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

Article abstract of DOI:10.1021/jacs.6b04910

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 K_d 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.

Full text of DOI:10.1021/jacs.6b04910

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Article
A Fluorescence Polarization Assay for Binding to
Macrophage Migration Inhibitory Factor and Crystal
Structures for Complexes of Two Potent Inhibitors
Jose A. Cisneros, Michael J Robertson, Margarita Valhondo, and William L Jorgensen
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04910 • Publication Date (Web): 14 Jun 2016
Downloaded from http://pubs.acs.org on June 15, 2016
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A Fluorescence Polarization Assay for Binding to Macrophage Migration
Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors
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José A. Cisneros, Michael J. Robertson, Margarita Valhondo, and William L Jorgensen,*
Department of Chemistry, Yale University, New Haven, Connecticut 06520ꢀ8107, United States
S Supporting Information
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Received: February 00 2016
ABSTRACT: Human macrophage migration inhibitory factor (MIF) is both a ketoꢀenol
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
Lꢀdopachrome methyl ester or 4ꢀhydroxyphenyl 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
labelled MIF inhibitors and their use in the first fluorescence polarizationꢀbased assay to measure
directly binding of inhibitors to the active site. The assay allows the accurate and efficient
identification of competitive, nonꢀcompetitive, and covalent inhibitors of MIF in a manner that
can be scaled for highꢀthroughput 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. Xꢀray crystal structures for two of the most potent
compounds bound to MIF are also reported here. Striking combinations of proteinꢀligand
hydrogen bonding, arylꢀaryl, and cationꢀπ 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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INTRODUCTION
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Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is involved in
numerous inflammatory and autoimmune diseases including rheumatoid arthritis, diabetes,
sepsis, and acute respiratory distress syndrome.1ꢀ4 Release of MIF from activated cells such as
macrophages and Tꢀcells in turn promotes release of other inflammatory cytokines. MIF is also
overꢀexpressed in many cancer cells where it enhances cell proliferation by inhibiting
accumulation of the tumor suppressor p53.5 The complex biological activities of MIF as a
cytokine are modulated by its binding to the cellꢀsurface receptors CD74, CXCR2, and CXCR4.
MIF is a homotrimeric protein with 342 residues, which also displays enzymatic activity as a
ketoꢀenol tautomerase. There are three identical active sites at the interfaces of the monomer
subunits. The enzymatic activity appears to be vestigial in humans; however, nonꢀphysiological
substrates including Dꢀdopachrome methyl ester (DOPA) and hydroxyphenyl pyruvic acid (HPP)
have been identified and form the bases for the most common assays.6,7 Though inhibition of the
tautomerase activity does not guarantee inhibition of biological function, many studies have
supported a correlation.8,9 A recent report has further strengthened the view that MIFꢀCD74
binding occurs near the tautomerase sites and that the protrusion of inhibitors outside the active
sites leads to reduced biological activity.10
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Most studies to identify MIF inhibitors have screened compound libraries using the
DOPA or HPP tautomerase assays.4,9,11ꢀ14 IC50 or Ki values are reported for inhibition of the
tautomerization of these substrates. As discussed previously,15 execution of these assays is
complicated by multiple factors including the lightꢀsensitivity of DOPA, the slow rate of
tautomerization of HPP, spectral interference of inhibitors and products, choice of protein
concentration, and short times for the linear range of product formation in both cases. There has
been limited report on activities of consensus reference compounds in the screening studies
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except for the isoxazoline (R)ꢀISOꢀ1.16 The IC50 results for it, which range from 7 µM to >100
µM, reflect the difficulties in obtaining consistency.9,16,17 We also reinvestigated the
chromenone Oritaꢀ13, which had been the most active compound in the journal literature with a
reported Ki of 0.038 µM in the DOPA assay.11 However, while Ki results should be independent
of the substrate, repeated testing in our HPP assay yielded modest Ki values of 13ꢀ22 µM.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.19 Displacement of a ligand by a fluorescent
probe yields a readily quantified increase in fluorescent polarization that reflects the fraction of
bound ligand. The usual advantages of FP assays apply including use of standard microplate
readers, direct determination of Kd values with no need for substrates or radiolabelled reagents,
and the ability to reanalyse the assay plates.19 In contrast, for the tautomerase assays, the
measurements of product formation can only be made once in the first seconds after the addition
of the substrates. Furthermore, since the present tracers have lowꢀnanomolar affinity for MIF,
only small amounts of the protein are required. In the course of this work, we also determined the
crystal structures of the complexes for two MIF ligands with particularly high affinities. The
results reveal common structural features for achieving strong binding with MIF.
