Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Article (PDF Available)inJournal of Biological Chemistry 285(34):26581-98 · August 2010with27 Reads
DOI: 10.1074/jbc.M110.113951 · Source: PubMed
Abstract
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.
Identification and Characterization of Novel Classes of
Macrophage Migration Inhibitory Factor (MIF) Inhibitors
with Distinct Mechanisms of Action
*
S
Received for publication, February 16, 2010, and in revised form, May 22, 2010 Published, JBC Papers in Press, June 1, 2010, DOI 10.1074/jbc.M110.113951
Hajer Ouertatani-Sakouhi
, Farah El-Turk
, Bruno Fauvet
, Min-Kyu Cho
§
, Damla Pinar Karpinar
§
, Didier Le Roy
,
Manfred Dewor
, Thierry Roger
,Ju¨ rgen Bernhagen
, Thierry Calandra
, Markus Zweckstetter
§
,
and Hilal A. Lashuel
‡1
From the
Laboratory of Molecular Neurobiology and Functional Neuroproteomics, Brain Mind Institute, Ecole Polytechnique
Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, the
§
Department of NMR-based Structural Biology, Max Planck
Institute for Biophysical Chemistry, 37077 Goettingen, Germany, the
Department of Medicine, Infectious Diseases Service, Centre
Hospitalier Universitaire Vaudois and University of Lausanne, CH-1011 Lausanne, Switzerland, and the
Department of
Biochemistry and Molecular Cell Biology, Institute of Biochemistry and Molecular Biology, Rheinisch-Westfa¨lische, Technische
Hochschule (RWTH) Aachen University, Aachen 52074, Germany
Macrophage migration inhibitory factor (MIF), a proinflam-
matory cytokine, is considered an attractive therapeutic target
in multiple inflammatory and autoimmune disorders. In addi-
tion to its known biologic activities, MIF can also function as a
tautomerase. Several small molecules have been reported to be
effective inhibitors of MIF tautomerase activity in vitro. Herein
we employed a robust activity-based assay to identify different
classes of novel inhibitors of the catalytic and biological activi-
ties of MIF. Several novel chemical classes of inhibitors of the
catalytic activity of MIF with IC
50
values in the range of 0.2–15.5
M were identified and validated. The interaction site and
mechanism of action of these inhibitors were defined using
structure-activity studies and a battery of biochemical and bio-
physical methods. MIF inhibitors emerging from these studies
could be divided into three categories based on their mechanism
of action: 1) molecules that covalently modify the catalytic site at
the N-terminal proline residue, Pro
1
; 2) a novel class of catalytic
site inhibitors; and finally 3) molecules that disrupt the trimeric
structure of MIF. Importantly, all inhibitors demonstrated total
inhibition of MIF-mediated glucocorticoid overriding and AKT
phosphorylation, whereas ebselen, a trimer-disrupting inhibitor,
additionally acted as a potent hyperagonist in MIF-mediated che-
motactic migration. The identification of biologically active com-
pounds with known toxicity, pharmacokinetic properties, and bio-
logical activities in vivo should accelerate the development of
clinically relevant MIF inhibitors. Furthermore, the diversity of
chemical structures and mechanisms of action of our inhibitors
makes them ideal mechanistic probes for elucidating the structure-
function relationships of MIF and to further determine the role of
the oligomerization state and catalytic activity of MIF in regulating
the function(s) of MIF in health and disease.
Macrophage migration inhibitory factor (MIF)
2
was discov-
ered in the 1960’s as a T-lymphocyte product that inhibits the
random migration of macrophages during delayed-type hyper-
sensitivity responses (1, 2). Two decades later, a human MIF
gene was cloned (3). Yet, the biological activity of MIF remained
ambiguous until the production of bioactive MIF and anti-MIF
antibodies (4). Various biological activities have been attributed
to MIF, which is recognized as a major regulator of inflamma-
tion and a central upstream mediator of innate immune
responses (5, 6). MIF has broad regulatory properties and is
considered as a critical mediator of multiple disorders includ-
ing inflammatory and autoimmune diseases such as rheuma-
toid arthritis (7, 8), glomerulonephritis (9, 10), diabetes (11),
atherosclerosis (12), sepsis (13–15), asthma (16, 17), and acute
respiratory distress syndrome (18). Furthermore, recent stud-
ies have highlighted a role for MIF in tumorigenesis. Human
cancer tissues, including skin, brain, breast, colon, prostate,
and lung-derived tumors were observed to overexpress MIF,
and MIF levels correlated with tumor aggressiveness and
metastatic potential (19, 20). Therefore, MIF is considered a
viable therapeutic target for treating inflammatory diseases
and neoplasia.
In addition to its physiologic and pathophysiologic activities,
MIF is known to act as a tautomerase. This activity was initially
discovered during the investigation of melanin biosynthesis
(21). Subsequent studies revealed that MIF catalyzes the tau-
tomerization of
D-dopachrome methyl ester (22–24) and
bears high structure, but not sequence, homology with bac-
terial tautomerases 4-oxalocrotonate-tautomerase, 5-car-
boxymethyl-2-hydroxymuconate isomerase, and chorismate
mutase (25–27). X-ray crystallography, NMR, and biophys-
* This work was supported by grants from the Swiss Federal Institute of Tech-
nology Lausanne (to H. A. L., F. E., and H. O.-S.), Swiss National Science Founda-
tion Grants 310000-110027 (to H. A. L.) and 3100-118266 (to T. C.), and German
Research Council (DFG) Grants Be1977/4-1 and SFB542-A7 (to J. B.).
