Irregularities in enzyme assays: The case of macrophage migration inhibitory factor

Cisneros, José A.; Robertson, Michael J.; Valhondo, Margarita; Jorgensen, William L.

doi:10.1016/j.bmcl.2016.04.074

Cited by 22 publications

(46 citation statements)

References 25 publications

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“… 15 Extension of the comparisons to additional compounds from the literature has revealed a pattern of substantial inconsistencies in reports of activities from MIF tautomerase assays. 18 Therefore, we decided to pursue development of a direct binding assay that can overcome the problems with the tautomerase assays. Based on our recent finding of biaryltriazoles as potent MIF tautomerase inhibitors, we were able to design and synthesize fluorescent ligands that can be used as effective tracers in a fluorescence polarization (FP) assay.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…The IC 50 value of ISO-1 was within the range reported in literature. 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.…”

Section: Resultsmentioning

confidence: 87%

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor

Kok

Wapenaar

Wang

et al. 2018

Bioorganic & Medicinal Chemistry

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Macrophage migration inhibitory factor (MIF) is an essential signaling cytokine with a key role in the immune system. Binding of MIF to its molecular targets such as, among others, the cluster of differentiation 74 (CD74) receptor plays a key role in inflammatory diseases and cancer. Therefore, the identification of MIF binding compounds gained importance in drug discovery. In this study, we aimed to discover novel MIF binding compounds by screening of a focused compound collection for inhibition of its tautomerase enzyme activity. Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. The library was synthesized using versatile cyanoacetamide chemistry to provide diversely substituted chromenes. The screening provided inhibitors with IC's in the low micromolar range. Kinetic evaluation suggested that the inhibitors were reversible and did not bind in the binding pocket of the substrate. Thus, we discovered novel inhibitors of the MIF tautomerase activity, which may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.

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“…[6,7] Though many MIF tautomerase inhibitors have been discovered through screening of compound libraries,[6,8] lead optimization to give inhibitors with nanomolar potency has been limited. In fact we have tested the most promising compounds from the literature in a tautomerase inhibition assay[9] and only found compounds from one patent[10] and our biaryltriazole series[11] to have sub-micromolar Ki values. The results were confirmed by measurement of Kd values in a fluorescence polarization assay.…”

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confidence: 99%

“…It was decided to retest 5 in an HPP tautomerase assay using optimized protocols in our laboratory. [9] Though the Ki for 5 from this assay was only 113 μM, in view of its low molecular weight and possibilities for substitution in the phenyl ring, we were encouraged to perform structure-based, computer-aided lead optimization. [17] As detailed here, this has been successful in providing pyrazole derivatives with ca.…”

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

et al. 2018

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Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is implicated in the regulation of inflammation, cell proliferation, and neurological disorders. MIF is also an enzyme that functions as a keto-enol tautomerase. Most potent MIF tautomerase inhibitors incorporate a phenol, which hydrogen bonds to Asn97 in the active site. Starting from a 113-μm docking hit, we report results of structure-based and computer-aided design that have provided substituted pyrazoles as phenol alternatives with potencies of 60-70 nm. Crystal structures of complexes of MIF with the pyrazoles highlight the contributions of hydrogen bonding with Lys32 and Asn97, and aryl-aryl interactions with Tyr36, Tyr95, and Phe113 to the binding.

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Irregularities in enzyme assays: The case of macrophage migration inhibitory factor

Cited by 22 publications

References 25 publications

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

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

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor

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

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