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EXPERIMENTAL SECTION
Chemistry. We previously reported biaryltriazoles with the general structures 1 – 4 as
MIF tautomerase inhibitors with Ki values as low as 0.057 µM in the HPP assay.15 Xꢀray crystal
structures for complexes of the parent quinoline 3a (X = Y = H, Z = OH) and its analogue with X
= MOEO (methoxyethoxy) were also reported and confirmed the expected binding in the MIF
active sites with the X group on C6 of the quinoline protruding into the solvent.15 The analogues
of 1 – 4 utilized here were reported previously with the exception of 3j;15 their syntheses feature
a 1,3ꢀdipolar cycloaddition of a phenylazide with a substituted ethynylheterocycle obtained via a
Sonogashira coupling. We have also examined the journal and patent literature for other potential
strong binders. Compounds reported in a patent from workers at Novartis appeared particularly
promising.20 22 benzoxazinones were exemplified and it was stated that they had activities of 20
nM – 20 µM in an HPP tautomerase assay. The second example, which we refer to as NVSꢀ2 (X
= pꢀmethoxyphenyl), was singled out as having an IC50 of 20 nM. We resynthesized it along with
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examples 1 and 6, NVSꢀ1 (X = cyclohexyl), and NVSꢀ6 (X = pꢀhydroxyphenyl). As described in
the Supporting Information, the general procedure from the patent was followed; however, the
details on the protection of the 7ꢀhydroxy group were unclear. It was found that a tꢀ
butyldimethylsilyloxy group worked well (Scheme 1). NVSꢀ6 was obtained from 4ꢀmethoxyꢀ2ꢀ
hydroxybenzaldehyde and 4ꢀmethoxyaniline upon treatment with sodium borohydride, followed
by demethylation of both methoxy groups using pyridinium hydrochloride. Additional reference
compounds, ISOꢀ1, Oritaꢀ13, 4ꢀIPP, and Pontamine Sky Blue (PSB) were obtained from
commercial sources (AlfaꢀAesar, Tocris, Santa Cruz Biotechnology). Compound identity was
confirmed by NMR and mass spectrometry, and >95% purity of all assayed compounds was
established by HPLC.
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Scheme 1. Synthesis of NVSꢀ2.
Biology. The expression and purification of human MIF followed the previous
procedures.15 The protein has been prepared on two occasions and stored at ꢀ80 °C. Aliquots are
thawed for the assaying and crystal growth, and never refrozen. Activities were shown to be
consistent for different samples using multiple control compounds. The HPP tautomerase assay
was carried out as described before.15 Inhibitory activity is monitored by measuring formation of
the borate complex of the enol product at 305 nm using a Tecan Infinite F500 plate reader.
Details for the FP assay are described below and in the Supporting Information.
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Crystallography. Xꢀray crystal structures at 1.8ꢀÅ resolution were obtained for
complexes of NVSꢀ2 and 3ꢀ((2ꢀ(1ꢀ(3ꢀfluoroꢀ4ꢀhydroxyphenyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)quinolinꢀ5ꢀ
yl)oxy)benzoic acid (3i) with human MIF. Crystals of apo MIF were obtained by the hanging
drop method. Subsequently, 2 µL drops containing the apo MIF crystals were treated with 10
mM suspensions of the ligands in DMSO. In both cases, after several weeks, the initial protein
crystals cracked and dissolved, and new crystals formed (Figure S1). The crystals were
cryoprotected and data collection was performed on a Rigaku 007HF+ xꢀray source with a Saturn
944+ CCD detector at Yale. Data processing, phasing, model building, and refinement were
carried out as described previously.15 Crystals of NVSꢀ2 were found to occupy the P3121 space
group, while those of 3i were I222. Full details are provided in the Supporting Information. The
structures have been deposited with the RCSB Protein Data Bank with PDP IDs 5HVT and
5HVS, respectively.