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S7.
1
To whom correspondence should be addressed. Tel.: 41-21-693-96-91; Fax:
41-21-693-17-80; E-mail: hilal.lashuel@epfl.ch.
2
The abbreviations used are: MIF, migration inhibitory factor; LPS, lipopo-
lysaccharide; TNF, tumor necrosis factor; DMSO, dimethyl sulfoxide; ISO-1,
(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl
ester; HTS, high throughput screening; HCLP, hexachlorophene; MALDI-
TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass
spectrometry; PBS, phosphate-buffered saline; EMCH, 3,3-N-[e-maleimi-
docaproic acid)hydrazide; HSQC, heteronuclear single quantum coher-
ence; EPC, endothelial progenitor cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; NAPQI, N-acetyl-p-benzoquinone imine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 34, pp. 26581–26598, August 20, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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Supplemental Material can be found at:
ical solution studies demonstrate that MIF exists predomi-
nantly as a homotrimer (28, 29). The tautomerase active site
is formed at the monomer-monomer interfaces within the
trimer and involves residues from two adjacent subunits.
The N-terminal proline (Pro
1
) with an unusual pK
a
(5.6
0.1) (24) is commonly conserved in all of these tautomerases
and is essential for MIF tautomerase activity. Covalent mod-
ification of Pro
1
or its replacement by serine, alanine, or
glycine totally abolishes the tautomerase activity of MIF (23,
30). p-Hydroxyphenylpyruvate and phenylpyruvate were
identified as potential tautomerase substrates (31). How-
ever, the kinetic parameters and the separate localization of
these substrates from MIF suggest that these molecules are
not physiological substrates of MIF.
Although the relationship between the catalytic activity and
biological function of MIF is not yet fully understood, targeting
MIF tautomerase activity using small-molecule inhibitors has
emerged as an attractive strategy for inhibiting MIF proinflam-
matory activity and attenuating its biological activity in vitro
and in vivo (32, 33). The first MIF inhibitors were reported in
1999 while trying to elucidate the mechanism of MIF tautomer-
ase activity by testing the inhibitory effect of various structure
analogues of its substrate,
D-dopachrome methyl ester (34).
Since then, different classes of tautomerase inhibitors have
been developed and were later shown to modulate biological
activities of MIF mediated by both its ability to act on intracel-
lular and extracellular signaling pathways (33, 35). As of today,
11 distinct chemical classes of MIF inhibitors have been devel-
oped (36) using different approaches, including (i) active site-
directed targeting; (ii) rational drug design, i.e. screening mol-
ecules that share structure similarity with known MIF
tautomerase substrates and inhibitors; and (iii) virtual high
throughput screening and computer-assisted drug design
approaches. The majority of the inhibitors described to date
exert their effects either by competing with the substrate for the
catalytic site (e.g. ISO-1 and OXIM11) or via covalent modifi-
cation of the catalytic Pro
1
residue (NAPQI (37) and 4-iodo-6-
phenylpyrimidine (4-IPP) (33)). For example, Senter and col-
leagues (37) identified a class of acetaminophen derivatives
(NAPQI), which form a covalent complex with MIF by react-
ing with the catalytic proline residue. NAPQI was shown to
block the ability of MIF to override the immunosuppressive
effect of dexamethasone on LPS-induced TNF production by
monocytes. A series of MIF inhibitors based on modifica-
tions of the scaffold of (S,R)-3-(4-hydroxyphenyl)-4,5-dihy-
dro-5-isoxazole acetic acid methyl ester (ISO-1) were devel-
oped (38 41). ISO-1 and some of its derivatives were shown
to block MIF tautomerase activity, inhibit TNF secretion
from macrophages upon stimulation with LPS, and increase
survival in models of sepsis (35, 42).
Recent studies from our group demonstrate that subtle con-
formational changes induced by mutations distal to the active
site result in significant inhibition of MIF tautomerase activity
(43, 44). These findings indicate that “allosteric” inhibition of
MIF enzymatic activity is possible, but may not be realized by
current efforts focused on targeting the active enzymatic site of
MIF using rational drug design strategies. Motivated by these
findings, we sought to develop a high throughput screening
(HTS) assay that would facilitate the identification of different
classes of MIF inhibitors, including novel irreversible, compet-
itive, and allosteric inhibitors, as well as molecules that may
inhibit MIF by blocking and/or disrupting its oligomerization
(i.e. trimer formation). To achieve this goal, we developed a
robust tautomerase activity-based HTS assay and screened
two chemical libraries containing a total of 15,440 com-
pounds. Twelve novel classes of MIF inhibitors were identi-
fied with IC
50
values in the range of 0.2–15.5
M. Using
structure-activity studies, and a battery of biochemical and
biophysical methods, we were able to define the mechanism
of action for each of the three classes of inhibitors. These
results and their implications for developing therapeutic
strategies targeting MIF and elucidating the biochemical and
structural basis underlying its activities in health and disease
are presented and discussed.
EXPERIMENTAL PROCEDURES
Chemical Libraries
The NINDS Custom Collection II library from Microsource
Discovery Systems, Inc. and the Maybridge library were tested.
