Structure-Activity Relationships and Anti-inflammatory Activities of N-Carbamothioylformamide Analogues as MIF Tautomerase Inhibitors

Article abstract of DOI:10.1021/acs.jcim.5b00445

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is an attractive therapeutic target for the treatment of inflammatory diseases. In our previous study, 3-[(biphenyl-4-ylcarbonyl)carbamothioyl]amino benzoic acid (compound 1) was discovered as a potent inhibitor of MIF by docking-based virtual screening and bioassays. Here, a series of analogues of compound 1 derived from similarity search and chemical synthesis were evaluated for their MIF tautomerase activities, and their structure-activity relationships were then analyzed. The most potent inhibitor (compound 5) with an IC₅₀ of 370 nM strongly suppressed lipopolysaccharide (LPS)-induced production of TNF-α and IL-6 in a dose-dependent manner and significantly enhanced the survival rate of mice with LPS-induced endotoxic shock from 0 to 35% at 0.5 mg/kg and to 45% at 1 mg/kg, highlighting the therapeutic potential of the MIF tautomerase inhibition in inflammatory diseases.

Full text of DOI:10.1021/acs.jcim.5b00445

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Article
Structure-activity relationships and anti-inflammatotry activities of N-
carbamothioylformamide analogues as MIF tautomerase inhibitors
Yu Zhang, Lei Xu, Zhiqiang Zhang, Zhiyu Zhang, Longtai Zheng, Dan
Li, Youyong Li, Feng Liu, Kunqian Yu, Tingjun Hou, and Xuechu Zhen
J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.5b00445 • Publication Date (Web): 19 Aug 2015
Downloaded from http://pubs.acs.org on August 25, 2015
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Structure-activity relationships and anti-inflammatotry
activities of N-carbamothioylformamide analogues as MIF
tautomerase inhibitors
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Yu Zhanga#, Lei Xub,c#, Zhiqiang Zhanga, Zhiyu Zhanga, Longtai Zhenga, Dan Lib,
Youyong Lid, Feng Liua, Kunqian Yue, Tingjun Houa,b,d*, Xuechu Zhena*
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aJiangsu Key laboratory of Translational Research and Therapy for
NeuroꢀPsychoꢀDiseases & Department of Pharmacology, College of Pharmaceutical
Sciences, Soochow University, Suzhou, Jiangsu 215123, China
bCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang
310058, China
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cInstitute of Bioinformatics and Medical Engineering, School of Electrical and
Information Engineering, Jiangsu University of Technology, Changzhou 213001,
China.
dInstitute of Functional Nano & Soft Materials (FUNSOM), Soochow University,
Suzhou, Jiangsu 215123, China
eState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 200031, China
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#Equivalent authors
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Corresponding author:
Xuechu Zhen
Eꢀmail: zhenxuechu@suda.edu.cn
Phone: +86ꢀ512ꢀ6588ꢀ0369
Tingjun Hou
Eꢀmail: tingjunhou@zju.edu.cn or tingjunhou@hotmail.com
Phone: +86ꢀ517ꢀ8820ꢀ8412
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Abstract
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Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is an
attractive therapeutic target for the treatment of inflammatory diseases. In our
previous study, 3ꢀ[(biphenylꢀ4ꢀylcarbonyl)carbamothioyl]amino benzoic acid
(compound 1) was discovered as a potent inhibitor of MIF by dockingꢀbased virtual
screening and bioassays. Here, a series of analogues of compound 1 derived from
similarity search and chemical synthesis were evaluated for their MIF tautomerase
activities, and their structureꢀactivity relationships (SARs) were then analyzed. The
most potent inhibitor (compound 5) with an IC50 of 370 nM strongly suppressed
LPSꢀinduced production of TNFꢀα and ILꢀ6 in a doseꢀdependent manner, and
significantly enhanced the survival rate of mice with LPSꢀinduced endotoxic shock
from 0% to 35% at 0.5 mg/kg and to 45% at 1 mg/kg, highlighting the therapeutic
potentials of the MIF tautomerase inhibition in inflammatory diseases.