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RESULTS AND DISCUSSION
Design of fluorescent ligands. The fluorescent ligands were designed based on the
biaryltriazoles previously reported by our group.15 As summarized in Scheme 2, starting with 3b,
which has a Ki of 0.77 µM in the HPP assay, a fluorine was added adjacent to the hydroxyl group
to yield 3c and a Ki of 0.33 µM. The improvement can be attributed to the fluorine enhancing the
acidity of the hydroxyl group, which forms a hydrogen bond with Asn97, and/or filling a small
hydrophobic space contacting Met101. Compound 3c also bears an amino group that was reacted
with the isothiocyanate derivative of fluorescein (FITC) to yield the fluorescent analogue A,
which was subsequently found in saturation experiments described below to have a Kd of 0.181
µM. Exploration to further improve the affinity of the fluorescent ligand included expansion of
the spacer between the quinoline ring and the fluorophore. This led to the fluorescent ligand B,
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Scheme 2. Design of fluorescent ligands A and B
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which has a Kd of 0.056 µM and was prepared from the carboxylic acid 3j (Scheme 3). The
increased potency is beneficial in allowing use of a lower protein concentration in the FP assay
since there is less unbound tracer lowering the FP value. Based on the previous15 and current
crystallographic results and the consistent structureꢀactivity data (SAR), it is fully expected that
these fluorescent ligands, derived from competitive inhibitors, bind to the active sites of MIF.
Therefore, they can be used to identify not only compounds that competitively bind to the
tautomerase sites, but also compounds that can bind to allosteric sites that may disrupt or block
the active site and keep the tracer from binding.
Scheme 3. Complete structure of fluorescent MIF ligand B used in the FP assays.
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FP assay development. The assay was developed in a 96ꢀwell format. To determine if
the fluorescent ligands bind to the protein and their affinity, binding saturation experiments were
carried out. After determining the lowest concentration of the tracer that gives a consistent FP
value, this concentration (0.004 µM) was fixed and increasing amounts of MIF (0 to 1.35 µM)
were added, which caused increase in the FP values around 4ꢀfold (Figure 1A). In order to
determine if any nonꢀspecific binding occurs, the assay was also carried out in the presence of a
high concentration of a highꢀaffinity ligand (NVSꢀ2), which binds to the active site based on the
crystal structure presented below. Ligand B shows very high specificity since there is no variation
in the FP value from that of the free tracer in the presence of 10 µM NVSꢀ2 (Figure 1A, bottom
curve). At high MIF and NVSꢀ2 concentrations there is no indication that the tracer participates
in any binding that would be signalled by an increase in the FP value. Thus, the fluorescent
ligand binds to the tautomerase sites, and the fraction of ligand bound to ligand total can be
calculated with the highest and lowest FP values obtained in the saturation experiments. Using
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Figure 1. Determination of binding affinity of tracers A and B through saturation experiments. (A)
Variation of FP values for both tracers as a function of MIF concentration. Bottom curve indicates the
high specificity of ligand B towards the active site of MIF. (B) Kd determination for ligands A and B.
Lb/Lt = ratio of ligand bound to the total. Data shown from quadruplicate experiments in three
independent assays. Mean ± SEM plotted for all data.
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GraphPad Prism 6, Kd values are readily determined by plotting this fraction vs. the concentration
of the protein and fitting the results to the Hill equation. From Figure 1B, tracer B has greater
affinity (Kd = 0.056 ± 0.002 µM) than tracer A (Kd = 0.181 ± 0.034 µM).
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Competition assays. In order to develop a competitive FP assay to determine the
affinities of unlabelled compounds, tracer B was selected since its higher affinity allows use of a
smaller amount of protein to give a sufficient difference in FP values. At 0.056 µM MIF, the
ꢁmP between positive and negative controls is ca. 80 mP. Thus, we settled on standard assay
conditions of 0.004 µM tracer B and 0.056 µM MIF.