These libraries were composed of 1,040 and 14,400 biologically
active chemical molecules, respectively. The compounds were
arrayed in 384-well plates at a final concentration of 10
M and
a final DMSO concentration of 1%.
Compounds Used for Follow-up Studies
All hits generated from the Maybridge library were pur-
chased from Maybridge. Hexachlorophene (HCLP) and its
analogues (dichlorophene, bithionol, bis(2-hydroxyphenyl)-
methane, 2,2-diaminodiphenyl sulfide, 4,4-dichlorobenzo-
phenone, 2,2-sulfinyl-bis(4,6-dichlorophenol), 3,4-dihy-
droxy benzophenone, igrasan, benzophenone, and emodin)
were purchased from Sigma and Fluka and were of the high-
est purity available, whereas the analogue MDPI 894 was
purchased from Molecular Diversity Preservation Interna-
tional (MDPI), Basel, Switzerland.
Expression and Purification of Human MIF and Its Mutants
(C56S, C59S, C80S, and N110C)
MIF was expressed by heat shock transformation of the
BL21/DE3 Escherichia coli strain (Stratagene) with the bacte-
rial expression vector pET11b containing the human (MIF)
gene under control of the T7 promoter. Four hours post-induc-
tion, the cells were harvested, re-suspended in lysis buffer (50
m
M Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgAc, 0.1% NaN
3
),
sonicated, and centrifuged at 14,000 g for 20 min. The clari-
fied cell lysate was filtered, injected onto a MonoQ anion
exchange column (HiPrep 16/10 Q FF, GE Healthcare), and
eluted with a linear NaCl gradient in the elution buffer (25 m
M
Tris-HCl, pH 7.4, 150 mM NaCl). The flow-through fractions
containing MIF were pooled and loaded onto a Superdex 75
16/60 (HiLoad 16/60, Superdex 75, GE Healthcare) gel filtra-
tion column. Fractions corresponding to MIF were combined,
dialyzed against 1 PBS, and filtered through a 0.2-
m filter.
Recombinant MIF used for cellular studies was subjected to
LPS removal as described previously (45). Briefly, bacterial cell
MIF Inhibition
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lysate was injected onto an anion exchange column. The flow-
through fractions containing MIF were applied to C8 Sep-Pak
cartridges (Waters Associates) and MIF was eluted with 60%
acetonitrile/water, lyophilized, and renaturated. The LPS con-
tent was quantified using the chromogenic Limulus amoebo-
cyte assay.
MIF mutants (C56S, C80S, and N110C) were expressed and
purified as described for the wild type human MIF. C59S and
C56S/C59S mutants were expressed and purified as previously
described by Kleemann et al. (46). Uniformly
15
N-labeled pro
-
tein samples were prepared for NMR experiments by culturing
the bacteria in M9 minimal medium containing
15
N-ammo
-
nium chloride (1 g/liter) as the only nitrogen source.
Assay Design to Screen for Potential Inhibitors of MIF
Tautomerase Activity
The assay principle is based on measuring the absorbance of
D-dopachrome methyl ester (red), which is transformed by
enzymatically active MIF to the enol form that is colorless (Fig.
1). In the absence of MIF, the keto form of the substrate shows
an absorbance at 475 nm that diminishes upon addition of MIF
(supplemental Fig. S1). Keto-enol tautomeric conversion of
D-dopachrome methyl ester by MIF was optimized in cuvette
format as described previously (23). A fresh stock solution of
D-dopachrome methyl ester was prepared through oxidation of
L-3,4-dihydroxy phenylalanine methyl ester with sodium meta-
periodate.
L-3,4-Dihydroxy-phenylalanine methyl ester (4 mM)
was first diluted in 5 ml of distilled water, then the appropriate
amount of sodium metaperiodate was added to a final concen-
tration of 8 m
M. The solution was mixed, incubated for 5 min at
room temperature, protected from light and then kept on ice.
D-Dopachrome methyl ester stability over time was evaluated at
different temperatures. Complete stability was only observed
when the substrate was stored at 4 °C. However, a decrease in
absorbance was easily noticed at room temperature or 37 °C
(supplemental Fig. 1A).
The tautomerase enzymatic activity was measured in 50 m
M
potassium phosphate buffer, 0.5 mM EDTA, pH 6.5. MIF (100
n
M) dialyzed in 1 PBS was added to the cuvette and the
decrease in absorbance at 475 nm was followed for 3 min using
a CARY 100 Bio UV-visible spectrophotometer. To perform
this assay in an HTS format, MIF (500 n
M,6
l) was added to
384-well plates (Nunc, Black wall, clear bottom) containing 50
m
M potassium phosphate buffer, 0.5 mM EDTA, pH 6, and the
compounds of interest at a final concentration of 10
M. MIF
and compounds were mixed and incubated for a fixed time
period of 15 min at room temperature before initiating the
reaction by addition of a fresh stock solution of
D-dopachrome
methyl ester, which was prepared as described above (final
reaction volume 80
l). The plates were centrifuged for 2
min at 2000 g to remove air bubbles and the absorbance
was measured at 475 nm using a Saphire II Tecan reader
(supplemental Fig. S2A). As a pos-
itive control, we initially used
ISO-1 and later switched to
ebselen (10
M), which gave com-
plete inhibition of the enzymatic
activity of MIF. The entire assay
was performed using the Biomek 3000 Laboratory Automa-
tion Work station from Beckman Coulter, and the entire
procedure was carried out in the dark. All assays were carried
out in duplicate and hit compounds were retested individu-
ally to eliminate any false positives.