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Macrophage migration inhibitory factor (MIF), a multiꢀfunctional protein,1 has been
regarded as an attractive therapeutic target for the treatment of sepsis2 and other
inflammatory diseases.3, 4 Previous studies illustrated that the inhibition of MIF
activity in vivo could attenuate the lethality of endotoxemia and sepsis in rodents.2, 5, 6
MIF exists as a homotrimer with three identical monomers, and the catalytic active
site is located between two adjacent monomers of the homotrimer.7 In addition, MIF
functions as a Dꢀdopachrome tautomerase,8 a phenylpyruvate tautomerase,9 or a
thiolꢀprotein oxidoreductase.10 Despite relentless efforts, there is no clearꢀcut evidence
between the catalytic activity and biological function of MIF.11 However, targeting
MIF tautomerase activity through smallꢀmolecule inhibitors has been proven to be an
attractive strategy for inhibiting MIF proinflammatory activity and attenuating its
biological activity in vitro and in vivo.11, 12
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To date, a variety of smallꢀmolecule MIF tautomerase inhibitors have been
reported by substrate analog screening, structureꢀbased virtual screening and
highꢀthroughput screening.1, 11, 13ꢀ15 Though these inhibitors provide a proof of
concept for therapeutic utility against MIF, most of them are not ideal for
pharmaceutical development owing to low potency or irreversible inhibition on MIF
tautomerase activity. For example, the prototypical MIF inhibitor ISOꢀ1 shows only
micromolar potency with respect to MIF inhibition.16 The first identified MIF
inhibitor NAPQI,17 a metabolite of acetaminophen, forms a covalent bond with the
MIF Nꢀterminal proline residue (Pro1), as well as 4ꢀiodoꢀ6ꢀphenylpyrimidine
(4ꢀIPP)18 and isothiocyanates (ITC).19 Recently, by combining dockingꢀbased virtual
screening and in vitro bioassays, 3ꢀ[(biphenylꢀ4ꢀylcarbonyl)carbamothioyl]amino
benzoic acid (presented as compound 1 in this study) was identified to be a
competitive inhibitor of MIF (IC50 = 550 nM), and was found to effectively inhibit the
biological functions of MIF.20 However, the structureꢀactivity relationships (SARs) of
the analogues of compound 1 have not been characterized and its in vivo potency has
not been tested. Therefore, in this study, a number of analogues of compound 1 were
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purchased or chemically synthesized, and their MIF tautomerase activities were
evaluated. In total, 31 compounds were found to be potent tautomerase inhibitors of
MIF with IC50 values below 10 ꢀM (six with IC50 < 1 ꢀM). The strong
antiꢀinflammatory potency of the most potent inhibitor, compound 5 (IC50 = 370 nM),
as evidenced by suppressing lipopolysaccharide (LPS)ꢀinduced macrophage
activation in vitro, was verified by ELISA, quantitative realꢀtime PCR and western
blot. Furthermore, the therapeutic importance of the MIF inhibition by compound 5
was demonstrated by increasing the survival rate of mice with LPSꢀinduced endotoxic
shock from 0% to 35% at 0.5 mg/kg and to 45% at 1 mg/kg, respectively.
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120 Materials and Methods
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Analogues Chosen by Similarity Search and Molecular Docking
Because the commercial chemical databases are usually enriched with analogues by
the nature of their synthetic routes, the analogues of compound 1 were identified from
the ChemBridge and Specs databases by similarity search based on the Tanimoto
similarity coefficients21 computed from the MACCS Structural fingerprints in MOE.22
Tanimoto similarity coefficients higher than 0.85 and 0.80 were set as the cutoffs for
the ChemBridge and Specs databases, respectively.
Then, the molecules selected by similarity search were processed with the
program LigPrep in Schrödiger23 to generate the tautomers and protonation states at
pH = 7.4 (up to 30 stereoisomers for each compound with chiral centers), and then
docked into the binding site of MIF. The crystal structure of MIF complexed with
pꢀhydroxyphenyl pyruvate (HPP) (PDB entry: 1CA7)24 was selected as the template
for molecular docking. The ligand located in the active site between chain A and
chain B was retained, and the other two ligands were deleted. The Nꢀterminal proline
residue (P1) was protonated in terms of the experimental measurement and theoretical
calculations.25, 26 Hydrogen and other missing atoms were added, and the protein was
energyꢀminimized using OPLS2005 force field27 with the Protein Preparation Wizard
module in Schrödinger (version 9.0). All structures were docked into the active site of
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MIF and scored by the extra precision (XP) scoring function of Glide. In the docking
process of Glide, the protein conformation is fixed and each docked ligand is flexible.
As for the grid generation and ligand docking, the default Glide settings were
employed. The top scored molecules were assessed by the REOS rules28 and the
drugꢀlikness filters developed in our group29ꢀ31 to remove compounds with toxic,
reactive or otherwise undesirable moieties, and finally 46 compounds were chosen
and purchased.
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Synthetic Chemistry
The importance of the halogen substitution on MIF tautomerase activity has been
reported in the previous study, where the substitution of monoꢀfluorination onto the
ortho position of the phenolic group of ISOꢀ1 could improve the inhibition activity
against MIF up to 41%.32 In order to study the effect of the halogen substitution on
1
bioactivity, 11 analogues of compound 1 were synthesized. H NMR and 13C NMR
spectra were acquired on 400 MHz (Varian) spectrometers. Chemical shifts were
given in ppm through tetramethylsilane (TMS) as internal standard. At last, the
compounds were purified using silica gel 100ꢀ200 mesh for column chromatography.
The synthetic routes for the pursed series are depicted in Schemes 1ꢀ2. A mixture of
4ꢀBromobenzoic Acid Methyl Ester (645 mg, 3.0 mmol, 1.0 eq.), aryl boronic acid
(3.6 mmol, 1.2 eq.), Pd(PPh3)4 (69 mg, 0.05 mmol, 0.02 eq.), and Na2CO3 (699 mg,
6.6 mmol, 2.2 eq.) in dioxane/H2O (7:1, v/v) (16 mL) was stirred at 100 °C for 1 h.
The solvent was removed under reduced pressure and the residue was diluted with
water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extracts
were washed with brine, dried over Na2SO4, filtered and concentrated. The residue
was purified by flash column chromatography (hexane/EtOAc 50:1) to give the
corresponding ester. A solution of unsaturated acids (2 mmol) in dichloromethane (10
mL) was added oxalyl chloride (1.02 g, 8 mmol), followed by adding 2 drops of DMF.
The mixture was stirred at room temperature for 1 h, and then excess oxalyl chloride
and dichloromethane were removed in vacuo to provide crude acyl chloride. A
suspension of benzoyl chloride (3 mmol) and sodium thiocyanate (486 mg, 6 mmol)
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in acetonitrile (9 ml) was stirred for 1 h at room temperature. Then, sodium chloride
was filtered, and anthranilic acid was added to the filtrate. The mixture was stirred for
1ꢀ2 h, methanol (10 ml) was added to the solvent and stirred for 10 min, and then the
solvent was filtrated and the solid was collected. The detailed spectral data are
provided at Part 1 of the Supporting Information.