The compounds listed in Table 1 were assayed. Most are known nonꢀcovalent
tautomerase inhibitors of MIF including 11 biaryltriazoles,15 (R)ꢀISOꢀ1,16 and Oritaꢀ13.11 In
addition, we added an inactive analogue 3e, where the 4ꢀmethoxy group disrupts the
characteristic hydrogen bond with Asnꢀ97,15 3j, and the three NVS compounds.20 We also
assayed 4ꢀIPP, a wellꢀknown covalent MIF inhibitor,21 and the azo dye pontamine sky blue
(PSB), which is reported to bind on the surface of MIF and inhibit both the tautomerase activity
of MIF and its binding to CD74 (IC50 = 0.81 µM).22
For the FP assay, compounds were incubated at room temperature with human MIF for
20 min, followed by the addition of tracer B. This order of addition is needed because of the high
affinity of the tracer; otherwise, it takes modest inhibitors like ISOꢀ1 1ꢀ2 hours to reach
equilibrium for displacing the bound tracer. A standard buffer solution was used composed of 20
mM HEPES, 150 mM NaCl, and 0.01% Tweenꢀ20 with a pH of 7.4. Fluorescence polarization
was monitored for 1 hour with λexc = 485 ± 20 nm and λem = 535 ± 25 nm. The IC50 of each
compound for reducing the fluorescence polarization was determined by fitting the data to a nonꢀ
linear regression for log concentration vs. response (Figure 2). The IC50 is then transformed into
the corresponding Kd via Eq 1,22 where
is the Kd of the tracer (0.056 µM), Lt and Lb are the
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Table 1. Results for Tautomerase Inhibition (Ki) from the HPP Assay and for Binding (Kd)
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from the Fluorescence Polarization Assaya
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Compd
1a
X
Y
Z
Ki (µM)
37
Kd (µM)b
30% (25 µM)
1.75
ꢀ
ꢀ
OH
ꢀ
ꢀ
OH
8.8
2a
H
H
OH
0.59
0.77
0.33
8.9
0.260
3a
AEOEO
H
OH
0.348
3b
AEOEO
H
3ꢀF,4ꢀOH
F
0.163
3c
MOEO
H
(>1.5)
NA
3d
MOEO
H
OMe
OH
NA
3e
MrEOEO
H
0.41
0.15
0.11
0.057
0.034
1.48
1.90
1.70
17
0.211
3f
MrEOEO
H
3ꢀF,4ꢀOH
3ꢀF,4ꢀOH
0.152
3g
H
5ꢀpꢀCOOHꢀOPh
0.110
3h
H
5ꢀmꢀCOOHꢀOPh 3ꢀF,4ꢀOH
0.071
3i
O(CH2)3COOH
H
H
ꢀ
3ꢀF,4ꢀOH
0.063
3j
H
OH
0.347
4a
OCH3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1.71
5a
COOH
ꢀ
1.15
5b
ꢀ
ꢀ
20% (100 µM)
24
Oritaꢀ13
(R)ꢀISOꢀ1
NVSꢀ1
NVSꢀ2
NVSꢀ6
4ꢀIPP
PSB
ꢀ
ꢀ
24
cyclohexyl
ꢀ
0.569
0.027
0.185
(4.5)c,d
ND
0.456
pꢀOMeꢀPh
ꢀ
0.055
pꢀOHꢀPh
ꢀ
0.159
(0.446)c
ꢀ
ꢀ
ꢀ
ꢀ
49% (200 µM)
a
MOEO = methoxyethoxy; AEOEO = aminoethoxyethoxy; Mr = Nꢀmorpholinyl. NA = not
b
c
active; Kd or % bound at indicated concentration in parentheses. Covalent inhibitor: result is
timeꢀdependent. d IC50 from ref. 9.
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1.0
0.5
0.0
1.0
0.5
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0.0
1.0
-1.5
-1.0
-0.5
0.0
0.5
-0.5
0.0
0.5
1.0
1.5
2.0
log [3b]
log [ISO-1]
1.0
0.5
0.5
0.0
-2.0
-1.5
-1.0
-0.5
0.0
0.0
0.5
1.0
1.5
2.0
log [PSB]
log [3h]
Figure 2. Determination of IC50 for representative compounds. Plotted data from quadruplicate
experiments in three independent assays, mean ± SEM.
total and bound concentrations of the tracer, and Pt is the total MIF concentration. Full
experimental details are provided in the Supporting Information.
(1)
The results from the HPP and FP assays are recorded in Table 1. In principle, the Ki and
Kd values for a nonꢀcovalent inhibitor that binds to the tautomerase sites should be the same
under identical assay conditions. However, variations can arise from multiple sources. For
example, allosteric binding might or might not lead to tautomerase inhibition and/or
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displacement of the fluorescent ligand. In addition, the assay conditions are not identical. An
important consideration is the pH. The tautomerase assays with both HPP and DOPA are carried
out at pH 6ꢀ6.1, the optimal conditions for the substrates, while we have performed the FP assay
at physiological pH, 7.4. pH affects the protonation of functional groups in fluorophores, and
therefore the amount of fluorescence. In general, acidic pH is worse than alkaline for
fluorescence assays. pH 7.4 keeps the fluorescecent properties of fluorescein in an optimal range
and enhances the biological relevance.24 There is also influence of the pH on the proteinꢀligand
binding, which we examined for tracer A (Figure 3). As shown, increasing the pH from 5.1 to 7.4
clearly enhances the binding of A, and presumably the other biaryltriazoles, to MIF; the effect is
likely associated with progressive deprotonation of the catalytic Pro1, which has a pKa of ca.