Data Analysis
The Z value was calculated according to the method devel-
oped by Zhang et al. (47),
Z 1
3 S.D. positive control 3 S.D. negative control
mean positive control mean negative control)
(Eq. 1)
where S.D. positive control is the standard deviation (n 16) of
the positive control (with 10
M ebselen), S.D. negative control
is the standard deviation (n 24) of the negative control (MIF
in the absence of inhibitor), mean positive control is the mean
(n 16) of the positive control (with 10
M ebselen), and the
mean of the negative control is the mean (n 24) of the nega-
tive control (MIF in the absence of inhibitor).
IC
50
and K
i,app
Assessment
For the determination of IC
50
values, the D-dopachrome was
used as a substrate; the hit compounds were distributed in 384-
well plate at 12 dilutions starting from 0 to 50
M. One percent
DMSO was used as a final concentration in the reaction buffer;
the assay was performed in triplicate as described above and
each sample was evaluated in duplicate.
K
i,app
values were obtained by plotting the relative initial
velocities as a function of the inhibitor concentration. MIF was
preincubated with 12 concentrations of hit compounds ranging
from0to50
M for 15 min, followed by the addition of 2 mM
HPP and the absorbance at 300 nm was measured for 3 min.
Data were fitted to the simple inhibition expression using Sig-
maplot software: V
i
(V
0
/(1 (I/K
i,app
)
n
)) bkg; where V
0
,
velocity at [I] 0 and n is the Hill coefficient (48). The percent
of inhibition was calculated relative to the DMSO vehicle
control and blank control as: % inhibition 100 (100
(test compound value average of blank control)/(average of
DMSO control average of blank control)).
MALDI-TOF MS Measurements to Examine Possible Protein
Modifications
Matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF MS) was performed to exam-
ine any inhibitor-induced protein modifications. The matrix
solution was prepared by dissolving 14 mg/ml of sinapinic acid
solution in 0.1% trifluoroacetic acid/acetonitrile (1:1). A thin
matrix layer was generated on the mirror-polished target using
a gel loading tip. One microliter of sample (15
M MIF incu-
bated with 10
M hit compounds for 1 h) was mixed with 1
lof
FIGURE 1. MIF catalyzed tautomerization of D-dopachrome.
MIF Inhibition
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sinapinic acid matrix solution, and 0.8
l of this mixture was
deposited on top of the thin layer and allowed to air dry. The
samples were analyzed with a 4700 MALDI-TOF/TOF instru-
ment (Applied Biosystems).
Protein Digestion Mass Spectrometry Analysis to Identify the
Modified Residue(s)
Twenty microliters of MIF sample previously incubated for
1 h with inhibitors at 10
M were digested overnight with tryp-
sin, 1:50 (Promega). The digestion was stopped with 1
lof10%
formic acid and stored at 4 °C until further use. The samples
were analyzed by MALDI-MS on a 4700 MALDI-TOF/TOF
instrument (Applied Biosystems) or an Axima CFR plus instru-
ment (Shimadzu) without further purification. One microliter
of sample was mixed with 1
l of 2,5-dihydroxybenzoic acid (20
mg/ml in 1% phosphoric acid/acetonitrile (1:1)), and 0.8
l of this
mixture was deposited on the target and allowed to air dry.
Probing the Effects of the Hit Compounds on MIF
Oligomerization by Analytical Ultracentrifugation
Analytical ultracentrifugation experiments were performed
using purified and dialyzed MIF samples (10
M) preincubated
with ebselen for1hataninhibitor concentration correspond-
ing to 100% inhibition. Radial UV scans were recorded on a
Beckman Optima XL-A at a wavelength of 277 nm. Sedimenta-
tion velocity experiments were carried out at 20 °C using 380
400
l of protein solution. Data were recorded at rotor speeds
of 50,000 rpm in continuous mode, with a step size of 0.003 cm.
The experimentally determined partial specific volume of 0.765
ml/mg was used to calculate the molecular weight of wild-type
MIF (49). The sedimentation velocity profiles were analyzed as
a c(s) distribution of the Lamm equation using SEDFIT (50). To
obtain the molecular weights, molar mass distributions c(M)
were obtained by transforming the corresponding c(s) using
SEDFIT.
MIF Aggregation Studies
Wild type human MIF at a concentration of 15
M in 1 PBS
was incubated with different ebselen concentrations ranging
from 0.01 to 1 m
M for1hatroom temperature, and then filtered
with a 0.2-
m filter (Millipore). The retentate was solubilized
in 0.1% SDS. Both the supernatant and the resolubilized aggre-
gates were analyzed in a 15% SDS gel. To further examine cys-
teine-induced aggregation by ebselen, either the N110C stable
cross-linked trimer or the alkylated wild type human MIF or
cysteine mutants were tested.
Cysteine Alkylation
To determine whether some of the hit compounds induce
their effect via modification of cysteine residues (i.e. Cys
56
,
Cys
59
, and Cys
80
), all free thiols were blocked by covalent mod
-
ification with maleimide (3,3-N-[e-maleimidocaproic acid)hy-
drazide, trifluoroacetic acid salt (EMCH)) (Pierce). Fifteen
micromolar wild type or mutant forms of human MIF in 1
PBS, pH 7.4, was incubated with 10 m
M EMCH fo r1hatroom
temperature. The unreacted EMCH was removed using PD-10
desalting columns (Pierce). The collected fractions were ana-
lyzed in an SDS-PAGE gel and the concentration was assessed
using a UV spectrophotometer.
Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectra were acquired at 27 °C on a Bruker Avance III
600 MHz NMR spectrometer. 500
M wild type human MIF
samples were prepared in 20 m
M Na
2
HP0
4
, 0.5 mM EDTA,
0.02% NaN
3
, pH 7.0, and 10% D
2
O. A specific amount of each
compound was prepared in DMSO and mixed with the above
sample to give a final 1% DMSO concentration in the total sam-
ple volume. Two-dimensional
1
H-
15
N heteronuclear single
quantum coherence (HSQC) spectra were recorded using 48
scans per increment with 256 1024 complex data points in F
1
and F
2
dimensions and relaxation delay of 1.0 s. Spectra were
processed with Topspin (Bruker Biospin, Germany) and
NMRPipe (51). Visualization and manipulation was performed
using Sparky 3.110.
3
Averaged chemical shift deviation was cal-
culated with Equation 2.
dN
5
2
dH
2
(Eq. 2)
Biological Assays
Glucocorticoid Overriding Assay—Mouse RAW 264.7
macrophages (TIB-71, ATCC) were grown in RPMI 1640
medium containing 2 m
M glutamine and 10% fetal calf serum.
MIF was incubated with the inhibitors for 15 min at room tem-
perature. RAW 264.7 macrophages (5 10
4
cells/well in
96-well plates) were preincubated fo r 1 h with 10
7
M dexa
-
methasone, dexamethasone plus murine MIF (100 ng/ml; 8
n
M), or dexamethasone plus MIF and Hit compounds at 10
M
before the addition of 100 ng/ml of Salmonella minnesota Ultra
Pure LPS (List Biologicals Laboratories). TNF in cell culture
supernatants collected after 48 h was measured by enzyme-
linked immunosorbent assay (BD Biosciences).
Phosphorylation of AKT by MIF—Confluent HeLa cells
(CCL-2) were grown in Dulbecco’s modified Eagle’s medium
containing 10% fetal calf serum. Cells were incubated for 2 h
with human MIF (50 ng/ml; 4 n
M) in the presence of hit com-
pounds at final concentrations of 1 and 10
M. Phosphorylation
of AKT at Ser
473
was measured using the Alpha screen SureFire
phosphokinase kit, according to the protocol provided by the
manufacturer (PerkinElmer Life Sciences) (53).
MIF-triggered Chemotaxis Assay—Chemotactic assays were
performed with primary human endothelial progenitor cells
(EPCs). EPCs were isolated from the mononuclear cell fraction
obtained by density gradient centrifugation from human blood
essentially as previously described (54). Briefly, “buffy coats”
were obtained from volunteers in accordance with the local
ethics committee. CD34 cells were enriched from mononu-
clear cells by Biocoll density gradient centrifugation and CD34-
specific magnetic separation. CD34 cells were plated on
fibronectin-coated 6-well plates and cultured in endothelial
growth medium (MV2). Endothelial progenitor cells were har-
vested on day 14 and their identity verified by fluorescence-
3
T. D. Goddard and D. G. Kneller (2010) SPARKY 3, University of California, San
Francisco, CA.
MIF Inhibition
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activated cell sorter analysis to stain for lectin-fluorescein iso-
thiocyanate, DiI-conjugated acLDL, CD31, and VEGFR-2 (KDR)
at a rate of 90%. EPC chemotaxis was evaluated using Tran-
swell cell migration chambers in combination with FluoroBlok
inserts (BD Biosciences; 8-
m pore size) in a 24-well plate for-
mat. Lower chambers contained 10 ng/ml of recombinant
human MIF or (100 ng/ml; 0.8 n
M) LPS in medium containing
0.5% bovine serum albumin as the chemoattractant. Calcein-
stained (Calbiochem) EPCs were preincubated with 10
M
compound and placed into the upper chambers (50,000 cells
per insert). Fluorescence signals representing the migration of
calcein-stained cells into the bottom chambers were deter-
mined 3 h later using a fluorescence microplate reader. As day
14 EPCs express CD14, LPS (100 ng/ml) was used as a positive
control. The percentage of cells migrated to the lower chamber
was determined after 3 h.
Compound Toxicity Studies Using MTT Assay—RAW 264.7
macrophages (5 10
4
cells/well in 96-well plates) were incu
-
bated with 3, 10, and 20
M of each compound. Twenty-four
hours later, toxicity was assessed using the MTT assay (Sigma).
Absorbance was recorded at 405 nm with 590 nm as a reference
wavelength.
RESULTS
High Throughput Screening Identifies 18 Novel Inhibitors of
MIF Tautomerase Activity
The tautomerase assay is a well established method to assess
MIF-specific enzymatic activity, and has yielded reproducible
results in different laboratories (22, 23, 55). We optimized the
conditions for performing this assay in a high throughput (384-
well plates) format. Two known non-physiological MIF sub-
strates (
D-dopachrome and phenylpyruvate) were evaluated.
D-Dopachrome was chosen for screening because it absorbs at
475 nm, a wavelength at which small molecules do not absorb,
which is not the case for HPP (300 nm). As a positive control, we
initially used ISO-1, a known inhibitor of the enzymatic activity
of MIF (35). A validation assay was performed to test the
screening conditions in an automated manner and determine
the robustness and reproducibility of the assay. Intra-plate and
inter-plate variations were determined using single point mea-
surements and Z values of 0.74 and 0.91 were obtained.