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In total, there are 51 compounds in Tables 1~3. Compound 1 is the lead
compound identified from our previous study,20 compounds 2ꢀ11, 15ꢀ34 and 43ꢀ51
were purchased from the ChemBridge and Specs databases, and the others were
chemically synthesized.
Assay for MIF Tautomerase Activity
The following protocol was adapted from Tao et al.33 Fresh solution of Lꢀdopachrome
methyl ester was prepared by mixing equal volumes of Lꢀ3,4ꢀdihydroxyphenylalanine
methyl ester (6 mM), sodium metaperiodate (12 mM) and incubated for 20 min at
room temperature. The dopachrome solution was prepared before the inhibition assay
owing to its relative instability. Then, hMIF (120 nM) was added to a 96ꢀwell plate
containing Lꢀdopachrome (30 ꢀL) in 10 mM potassium phosphate buffer and 0.5 mM
EDTA at pH = 6.2. The inhibitory effect of each compound against the tautomerase
activity of MIF was measured by adding various concentrations of the compound to
the 96ꢀwell plate containing hMIF (120 nM) and incubating for 30 min followed by
the addition of Lꢀdopachrome methyl ester. The absorbance was measured by
monitoring the decrease in absorbance at 475 nm for 3 min with a Tecan Infinite
M1000 microplate reader (Tecan Group Ltd.).
Cell Culture and Cell Viability Assay
The RAW 264.7 cell line was purchased from the American Type Culture Collection
(ATCC). The cells were maintained in complete DMEM supplemented with 10%
heatꢀinactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100
mg/mL) in a 37 °C, 5% CO2 incubator. The cells were attached to the culture plate at
least for 24 h prior to allowing various experiments. Cell viability was determined by
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3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay. RAW
264.7 macrophages were seeded into 96ꢀwell plates (5 × 104 cells/well). After
appropriate treatment, the cells were incubated in MTT solution at 37 °C for 4 h, and
then 100 ꢀL of DMSO was added to each well to dissolve the violet formazan crystals
for 5 min as previously described 18. The optical density was measured at 540 nm on a
Microplate Reader (Infinite M200 PRO, Tecan, Switzerland).
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Measurement of Intracellular Nitric Oxide (NO)
RAW 264.7 macrophages were seeded into 96ꢀwell plates, and culture supernatants
were collected after treatment with LPS or compounds. Concentration of NO was then
determined by using the Griess reagent as described previously.34 Briefly fifty
microliters of Griess reagent (containing 0.2% w/v Nꢀ(1ꢀnapthyl) ethylenediamine
dihydrochloride and 2% w/v sulfanilamide in 5% v/v H3PO4) were mixed with same
volumes of cell culture conditioned medium, and then incubated at room temperature
for 10 min in the dark. The absorbance of mixture was measured by a 96ꢀwell
microplate reader (Tecan Systems, Inc.) at 570 nm.
Enzyme-linked Immunosorbent Assay (ELISA)
The levels of TNFꢀα and ILꢀ6 in the cell culture medium were determined by specific
ELISA kits according to the manufacturer’s instructions.35
RNA Extraction and Real-time Quantitative PCR (RT-qPCR)
Total RNA was extracted using the TRIzol reagent according to its manufacturer’s
instructions (TaKaRa, Dalian, China). The obtained total RNA was used for synthesis
of cDNA using the ImPromꢀIITM Reverse Transcription System (Promega, USA).
Quantitative real time PCR was performed using Maxima SYBR Green/ROX qPCR
Master Mix. The specific primers used for cDNA amplification are following: iNOS
forward, CAG GAG GAG AGA GAT CCG ATT TA, and iNOS reverse, GCA TTA
GCA TGG AAG CAA AGA; TNFꢀα forward, CAT CTT CTC AAA ATT CGA GTG
ACA A, and TNFꢀα reverse, TGG GAG TAG ACA AGG TAC AAC CC; ILꢀ1β
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forward, TCC AGG ATG AGG ACA TGA GCA C, and ILꢀ1β reverse, GAA CGT
CAC ACA CCA GCA GGT TA; GAPDH forward, TGT GTC CGT CGT GGA TCT
GA, and GAPDH reverse, TTG CTG TTG AAG TCG CAG GAG; MIF forward,
CGT GCC AGA GGG GTT TCT GT, and MIF reverse, GTT CTG GGC ACC ACC
GAT CT; HOꢀ1 forward, CAC GCA TAT ACC CGC TAC CT, and HOꢀ1 reverse, TCT
GTC ACC CTG TGC TTG AC. The relative level of gene expression was normalized
to the level of GAPDH and calculated using the 2 ꢀ(ꢁꢁCT) formula.