5.6.25
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Another issue in FP assays is possible alteration of the fluorescence by test compounds.
The fluorescent properties of the tracer in the presence of the compounds, but in the absence of
protein, should be checked before assaying. In the present cases, some compounds have no effect
Figure 3. Influence of the pH in the binding of tracer A to MIF. Lb/Lt = ratio of ligand bound to the
total. Data shown from quadruplicate experiments in two independent assays. Mean ± SEM plotted for
all data.
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on FP values even at high concentrations, e.g., ISOꢀ1 and PSB. However other compounds,
specifically 1a, 3d, and Oritaꢀ13, did yield effects. In general, the undesirable effects decrease
with the concentration such that concentrations of test compounds below 1ꢀ3 µM do not cause
interference. However, this modulation of the fluorescence does not allow determination of the
the affinity of those compounds for which the limiting concentration is lower than their Kd
values. Another issue that can affect fluorescence is aggregation. Compounds that form
aggregates or precipitates can yield misleading FP values. Overall, the high concentrations
needed to determine the affinity of weak ligands can be problematic due to potential nonꢀspecific
binding of the ligand to the tracer.24 The use of detergents in the FP buffers such as Tweenꢀ20
helps to avoid aggregation; however, at high concentrations, this issue may still arise. Other
assays including the tautomerase one are also prone to aggregation problems at high
concentrations.26
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In spite of the caveats, we do observe similar results with our tautomerase and FP assays
for the compounds in Table 1. This is especially true for the most potent compounds including 3g
ꢀ 3j and the three NVS compounds, and the order of activities is well preserved. The consistency
between the present Ki and Kd results supports the accuracy of both assays and contrasts the
inconsistencies in prior reports for the tautomerase activities of reference compounds.18
Progression from 1a (3ꢀpyridinyl) to 2a (2ꢀpyridinyl) to 3a (2ꢀquinolinyl) increases tautomerase
inhibition in parallel with binding to MIF. The effects of substituents for the quinoline series,
3a – 3i, are also in good accord. The replacement of the phenolic hydroxyl group by a methoxy
group in 3e abolishes the binding and tautomerse inhibition, while the addition of a fluorine next
to the hydroxyl group improves the Ki and Kd values by a factor of two for 3b/3c and 3f/3g. For
compound 3d the phenolic OH is replaced by F leading to weaker inhibition, again due to loss of
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hydrogen bonding with Asn97.15 In this case for the FP assay, no inhibition is evident up to 1.5
µM. Above this point, there is interference with the fluorescence, so a Kd value could not be
determined. The two assays also agree essentially quantitatively that the most potent triazole
derivatives, 3i and 3j, incorporate an oxybenzoic acid substituent at the 5ꢀposition in the
quinoline ring or an oxybutanoic acid substituent at the 6ꢀposition yielding Ki and Kd values of
0.034 ꢀ 0.071 µM. Insights on the structural origins of this effect are provided below.
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Turning to the reference compounds, as noted above, Oritaꢀ13 with a Ki of 17 µM is
much less active in our HPP assay15 than expected from the original report of 0.038 µM in the
DOPA assay.11 The modest activity is supported by the present FP results in which only 20%
binding is found at 100 µM. (R)ꢀISOꢀ1 is unequivocally more active in the FP assay with an
average Kd of 24 µM, from three separate measurements of 21, 22, and 30 µM. Our group and
others have assayed (R)ꢀISOꢀ1 multiple times yielding IC50 and Ki values ranging from 7 to >100
µM. In three separate measurements with the present protocols, we obtained Ki values of 21, 24,
and 28 µM for (R)ꢀISOꢀ1, also averaging 24 µM. These two cases reflect that significant
variations are more probable for reports of activities of less potent compounds, likely for the pH,
aggregation, and spectral interference issues noted above.
In view of the previous inconsistencies with published results,15,18 it was gratifying to find
that in our HPP assay, the NVS compounds are indeed potent tautomerase inhibitors with Ki
values of 0.569, 0.027, and 0.185 µM for NVSꢀ1, NVSꢀ2, and NVSꢀ6, respectively. The
activities of NVSꢀ2 and 3j are essentially identical at 0.03 µM, which establishes them as the
most potent tautomerase inhibitors to our knowledge. The Ki of 0.027 µM for NVSꢀ2 is very
similar to the previously reported IC50 of 0.020 µM (20 nM).20 In addition, the Kd values from
the FP assay are quantitatively similar to the Ki results. NVSꢀ2, 3i, and 3j with Kd values of 0.055
ꢀ 0.071 µM bind the most strongly to MIF among the compounds studied here.
