03 μM, which are ca.…”

“…[6,11,12] Though there are more than 125 approved drugs that contain a phenolic group including, for example, acetaminophen, albuterol, amoxicillin, raloxifene, and doxycycline, the oral bioavailability of phenols is well-known to often be unacceptably low owing to metabolic glucoronidation[14] and/or sulfation. [15] Thus, we set out to find a phenol-free series of MIF tautomerase inhibitors with low-nanomolar potencies.…”

“…In addition, the aqueous solubilities of several compounds were determined with a shake-flask procedure. [11,12,18] Saturated solutions are filtered and analyzed by UV-vis spectroscopy.…”

“…The resultsw ere confirmed by measurement of K d values in af luorescencep olarization assay. [12] Exemplary potent compounds are 1 (NVS-2 [10] )a nd 2 [11] with K i values of~0.03 mm,w hich are~1000-fold lower than for well-known MIF inhibitors such as 3 ((R,S)-ISO-1 [13] )a nd the chromen-4-one 4 [6a] (Figure 1).…”