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Western Blot Analysis
After appropriate treatment, RAW 264.7 macrophages were collected and washed
three times with iceꢀcold PBS. And the cells were lysed with RIPA lysis buffer [50
mM Tris [pH 8], 1% Triton Xꢀ100, 0.1% SDS, 0.5% sodium deoxycholate, 150 mM
NaCl and a protease inhibitor cocktail (Roche Boehringer Mannheim Diagnostics,
Mannheim, Germany)]. Protein concentration was measured with protein extraction
kit and BCA protein assay kit (BioꢀRad, Hercules, CA). The samples were boiled for
5 min which contained 1% βꢀmercaptoethanol followed by separation with
SDSꢀPAGE, then the proteins were transferred to polyvinylidene difluoride (PVDF)
membranes (Millipore, Billerica, MA), blocked in PBSꢀT (PBS plus 0.1% Tweenꢀ20)
containing 5% skim milk at room temperature for 90 min. The membrane was probed
with primary antibodies [polyclonal rabbit antiꢀmouse iNOS (Abcam, NT); polyclonal
rabbit antiꢀmouse COXꢀ2(Santa Cruz Biotechnology); monoclonal antiꢀrabbit MIF
(Abcam, NT); and monoclonal antiꢀaꢀtubulin mouse ascites fluid (Sigma)] at 4 °C
overnight. After washing the membrane with PBSꢀT, the membrane was incubated
with HRPꢀconjugated secondary antibodies (SigmaꢀAldrich) at room temperature for
90 min respectively. The protein bands were visualised using enhanced
chemiluminescence (ECL) (Pierce Biotechnology Inc., Rockford, IL), with a
ChemiScope 3300 Mini (CLINX, Shanghai, China) and the relative signal intensity
was quantified by densitometric analysis (Quantity One).
Experimental Endotoxic Shock Model
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LPS (Escherichia coli 0111: B4, Catalog no.Lꢀ2630, Lot no.78H4086, Sigma–Aldrich,
St. Louis, MO, USA) was dissolved in saline for administration to C57 BALB/c mice.
Mice were injected intraperitoneally with LPS at a dose of 4.5 mg/kg and in the
volume of 0.1 ml/10g body weight to induce endotoxin shock model. Mice received
intraperitoneal administrations of compound 5 (0.5 mg/kg or 1 mg/kg) which prepared
in 2% dimethyl sulfoxide/2% Tween 80/2% Cremophor EL in saline either or vehicle
1 h prior to LPS injection. Survival was monitored at 1, 2, 3, 4, 5 and 6 days postꢀLPS
challenge. The number of animals used in each experimental group was 20. The mice
that survived over 6 days were euthanized by cervical dislocation. All experimental
procedures utilizing mice were in accordance with National Institute of Health
guidelines. The animal study was approved by the Institutional Review Board of
Soochow University.
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Histological and Statistical Analysis
The lung tissues were collected at 12 h after LPS challenge for histological
assessment. Lung tissues were fixed in formalin and subsequently embedded in
paraffin then stained with hematoxylin & eosin (H.E) for histopathological evaluation
under light microscope (Olympus, Japan). Results were expressed as the mean ± SD
using GraphPad Prism 5.0. Oneꢀway ANOVAs followed by Bonferroni tests were
utilized for multipleꢀgroup comparisons. A value of p < 0.05 was considered
statistically significant.
281 Results and Discussion
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SAR Analyses for Analogues of Compound 1
The commercial chemical databases are usually enriched with analogues by the nature
of their synthetic routes, and therefore, in this study, based on similarity search and
molecular docking, 16 analogues from ChemBridge and 30 analogues from Specs
were purchased for biological assays.
Seven out of the 46 compounds were not tested owing to poor solubility in
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DMSO/ethanol/water, and the other compounds were then assayed for their MIF
tautomerase activities with Lꢀdopachrome as the substrate. The prototypical MIF
inhibitor (compound 1), our previously reported tautomerase inhibitor of MIF,20 was
used as the reference control, and DMSO (1%, v/v) used as the vehicle control. The in
vitro MIF tautomerase assay showed that 28 analogues have IC50 values below 10 ꢀM
and 5 below 1 ꢀM. All these compounds had one common key structural feature: two
aromatic rings linked by Nꢀcarbamothioylformamide in the middle position.
According to the structural differences, these analogues could be roughly classified
into three categories (Tables 1~3): analogues with biphenyl (2ꢀ11), analogues with
benzene (15ꢀ34) and analogues with diphenylmethane (43ꢀ51). The SAR analyses of
these compounds could provide important insights of the essential structural features
for effective MIF inhibitors.
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Analyses of the 10 compounds in Table 1 illustrated some noteworthy
observations of the SARs: (1) the substitution of the carboxy group on R2 was critical,
and the MIF inhibitory activity became worse when the position of carboxy was
changed from meta to para (1 vs 3); (2) the introduction of a chlorine atom (R4)
slightly promoted the potency (1 vs 8), and the replacement of chlorine with the
methyl or methoxyl group decreased the potency, which might be explained by the the
changes in the density distribution of the electron cloud of the benzene ring resulted
from electronꢀwithdrawing group in comparison with electronꢀdonating group (2, 8
and 10); (3) when only R3 was changed by replacing the hydrogen atom with the
hydroxy, acetylamino, carboxy or carbomethoxy group, the activity decreased
significantly (5, 4, 3 and 6), for instance, compound 5 showed the best MIF inhibitory
potency. Thus, the introduction of hydroxy on R2 could enhance the inhibitory activity
on MIF.
In addition, the key observations of the SARs from the 20 compounds in Table 2
were summarized as follows: (1) upon the replacement of hydrogen with a methyl
group on R6, improvement of the inhibitory activity was observed (16 vs 18).