An FDA-approved library (NINDS Custom Collection II)
composed of 1,040 bioactive compounds containing a diverse
set of drugs, 85% of which are marketed drugs with a wide range
of therapeutic usage including anti-inflammatory and analge-
sia, and the Maybridge library composed of 14,400 compounds
were screened. The compounds were dissolved in DMSO to
yield a final concentration of 10
M. DMSO alone at a final
concentration of up to 2% had no effect on MIF tautomerase
activity (supplemental Fig. S1B). The inhibitor screening assay
was performed as follows: MIF was added to 384-well plates
containing the compounds in reaction buffer and incubated for
15 min at room temperature. The enzymatic reaction was ini-
tiated by adding the
D-dopachrome substrate. In the positive
control wells, with no enzyme, or with compounds only, the
absorbance of the substrate at 475 nm was 0.9 –1, whereas in
the presence of enzyme the absorbance rapidly decreased (3
min) to an OD value of 0.1 (supplemental Fig. S2A). In our
preliminary screens, ebselen was identified as a potent inhibi-
tor, which fully inhibited MIF tautomerase activity at a con-
centration of 10
M, making it more effective than ISO-1.
Thus, ebselen was used as a positive control (10
M)inall
subsequent screens. The Z value for the FDA and May-
bridge library screenings were 0.74 and 0.91, respectively
(supplemental Fig. S2B). These data suggested that the assay
is quite robust and reproducible. The hit compounds dem-
onstrating greater than 50% inhibition of MIF activity were
retested manually to exclude false positives. Of the 15,440
compounds tested, only 18 were found to be active (Fig. 2
and Table 1) and demonstrated more than 70% inhibition in
the primary assay. To determine the IC
50
values, we per
-
formed concentration-dependent studies by testing 12 dif-
ferent dilutions ranging from 0 to 50
M and obtained IC
50
values in the range of 0.2–15.5
M (Fig. 3).
MIF Antagonists: Mode of Action
Inhibition of the tautomerase activity of MIF can occur via at
least five different mechanisms: 1) binding to the active site; 2)
allosteric inhibition; 3) covalent modification of active site res-
idues; 4) disruption of the active site through compound-in-
duced dissociation of the active trimer; and 5) stabilization of
the MIF monomer and prevention of its re-association to form
the active trimer. The inhibitors identified in our HTS were
subjected to biochemical and biophysical studies to understand
their mechanism of action.
The Compounds 1, 2, 4, 5, 6, 7, 8, 10, 12, 13, and 14 Inhibit MIF
Tautomerase Activity by Selective Modification of the
Catalytic N-terminal Proline Residue
To determine whether MIF inhibitors covalently modify
MIF, the compounds were incubated for1hatroom tempera-
ture with 15
M human MIF (in 25 mM Tris buffer, pH 7.4) and
subjected to analysis by MALDI-TOF mass spectrometry.
Upon incubation with 10
M of corresponding compounds 1, 2,
4, 5, 6, 7, 8, 10, 12, 13 and 14, we observed a shift in the mono-
meric molecular mass consistent with a single modification of
MIF (supplemental Fig. S3). To identify the site of covalent
modifications, the modified MIF was subjected to proteolytic
digestion by trypsin and peptide mapping by mass spectrome-
try. In the case of compounds 2, 6, 8, and 12, we observed a
peptide fragment with molecular mass corresponding to that
comprising N-terminal residues PMFIVNTNVPR (molecular
mass 1287.6 Da) plus one molecule of inhibitor. MS/MS anal-
yses and sequencing of the modified peptide fragments revealed
that modification by these compounds takes place exclusively
at the N-terminal proline residue.
Dissociation Constant (K
D
) Reveals a Strong Binding Behavior
for HCLP and Compound 9
No covalent modifications were observed when compounds
3, 9, 11, and HCLP were incubated with MIF, suggesting that
the inhibitory activity of these compounds is mediated by non-
covalent interactions with the protein. To evaluate the strength
of binding, the K
D
for each compound was measured using
fluorescence spectroscopy. MIF at 5
M was incubated with
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increasing concentrations of compounds and binding was eval-
uated by monitoring changes in the tryptophan emission max-
imum upon MIF binding to compounds. The maximum fluo-
rescence emission was plotted as a function of compound
concentration and normalized. HCLP and compound 9 showed
K
D
values of 1.5 0.1 and 2.11 0.15
M, respectively. Com
-
pounds 3 and 11 exhibited K
D
values of 6.28 1.02 and 24.45
6.74
M, respectively (Fig. 4).
Elucidating the Structural Basis Underlying the Inhibitory
Activity of Ebselen, HCLP, and Compounds 3, 9, and 11
Using NMR
To determine the structural basis for the inhibition of human
MIF enzymatic activity, NMR titrations were performed with
ebselen, HCLP, and compounds 3, 9, and 11.