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Covalent and allosteric inhibition. The FP assay also allows discrimination of covalent
and nonꢀcovalent inhibitors. The formation of a covalent bond between an inhibitor and a residue
in a binding site is expected to occur more slowly than establishment of a nonꢀcovalent complex.
Consequently, the activity of the covalent binder will appear to increase with increasing time. We
monitored the FP for 1 hour after the 20 min incubation period with a nonꢀcovalent inhibitor (2a)
and the covalent inhibitor 4ꢀIPP.21 As seen in Figure 4, there is a clear difference between the
results. The nonꢀcovalent interactions of 2a are formed quickly and the equilibrium between the
bound and unbound tracer is time independent. On the other hand, in the case of 4ꢀIPP the FP
results are time dependent showing the expected decrease in fluorescence polarization (increase
in unbound tracer) with increasing time.
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Finally, PSB has been reported as an allosteric inhibitor of MIF tautomerase activity as
well as an agent able to block the binding of MIF to CD74 with an IC50 value of 0.81 µM.22
Crystallographic results show PSB bound to the interface between two MIF trimers.22 According
to our FP results, the affinity of this compound is low with 49% binding of PSB at 200 µM.
Figure 4. Contrasting FP results for a nonꢀcovalent inhibitor 2a (left) and a covalent one, 4ꢀIPP (right).
The results for the covalent inhibitor depend on the detection time showing increasing amounts of
unbound tracer with increasing time. Mean ± SEM plotted for all data.
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However, concern can be expressed that the compound is aggregating at such concentrations;
when the amount of Tweenꢀ20 was reduced below 0.01%, the FP signal was lost. Though PSB
does not bind in the active site, in some multimeric form it may cover the active site and cause
partial exclusion of the tracer.
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Assay Quality. In order to provide a measure of the quality of HTS assays, Zhang et al.
introduced the Z’ factor, which is calculated from the mean signals of the positive and negative
controls (ꢂc+ and ꢂc–) and their standard deviations (σc+ and σc–) using the formula Z’ = 1 –
[3(σc+ + σc–)/|ꢂc+ – ꢂc–|].27 As the signal range increases and the variations decrease, Z’ tends to
the ideal limit of 1, while Z’ values above 0.5 are considered “excellent”.27 From four
independent measurements of the positive (tracer B plus MIF) and negative (tracer B alone)
controls for the present FP assay, the Z’ factor is 0.69.
Crystal structures. Xꢀray structures were obtained for crystals of two of the most potent
inhibitors identified here with human MIF. A closeꢀup of the structure in the vicinity of the
tautomerase site is shown for 3i in Figure 5. The general features are as expected from the prior
reports for 3a and its analogue with a MOEO group at C6 of the quinoline ring.15 The proteinꢀ
ligand complexation features multiple hydrogen bonds and arylꢀaryl interactions. All three
tautomerase sites of the MIF trimer are occupied by 3i. The ammonium group of Lys32 is
coordinated by the quinoline nitrogen, N3 of the triazole, and the carbonyl oxygen of Ile64 at
distances of 3.14, 3.97, and 2.79 Å, respectively. N2 of the triazole is also in a hydrogen bond
with the backbone nitrogen of Ile64 (2.93 Å), and the phenolic oxygen is 2.59 Å from the side
chain nitrogen or oxygen atom of Asn97. The new feature is the accommodation of the phenoxy
group on C5 of the quinoline ring. It is partially tucked into the pocket between Pro33 and Tyr36.