Moreover, the position of methyl had substantial influence on the inhibitory activity,
and the potency increased from meta to para (16 vs 20) while strongly decreased to
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ortho (23 vs 24); (2) when only R3 was changed by replacing hydrogen with the acetyl,
carbomethoxy, carboxy or carbethoxy group, the activity decreased gradually (25, 24,
18 and 28), and thus the introduction of acetyl on R3 might be beneficial to the
potency on MIF inhibition; (3) the substitution of carbomethoxy on R1 was important
for the inhibitory activity (compounds 33 and 34 with the IC50 values below 1 ꢀM),
and when only R3 was changed, the potency increased upon the replacement of
carboxy with the carbomethoxy group (18 vs 24). Generally, the introduction of the
carbomethoxy group was beneficial to the potency, in particular on R1.
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Analyses of the 9 compounds in Table 3 exhibited some interesting observations
of the SARs: (1) the substitution of carbomethoxy was beneficial, and compound 46
with carbomethoxy at the para position showed better inhibition than the
corresponding analogue 49 with carbomethoxy at the ortho position; (2) when only R3
was changed by replacing hydrogen with the phenylamino, benzamido, carbomethoxy,
carboxy or formylamino group, the activity decreased gradually (51, 47, 46, 43 and
48). Generally, the substituents of the aromatic group on R3 were critical to the
potency (47 and 51).
To determine the activity affected by the substitution of halogen atom, 11
compounds were synthesized and assayed for the MIF tautomerase inhibitory
activities. As shown in Tables 1 and 2, the introductions of the fluorine atoms on R5
and R6 showed unfavorable effect on the inhibitory activity in comparison with the
chlorine atoms (13 vs 14), as well as the trifluoromethyl group (12 vs 13).
Accordingly, we speculated that the introduction of the trichloromethyl group on R5
and R6 might be favorable to the inhibitory activity. In addition, the position of the
fluorine atom had significant impact on the MIF inhibitory activity, and the potency
dramatically increased from ortho to para to meta (42, 38 and 39). Consequently, the
introduction of the fluorine atom on R5 might be beneficial, whereas the introduction
of the fluorine atom on R4 resulted in a dramatic loss of the inhibitory activity. The
potency gradually increased with the substitution of the fluorine, chlorine or bromine
atom, respectively (38, 37 and 35), which might be explained by the inductive effect
that lead to the changes in the density distribution of the electron cloud of the benzene
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ring, thereby making strong πꢀπ interaction with Tyr95 and Phe113.
Predicted Binding Poses of Four Most Potent Inhibitors
The most potent inhibitor (compound 5) possesses a hydroxyl group at the para
position, in comparison with the carboxyl group at the meta position of the other three
compounds (8, 13 and 15). As shown in Figure 1a, the biphenyl group of compound 5
could form the arylꢀaryl interaction with Tyr95 and Phe113, and the cationꢀπ
interaction with the protonated Pro1 (Figure 1a). In addition, the hydroxyl group of
compound 5 could form the Hꢀbonds with Gly31 and Lys66, and the hydrophobic
interaction with Pro1, Trp108 and Phe113. Compounds 8 and 13 were the chlorine
derivatives of compound 1, though one substitutes in the benzoic acid side chain and
the other in the biphenyl group. The modeling results suggested that their docked
poses were quite similar, and their biphenyl rings were stabilized in a Tꢀshaped
geometry by forming a network of the πꢀπ interactions with Tyr95 and Phe113. The
carboxyl group of compound 8 was predicted to form the Hꢀbonds with Lys32 and
Lys66, and compound 13 formed the cationꢀπ interaction with the protonated Pro1.
Compared with the other three compounds (5, 8 and 13), compound 15 had a phenyl
group rather than a biphenyl side chain. However, its activity (IC50 = 660 nM) was
improved slightly in comparison with that of compound 1 with the biphenyl group
(IC50 = 720 nM), and its carbonyl group was predicted to form the Hꢀbonds with the
basic residues Lys32 and Lys66. Accordingly, we speculated that if the biphenyl
group of compound 5 was substituted by the benzyl group, and the chlorine atom was
introduced at the para position of the hydroxyl group, its potency might be promoted.
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Biological Activities
As an important proinflammatory cytokine, MIF can induce the secretion of itself and
other proinflammatory cytokines such as TNFꢀα, ILꢀ1b, ILꢀ6, and ILꢀ8, and stimulate
NO production.36, 37 Expression studies also identified that MIF secretion could be
induced by LPS, TNFꢀα and IFNꢀγ in RAW 264.7 macrophages.38 A separate line of
investigation implicated a strong relationship between the specific inhibition of MIF
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tautomerase activity and suppression of MIF proinflammatory activities, such as
ISOꢀ1 that could reduce inflammation induced by MIF.11, 16 It was also reported that
inhibition of MIF tautomerase activity not only suppressed MIF induced production
of proinflammatory cytokines but also reduced LPSꢀ triggered inflammatory
responses in vitro and in vivo.6, 39 Therefore, we investigated the antiꢀinflammatory
effects of MIF tautomerase inhibitors on LPSꢀinduced macrophage. Overꢀproduction
of NO has been used as a hallmark of inflammatory activation in LPSꢀtreated
macrophage.40 Thus, we examined the effect of inhibitors of MIF enzymatic activity
on NO production in LPSꢀinduced RAW 264.7 cells. All compounds with potent
tuatomerase activities (IC50 < 10 ꢀM) could attenuate LPSꢀstimulated NO production,
and three of them with IC50 < 720 nM exhibited the highest potency (Table 1 and
Figure 2). LPS markedly increased TNFꢀα and iNOS expression, which was
significantly inhibited by respective compound among them, compound 5 exhibited
the most potent activity. (Figure 2eꢀ2f). Therefore, we focused on the
antiꢀinflammatory activity of compound 5 in the following experiments.