Ebselen—An equimolar mixture of MIF with ebselen resulted
in severe resonance broadening throughout the whole se-
quence, indicative of conformational exchange (Fig. 5A). Most
drastic effects were seen for residues 3–5, 38–39, 49 –51,
61–62, 64, 99, 101, 106 –108, and 112. Most of these residues
are in the subunit-subunit interface. The
-sheet core of a
monomeric subunit that consists of four
-strands, as well as
the three
-strands coming from the other two subunits are
highly affected by ebselen (Fig. 5, B and C), which supports the
idea that a stable trimer dissociates into monomeric units upon
destabilization by ebselen. In agreement with the sedimenta-
tion velocity experiments (see below), the drop in signal inten-
sity could be due to a combination of both conformational
changes and aggregation of the dissociated monomeric sub-
units into larger moieties that are beyond the detection limit of
liquid state NMR. Among the three cysteines of MIF, only Cys
59
displays a minor chemical shift, such an effect is observed for
neither Cys
56
nor Cys
80
(supplemental Fig. S4A). Additionally,
the only residue that has surface accessibility is Cys
59
(supplemental Fig. S4, B-D), suggesting a role for this residue in
ebselen interaction. No change is observed for the
-helices.
HCLP—Titration of MIF with HCLP results in both reso-
nance broadening and chemical shift changes (Fig. 6A), indica-
tive of an intermediate exchange regime in the NMR time scale.
The residues that are most affected by HCLP are 3–5, 35–38,
64, 67, 95, 107–110, 112, and 114 (Fig. 6B). Similar to the MIF
inhibitor benzyl isothiocyanate (56) and HPP (31), these resi-
dues cluster around Pro
1
. Among these residues, Ile
64
and Tyr
95
are found in the active site.
Compounds 3, 9, and 11—Backbone amide signals of MIF
were monitored for hit compounds 3, 9, and 11. An equimolar
mixture of MIF and compound 3 resulted in chemical shift
deviations averaged on
1
H and
15
N dimensions in residues 2–3,
35–39, 49 –51, 63–66, 94 –96, 107–110, and 112–113 (Fig. 6, A
and B). Many of these residues are located in the subunit-sub-
unit interface and enzymatic active site (Fig. 6C). Upon addition
of compound 9, more than 0.75 molar ratio relative to MIF,
several peaks in the HSQC analysis disappeared, indicating
FIGURE 2. High throughput screening of the NINDS and Maybridge libraries resulted in the identification of 18 novel inhibitors. Chemical structures of
the primary and validated hits.
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TABLE 1
Summary table of Maybridge hit compounds code, name, structure, IC
50
, K
i,app
and the mass shift observed by mass spectrometry upon
30 min incubation with MIF covalent modifiers
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intermediate exchange of conformation. These peaks reap-
peared with an increasing molar ratio of compound 9, which
implies a higher affinity of this inhibitor to MIF. Addition of
compound 9 to MIF also induced a chemical shift deviation, but
with two sequential changes: first a milder interaction followed
by conformational changes in MIF. Up to a 0.5 molar ratio of
compound 9, resonances from residues 2–3, 35–37, 50, 58, 60,
61, 65– 66, 75, 81, 93, 95–96, 98, 107–108, and 110 –111, which
correspond to the subunit-subunit interface and enzymatic
active site were observed (Fig. 6, B and C). Although compound
11 had the lowest IC
50
value in the MIF tautomerase assay, its
binding to MIF resulted in an even larger number of residues
affected by slow conformational exchange, including residues
2–3, 35–37, 5860, 60, 65– 68, 72–78, 97–102, 105–106, and
112–114 (Fig. 6B). The residue stretches affected by slow con-
formational exchange are located in the subunit-subunit inter-
face and close to the active site (Fig. 6C).
Structure-Activity Relationship Studies for Hexachlorophene
Hexachlorophene did not lead to covalent modification of
MIF. Fortunately, several analogues of HCLP are commercially
available. Therefore, we performed structure-activity relation-
ship studies to better understand its mechanism of action and
to identify the chemical groups responsible for evoking tau-
tomerase inhibition. Eleven analogues of HCLP (Fig. 7A) were
selected and tested. Only MDPI894 (IC
50
,8
M), bithionol
(IC
50
, 6.5
M and K
i,app
, 4.74 0.85
M), and dichlorophene
(IC
50
,15
M and K
i,app
, 6.57 0.54
M) were observed to
exhibit significant inhibition of MIF compared with HCLP with
IC
50
, 3.5
M, and K
i,app
, 2.21 0.39
M (Fig. 7B). Compound 5,
bis(2-hydroxyphenyl)methane, which lacks any of the chlorine
substitutions in the phenol rings is inactive. Interestingly, the
addition of only two chlorine atoms at the 3,3 positions,
dichlorophene, was sufficient to transform this molecule into
an active inhibitor of MIF, albeit less active than HCLP.
Removal of either the hydroxyl groups or the 3,3 chlorine
atoms results in loss of HCLP inhibitory activity. Together, the
FIGURE 3. K
i,app
obtained by plotting relative initial velocities as a function of inhibitor concentration and data fitting to the simple inhibition
expression: V
i
(V
0
/(1 (I/K
i,app
)
n
)) bkg, where V
0
, velocity at [I] 0, and n the Hill coefficient. Each data point represents the mean S.D., n 4. The
data were analyzed using Sigma plot software and each experiment was done in triplicate.
FIGURE 4. Dissociation constant (K
D
) measurements for HCLP and com
-
pounds 3, 9, and 11 determined by monitoring MIF tryptophan fluores-
cence quenching at 295 nm as a function of increasing concentration of
MIF inhibitors.