The added van der Waals contacts can account to some extent for the increased potency of 3i
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Figure 5. Rendering from the 1.8ꢀÅ xꢀray crystal structure of 3i bound to human MIF. Residues near the
active site are illustrated with carbon atoms of 3i in yellow. Dashed lines indicate hydrogen bonds. The
PDB code is 5HVS.
over 3c and 3g, whose substituents at C6 of the quinoline are solventꢀexposed. There is also an
electrostatic benefit of the negative carboxylate group on the phenoxy ring in view of its
proximity to Lys32. Decreases in potency of 2ꢀfold to 10ꢀfold are found when the carboxylate
group is replaced by neutral or positively charged alternatives.15 Another new feature is that the
fluorine atom adjacent to the phenolic hydroxyl group is oriented down between Ile64 and
Asn97, making closest contact with Cγ of Met101 (3.07 Å). This was expected from molecular
modeling studies,15 but the ligands in the previous crystal structures lacked the fluorine atom. As
noted above, the twoꢀfold increases in potency obtained by addition of the fluorine atom may be
attributed to inductive enhancement of the hydrogen bond between the hydroxyl group and Asnꢀ
97 and/or the hydrophobic contact with Met101.
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The corresponding structure for NVSꢀ2 is illustrated in Figure 6. The interaction themes
are similar as for 3i with coordination of the Lys32 ammonium group by the carbonyl groups of
NVSꢀ2 (2.80 Å) and Ile64 (2.86 Å), a bifurcated hydrogen bond between the backbone NH of
Ile64 and the NVSꢀ2 carbonyl (3.20 Å) and ring oxygen (3.12 Å) atoms, and a hydrogen bond
between the hydroxyl group of NVSꢀ2 and Asn97 (2.62 Å). A striking additional feature is a
cationꢀπ interaction between the anisyl group of NVSꢀ2 and Lys32; the ammonium group is
positioned below the ring center with distances of 4.5 – 5.5 Å to all six carbon atoms, typical of
cationꢀπ interactions.28,29 As for 3i, there are also multiple arylꢀaryl interactions between the
inhibitors and Tyr36, Tyr95, and Phe113. The complementarity between NVSꢀ2 and the MIF
tautomerase site appears almost ideal with the remarkable multiꢀdentate coordination of Lys32,
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Figure 6. Rendering from the 1.8ꢀÅ xꢀray crystal structure of NVSꢀ2 bound to human MIF. The PDB
code is 5HVT.
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satisfaction of the hydrogenꢀbonding demands of Ile64 and Asn97, and the arylꢀaryl interactions
for Tyr36, Tyr95, and Phe113. The only part of the ligand that appears to not be engaged in
beneficial interactions is the terminal methoxy group, though its electronꢀdonating character may
enhance the hydrogenꢀbond accepting character of the carbonyl group in the oxazinone ring. This
notion is consistent with the 3ꢀfold stronger binding for NVSꢀ2 than for NVSꢀ6 in which the
solventꢀexposed methoxy group is replaced by hydroxy. The ca. 10ꢀfold weaker binding for the
cyclohexyl analogue NVSꢀ1 clearly results from loss of the cationꢀπ interaction with Lysꢀ32. In
view of its small size with a heavy atom count (HAC) of only 20, the ligand efficiency for NVSꢀ2
is high at 0.50 ((ꢀ2.3RT/HAC) log Kd).30 For the much larger 3i, the ligand efficiency is 0.30.
It should also be noted that, as in the case of 3i, all three tautomerase sites are occupied
by copies of NVSꢀ2. This may reflect the strength of the interactions or it may be regulated by
proteinꢀprotein packing. In the crystal structure for 3a bound to MIF (PDB ID: 4WR8),15 there is
only one tautomerase site occupied per MIF trimer; however, the proteinꢀprotein interface has
tight contact between the quinoline rings of the inhibitors protruding from the surfaces of pairs of
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Figure 7. Renderings from the crystal structures for 3i (left) and NVSꢀ2 (right) illustrating the difference
in protrusion from the MIF binding site. The protein is shown as a spaceꢀfilling surface. The orientation
of the terminal methoxy group of NVSꢀ2 varies depending on the binding site of the MIF trimer.
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MIF trimers. For the present cases, 3i extends much farther than NVSꢀ2 from the active site
(Figure 7), so it is likely that MIF signalling via binding to cellꢀsurface receptors including CD74
will be more impaired in the presence of 3i.22 Thus, higher ligand efficiency may not be desirable
in this case. We have also used the substituents at C5 and C6 of the quinoline to enhance the
solubility of the biaryltriazoles.15
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Combined Design. In view of the striking cationꢀπ interaction for NVSꢀ2 in Figure 6, a
new molecular design was sought to merge this feature with the triazolylphenol core of 3.
Structure building with the BOMB program31 suggested that replacement of the quinolinyl group
of 3 with a phenoxy group as in 5 would be appropriate (Figure 8). It is expected that the cationꢀπ
interaction is enhanced by substituents that would further increase the electronꢀrichness of the
phenoxy group. Thus, we had interest in preparing the 4ꢀmethoxy (5a) and carboxylic acid (5b)
derivatives. 5b is expected to be ionized to the carboxylate above pH 5.