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To further test the antiꢀinflammatory effect of compound 5, the production of NO,
TNFꢀα and ILꢀ6 were measured from LPS ꢀinduced in RAW 264.7 macrophage in the
presence or absence of compound 5. As shown in Figure 3, compound 5 strongly
inhibited NO production in LPSꢀstimulated RAW 264.7 in a doseꢀdependent manner
(Figure 3a). To exclude the possibility that the decrease of NO was due to cell death,
the cell viability was measured by MTT assay. Results showed that compound 5 at the
indicated concentrations (1ꢀ25 ꢀM) did not alter the cell viability (Figure 3b). TNFꢀα
and ILꢀ6 are proinflammatory cytokines that can initiate the inflammatory response
and mediate the development of chronic inflammatory diseases.41, 42 The productions
of TNFꢀα and ILꢀ6 in the culture supernatants were measured by ELISA. In
agreement with the results of NO, compound 5 markedly inhibited LPSꢀinduced
production of TNFꢀα and ILꢀ6 in a doseꢀdependent manner (Figure 3cꢀ3d).
Next, we measured the gene and protein expression of those proinflammatory
cytokines in RAW264.7 macrophages. As shown in Figure 4aꢀ4e, compound 5 could
dramatically inhibit the gene expression of iNOS, ILꢀ6, TNFꢀα, COXꢀ2, and ILꢀ1b in
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a doseꢀdependent manner in LPSꢀstimulated RAW 264.7 macrophages. Recent study
suggested that HOꢀ1 was one of the key defense molecules against LPSꢀinduced
sepsis and HOꢀ1 overexpression could protect against the systemic inflammatory
response.43ꢀ46 To determine whether compound 5 could attenuate inflammatory
response by inducing HOꢀ1 expression, the HOꢀ1 mRNA expression levels were also
measured. LPS resulted in an increase in HOꢀ1 mRNA levels, and pretreatment with
compound 5 (25 ꢀM) for 30 min before LPS further increased HOꢀ1 protein levels
(Figure 4f). In accordance with the mRNA expression, compound 5 also suppressed
LPSꢀinduced iNOS, COXꢀ2 expression in a doseꢀdependent manner in RAW 264.7
macrophages without altering MIF protein level (Figure 4g).
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Compound 5 elicts protection from LPS-induced Endotoxic Shock in mice.
Many studies have demonstrated that antiꢀMIF antibodies or MIF inhibitors could
attenuate inflammatory cascade and improve the survival rate in sepsis.2, 38, 47, 48 To
determine whether compound 5 also produced antiꢀinflammatory activity in vivo,
LPSꢀinduced experimental shock mouse model was employed. As shown in Figure 5a,
application of compound 5 could protect the BALB/c mice from endotoxic shock, as
evidenced by increasing the survival rate of mice from 0% to 35% at 0.5 mg/kg and to
45% at 1 mg/kg. It is known that LPS induces dramatic increases in inflammation
cytokines, such as TNFꢀα and ILꢀ6, and results in severe tissue damage, multiple
organ failure and eventually leading to death in sepsis.49, 50 We found that compound
5 significantly decreased the serum levels of TNFꢀα and ILꢀ6 in LPSꢀinduced
endotoxin model. These results clearly indicated that compound 5 attenuated
inflammatory responses in LPSꢀinduced sepsis mice by inhibiting the secretion of
proinflammatory cytokines. Tissue injury, especially lung, is a main cause of mortality
in LPSꢀinduced sepsis.51 Therefore, the lungs were examined histologically at the
points of 12 h after LPS treatment with or without compound 5 (1, 0.5 mg/kg, i.p.) 1 h
pretreatment. Histopathology of the lung sections were illustrated in Figure 5d.
Vehicle group showed integrate and clear alveolar spaces, while LPS alone treatment
showed an acute inflammatory response including infiltration of inflammatory cells,
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alveolar septum thickness, and swelling of the alveolar wall. However, the lung injury
was significantly alleviated in the mice with compound 5 (1, 0.5 mg/kg) pretreated
group. These findings highlighted that compound 5 reduced the mortality through
improving inflammatory responses.
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443 Conclusion
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In this study, the SARs for the 50 analogues of Nꢀcarbamothioylformamide were
analyzed. The results provided valuable information about the essential structural
features of effective MIF inhibitors. We further studied the biological functions of
compound 5, the most potent MIF inhibitor with IC50 = 370 nM, in antiꢀinflamatory
responses both in vitro and in vivo. The present data illustrated that targeting the
tautomerase active site of MIF by smallꢀmolecule inhibitors was an attractive strategy
for therapeutic interventions in the inflammatory diseases such as sepsis.
450
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452 Acknowledgment
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This study was supported by the National Science Foundation of China (21173156,
81130023, 81373382, 81372688 and 21302134), the National Basic Research
Program of China (973 program, 2012CB932600 and 2011CB5C4403). Supports
from Priority Academic Program Development of Jiangsu Higher Education Institutes
(PAPD), Project Funded by China Postdoctoral Science Foundation and Grant from
Jiangsu Science and Technology Commission (BY2011131) are also appreciated.
460 Supporting Information
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462
463
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465
Part 1. The detailed spectral data for Compounds 12~14 and 35~42; Table S1. The
number, docking score, source of database and IDs for the compounds shown in
Tables 1~3 from similarity search. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Medicines. J. Cheminformatics 2013, 5, 5.