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structure activity results shown in Fig. 7 highlight the impor-
tance of the 6,6 hydroxyl groups and 3,3 chloride atoms for
MIF inhibition. Furthermore, our results suggest that the
position of the remaining chlorine atoms on the aromatic
rings as well as the nature of the bridging atom influence the
potency of HCLP. The inhibitory activity of bithionol dem-
onstrates that replacing the bridging carbon atom of HCLP
by a sulfur and removal of the chlorine atoms at the 2,2
positions reduce the inhibitory potency of HCLP. Substitu-
tion of the bridging carbon by a sulfoxide results in loss of
inhibitory activity despite the presence of the chlorine atoms
at the 3,3, and 5,5 positions, suggesting that some confor-
mational flexibility between the two phenol rings is crucial to
allow these inhibitors to adopt the right conformation in the
active site.
Sedimentation Velocity Demonstrates That Only Ebselen
Induces Trimer to Monomer Dissociation
To determine whether any of the antagonists functioned by
disrupting the MIF trimer, we compared the quaternary struc-
ture distribution of human MIF in the presence or absence of
inhibitors by sedimentation velocity analytical ultracentrifuga-
tion. Human MIF preincubated with DMSO as a control sedi-
mented predominantly as a single species with a sedimentation
coefficient and molecular mass corresponding to that of the
trimer (s 3.15 0.1 S and 36,373 kDa) (Fig. 8A). In the pres-
ence of ebselen, at a concentration corresponding to the IC
50
(3
M, Fig. 8A), we observed an addi-
tional sedimenting species with an
average s value of 1.7 0.2 S and a
molecular mass of 12,428 kDa,
indicative of the presence of mono-
meric MIF (Fig. 8B). Studies at
higher ebselen concentrations were
not possible due to ebselen-induced
aggregation and precipitation of
MIF (Fig. 9A). None of the other
inhibitors was shown to disrupt the
MIF trimer.
Ebselen Inhibits MIF Tautomerase
Activity by Inducing Cysteine-
mediated Modification, Trimer
Dissociation, and Aggregation
of MIF
Ebselen is a well known anti-in-
flammatory and antioxidant drug
(57, 58). The therapeutic effects of
ebselen have been linked to its per-
oxidase activity; it has high chem-
ical reactivity toward hydroperox-
ides and thiols. Ebselen has been
used as a probe for characterizing
cysteine-targeted oxidation of thi-
oredoxin (59). In bovine systems,
free ebselen quickly reacts with
thiols to form two metabolites, an
ebselen-glutathione adduct and an
ebselen-cysteine adduct (60). MIF monomer has three cys-
teine residues at positions 56, 59, and 80. Previous studies
showed that mutating or alkylating the cysteine residues at
positions 56 or 59 led to a reduced conformational stability
of MIF (61).
To determine whether ebselen inhibition of MIF is medi-
ated by covalent modification of specific cysteine residues,
mass spectrometry analyses were carried out on MIF sam-
ples preincubated with increasing concentrations of ebselen
(0–10
M)for1hatroom temperature. MALDI-TOF anal-
ysis revealed a peak with a m/z 12620.48, which corre-
sponds to monomeric MIF with one bound molecule of
ebselen (molecular mass of 274 Da). However, we also
observed that ebselen induced rapid aggregation and precip-
itation of MIF. To quantify the degree of ebselen-induced
MIF aggregation, the samples were centrifuged at high speed
(13,000 g) and the supernatants and pellets were collected
and analyzed in SDS-PAGE gels. Fig. 9A demonstrates that
the amount of MIF remaining in solution decreased as the
concentration of ebselen increased, with complete precipi-
tation of MIF at ebselen concentrations of 15
M.
Blocking Cysteine Residues by Alkylation or Chemical
Cross-linking Prevents Ebselen-induced MIF Aggregation
MIF contains three free cysteines at positions 56, 59, and 80.
To assess whether ebselen-induced aggregation is mediated via
covalent modification of specific cysteine residue(s), wild-type,
FIGURE 5. Structural basis for the inhibition of MIF enzymatic activity by ebselen using NMR. A, the
two-dimensional
1
H-
15
N HSQC reference spectrum (black) was recorded with MIF in the presence of 1% DMSO.
The addition of ebselen to MIF at equimolar concentrations (red spectra) resulted in resonance broadening.
B, changes in NMR signal intensities in a
1
H-
15
N HSQC upon addition of ebselen. C and D, residues with peak
intensity ratios below 0.18 in B are highlighted in red on the three-dimensional structure of MIF (PDB code
1GD0). C and D are related to each other by 90°. Cyan, green, and pink show the three different subunits of MIF.
The catalytically active P1 residue is indicated in yellow.
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C56S, and C59S MIF were expressed, purified, and alkylated for
1 h at room temperature using 10 m
M maleimide. In the case of
wild-type MIF, a major peak of 13,020 Da, corresponding to
monomeric MIF with three alkylated cysteine residues (molec-
ular mass of maleimide is 225.4 Da) could be detected by MS,
confirming total alkylation of MIF under the native conditions
(Fig. 9B). After removal of excess maleimide, alkylated MIF was
incubated for1hatroom temperature with 100
M ebselen and
the sample mixtures were centrifuged at 13,000 g for 15 min.
The supernatant and the re-suspended pellet (in the original
working volume) were then analyzed in a 15% SDS gel to quan-
tify the amount of soluble and precipitated protein. As