The synthesis of compounds 5 is outlined in Scheme 4; full details are in the
Supplementary Information. Treatment of 4ꢀmethoxyphenol with trichloroethylene and base,
followed by dehydrohalogenation with nꢀbutyl lithium yielded the ethynyl ether,32 which was
converted to 5a by a oneꢀpot Cu(I)ꢀcatalysed “click” reaction using 4ꢀazidoꢀ2ꢀfluorophenol
generated in situ.15 However, synthesis of 5b required an oxazoline protecting group33 in view of
the step using nꢀbutyl lithium. The two compounds were then assayed and show good potency
with Ki and Kd values of 1 ꢀ 2 µM (Table 1). They are significantly more potent than (R)ꢀISOꢀ1
and the parent 2ꢀpyridyl analogue 2a. The small difference in the Ki values for the acid (5b) and
ether (5a) derivatives is consistent with what was observed for 3i (0.057 µM) and its
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methoxyethoxy analogue (0.082 µM).15 Thus, the quinolinyl and phenoxy motifs of 3 and 5
provide interesting polydentate alternatives for accommodation of Lys32 by incorporating azine
coordination or a cationꢀπ interaction (Figures 5 and 8).
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Figure 8. Computed structure for the complex of MIF and 5b as modelled with the BOMB program.
Anticipated hydrogen bonds and the cationꢀπ interaction are indicated with dashed lines.
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Scheme 4. Synthesis of compounds 5a and 5b.
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CONCLUSION
In this work, a fluorescence polarization assay was developed by designing and synthesizing two
fluoresceinꢀlabelled inhibitors of MIF. The high affinity of tracer B permits use of low
concentrations of both the tracer (0.004 µM) and the protein (0.056 µM) in the FP assay. The FP
assay shows multiple advantages over the traditional HPP and DOPA tautomerase assays used
for discovery of molecules that bind to MIF including ease of use and detection, stability of
reagents, and ready expansion to HTS formats. Both the HPP and FP assays were applied to 22
compounds including known nonꢀcovalent, covalent, and allosteric inhibitors of MIF. The results
for the nonꢀcovalent inhibitors show excellent accord between the measured Ki and Kd values
giving confidence in the viability of the present protocols for both assays. We also synthesized a
particularly interesting compound from the patent literature, NVSꢀ2; it and 3j are the most potent
inhibitors/binders in our assays. With Ki values of 0.03 µM, they are roughly 1000ꢀfold more
potent than the prototypical MIF inhibitor (R)ꢀISOꢀ1.
Furthermore, the FP assay was shown to perform well for the covalent inhibitor 4ꢀIPP,
which demonstrated the expected timeꢀdependence for its binding results. The weak allosteric
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inhibitor PSB also yielded a response in the FP assay showing some interference with binding of
the tracer at high concentrations. The assay results were much enhanced by obtaining xꢀray
crystal structures at 1.8ꢀÅ resolution for two of the most potent MIF inhibitors/binders, 3i and
NVSꢀ2. The structures confirmed that the anticipated binding of these molecules and the closely
related tracers occurs in the MIF tautomerase active sites. The structures also illustrate the
exquisite binding of these potent compounds to MIF featuring extensive hydrogenꢀbonding and
arylꢀaryl interactions along with a cationꢀπ interaction for the anisyl group of NVSꢀ2. A new
chemical series was then designed merging the triazolylphenol core of 3 with the cationꢀπ feature
of NVSꢀ2 to yield additional strong MIF inhibitors/binders, 5a and 5b. The present structural and
activity results along with the availability of the FP assay place further work on seeking
regulators of the activity of MIF on a firm foundation.
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ACKNOWLEDGMENTS
Gratitude is expressed to the National Institutes of Health (GM32136) for research support and
to the National Science Foundation (DGEꢀ1122492) for a fellowship to MJR. The authors also
thank Dr. Julian TiradoꢀRives and Prof. Richard H. G. Baxter for technical advice.
AUTHOR INFORMATION
Corresponding Author
william.jorgensen@yale.edu
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
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Supporting Information. Full synthetic procedures and spectral characterization data for all
intermediates and final compounds; crystallographic data; experimental details of the HPP and
FP assays. This material is available free of charge via the Internet at http://pubs.acs.org.
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