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33. Tao, L.; Zhang, F.; Hao, L.; Wu, J.; Jia, J.; Liu, J.ꢀy.; Zheng, L. T.; Zhen, X.,
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Response by Modulating Heme Oxygenaseꢀ1 Expression in Endotoxic Shock Model. Am. J.
Physiol. Gastrointest. Liver Physiol. 2007, 293, G1308ꢀG1314.
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Host to Endotoxin and ILꢀ1/tumor necrosis Factor. J. Immunol. 1992, 148, 3596ꢀ3603.
51. Miyashita, T.; Kakimoto, N.; Ishida, Y.; Hayashi, T.; Kimura, A.; Tsokos, M.; Kondo, T.,
Immunohistochemical Expression of Tumor Necrosis Factorꢀalpha in Sepsisꢀinduced Lung Injury.
Forensic. Sci. Med. Pathol. 2006, 2, 103ꢀ108.
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641 Legend of the figures
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
Figure 1. The binding pockets of MIF for compounds (a) 5, (b) 8, (c) 13, and (d) 15.
Figure 2. (aꢀd) Concentrationꢀdependent inhibition of MIF tautomerase activity by
three active molecules. (eꢀf) Three compounds were chosen to measure iNOS
expression and TNFꢀα release based on the inhibitory effect both on tautomerse
activity and NO production. All compounds show protective effect in LPSꢀstimulated
RAW 264.7 macrophages, with compound 5 of them exhibiting the highest potency.
Figure 3. Effects of compound 5 on the production of nitric oxide (NO), TNFꢀα and
ILꢀ6 in LPSꢀactivated RAW 264.7 macrophage cells. RAW 264.7 macrophage cells
were pretreated with or without the indicated concentration (1ꢀ25 ꢀM) of compound 5
for 30 min, followed by LPS (50 ng/mL) treatment for 24 h. (a) Amounts of NO in the
culture supernatants were measured using Griess reagent. (b) Cell viability was
determined by MTT assay. (c) The amounts of TNFꢀα and (d) ILꢀ6 in the supernatants
were measured using ELISA respectively. The data were expressed as percentage of
surviving cells over control cells. The data are the mean ± SD of 3 independent
experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with LPS only group.
Figure 4. Effects of compound 5 on gene expression of proinflammatory factors and
HOꢀ1 in LPSꢀtreated RAW 264.7 macrophages cells. RAW 264.7 macrophages cells
were treated with compound 5 (1ꢀ25 ꢀM) for 30 min, followed by LPS (50 ng/mL)
treatment. (aꢀf) After 6 h of LPS stimulation, the mRNA levels of iNOS, ILꢀ6, TNFꢀα,
COXꢀ2, ILꢀ1β and HOꢀ1 were determined by quantitative realꢀtime PCR assay. After
16 h of LPS stimulation, the protein levels of iNOS, COXꢀ2 and MIF were
determined by western blotting using respective primary antibodies. (g) The αꢀtubulin
was used as an internal control. Quantification of protein expression was performed
by densitometric analysis. (h) The data are the mean ± SD of 3 independent
experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with LPS only group.
Figure 5. Effects of compound 5 on LPSꢀinduced endotoxic shock. (a) Male Balb/C
mice were injected compound 5 (i.p. intraperitoneal injection) at indicated doses or
vehicle 1 h prior to administration of LPS (4.5 mg/kg, i. p.) for 3 days. Survival was
6
7
8
9
10
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16
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19
20
21
22
23
24
25
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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52
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9
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
monitored for 6 days. (n = 20 for each group). (b) Mice were pretreated with
compound 5 (1, 0.5 mg/kg, i.p) or vehicle 1 h prior to the administration of LPS (4.5
mg/kg, i.p.). Serum was collected at 12 h postꢀinjection of LPS. The serum level of
TNFꢀα and ILꢀ6 were measured using ELISA. Data are presented as means ± SD (n =
6 for each group). *p < 0.05, **p < 0.01, ***p < 0.001 compared with LPS only
group. (c) Mice were pretreated compound 5 (1, 0.5 mg/kg, i.p) or vehicle 1 h prior to
the administration of LPS (4.5 mg/kg, i.p.). 12 h after LPS challenge, mice were
sacrificed to obtain lung tissue for histological analysis. (d) H&E staining was
performed using lung tissue specimens from vehicle LPS + vehicle, LPS + 1 mg/kg
compound 5, LPS + 0.5mg/kg compound 5, mice, respectively (Magnification 400 ×).
10
11
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13
14
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17
18
19
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23
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25
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35
36
37
38
39
40
41
42
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700
Scheme 1. General procedure for the synthesis of 12ꢀ14.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
701
702
703
704
Scheme 2. General procedure for the synthesis of 35ꢀ42.
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
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736
737
738
Table 1. Experimentally determined halfꢀmaximal inhibitory concentrations (IC50) of
MIF tautomerase activity (TIC50) for biphenyl series, and NO production of
compounds with TIC50 < 10 ꢀM in LPSꢀactivated RAW 264.7 macrophage (NIC50).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
739
740
No.
R1
R2
R3
R4
R5
H
H
H
H
H
H
H
H
H
H
H
R6
H
H
H
H
H
H
H
H
H
H
H
TIC50 (µM)
0.72 ± 0.15
4.25 ± 0.63
11.66 ± 1.07
6.25 ± 0.80
0.37 ± 0.09
20.42 ± 1.31
8.42 ± 0.93
0.71 ± 0.15
8.36 ± 0.92
5.53 ± 0.74
8.63 ± 0.94
NIC50 (µM)
36.02 ± 1.56
49.93 ± 1.70
1
2
H
carboxy
carboxy
H
H
H
H
methyl
3
H
carboxy
H
4
H
H
acetylamino
H
33.51 ± 1.53
4.26 ± 0.63
5
H
H
hydroxy
H
6
H
H
carbomethoxy
H
7
H
hydroxymethyl
carboxy
H
H
H
H
H
H
H
H
H
H
25.57 ± 1.41
23.77 ± 1.38
26.57 ± 1.42
12.05 ± 1.08
13.10 ± 1.12
20.71 ± 1.32
23.24 ± 1.37
28.08 ± 1.45
8
H
Cl
9
carbomethoxy
H
10
11
H
H
H
H
H
carboxy
acetyl
carboxy
carboxy
carboxy
methoxyl
H
H
H
H
*
trifluoromethyl trifluoromethyl 1.48 ± 0.17
12
13
14
*
*
Cl
F
Cl
F
0.96 ± 0.02
2.48 ± 0.39
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
*Synthesized compounds
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5
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758
759
760
761
Table 2. Experimentally determined halfꢀmaximal inhibitory concentrations (IC50) of
MIF tautomerase activity (TIC50) for benzene series, and NO production of
compounds with TIC50 < 10 ꢀM in LPSꢀactivated RAW 264.7 macrophage (NIC50).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
762
763
No.
R1
H
R2
R3
R4
H
R5
R6
TIC50 (µM)
0.66 ± 0.18
1.33 ± 0.37
6.67 ± 0.82
9.77 ± 0.99
8.63 ± 0.94
28.33 ± 1.45
39.06 ± 1.59
2.74 ± 0.44
>100
NIC50 (µM)
12.61 ± 1.10
25.70 ± 1.41
45.98 ± 1.66
59.74 ± 1.78
25.82 ± 1.41
15
16
carboxy
H
H
methyl
H
H
carboxy
H
H
methyl
17 methyl
18
19 methyl
carboxy
H
H
H
phenoxymethyl
H
H
carboxy
H
H
H
carboxy
H
H
H
isopropyl
20
21
22
23
24
25
26
27
28
29
30
31
32
H
H
H
H
H
H
H
H
H
H
H
H
H
H
carboxy
H
methyl
H
Cl
carboxy
H
H
F
acetyl
H
H
H
H
16.39 ± 1.21
H
carbomethoxy
methyl
H
H
H
H
carbomethoxy
H
H
4.65 ± 0.67
2.73 ± 0.44
64.94 ± 1.81
3.75 ± 0.57
13.18 ± 1.12
10.39 ± 1.02
8.43 ± 0.93
5.00 ± 0.70
10.58 ± 1.02
0.81 ± 0.09
0.77 ± 0.11
16.69 ± 1.22
21.01 ± 1.32
24.25 ± 1.39
26.26 ± 1.42
12.23 ± 1.09
28.71 ± 1.46
61.15 ± 1.79
>100
40.35 ± 1.61
17.06 ± 1.23
H
acetyl
H
H
H
H
carbomethoxy
H
methyl
H
H
benzamido
H
H
methyl
32.52 ± 1.52
H
carbethoxy
H
H
H
H
acetylamino
H
methyl
H
H
carbamoyl
H
carbomethoxy
H
17.13 ± 1.23
20.85 ± 1.32
H
carbamoyl
H
H
carbomethoxy
H
acetylamino
H
H
methyl
33 carbomethoxy
34 carbomethoxy
H
H
H
H
H
H
H
H
H
H
H
H
H
methyl
55.24 ± 1.74
45.23 ± 1.66
H
H
methyl
H
H
35*
36*
H
H
H
H
H
H
H
H
carboxy
carboxy
carboxy
carboxy
carboxy
carboxy
carboxy
carboxy
H
Br
H
H
trifluoromethyl
37*
38*
39*
40*
41*
42*
H
H
Cl
F
H
H
H
F
H
H
H
H
H
Cl
H
Cl
F
H
764
765
766
767
*Synthesized compounds
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5
6
768
769
770
771
Table 3. Experimentally determined halfꢀmaximal inhibitory concentrations (IC50) of
MIF tautomerase activity (TIC50) for diphenylmethane series, and NO production of
compounds with TIC50 < 10 ꢀM in LPSꢀactivated RAW 264.7 macrophage (NIC50).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
772
773
No.
43
44
45
46
47
48
49
50
51
R1
R2
R3
R4
TIC50 (µM)
13.55 ± 1.13
7.95 ± 0.90
6.67 ± 0.82
5.51 ± 0.74
2.74 ± 0.44
19.80 ± 1.30
8.70 ± 0.94
3.63 ± 0.56
1.43 ± 0.15
NIC50 (µM)
H
H
carboxy
H
H
carboxy
H
H
H
59.74 ± 1.78
22.50 ± 1.35
13.43 ± 1.13
12.57 ± 1.10
H
H
H
H
H
carbomethoxy
benzamido
formylamino
H
H
H
H
H
H
H
H
carbomethoxy
H
carbomethoxy
H
H
carbomethoxy
H
18.66 ± 1.27
9.33 ± 0.97
15.51 ± 1.19
H
H
H
phenylamino
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
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42
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Figure 1
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Journal of Chemical Information and Modeling
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Figure 2
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Page 29 of 31
Journal of Chemical Information and Modeling
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Figure 3
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