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Ellagic Acid, a Dietary Polyphenol Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and its Proinflammatory Responses in Human Peripheral Blood Mononuclear Cells Souvik Sarkar, Asim A Siddiqui, Somnath Mazumder, Rudranil De, Shubhra J Saha, Chinmoy Banerjee, Mohd S Iqbal, Susanta Adhikari, Athar Alam, Siddhartha Roy, and Uday Bandyopadhyay J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00921 • Publication Date (Web): 01 May 2015 Downloaded from http://pubs.acs.org on May 5, 2015

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Journal of Agricultural and Food Chemistry

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Ellagic Acid, a Dietary Polyphenol Inhibits Tautomerase Activity of Human Macrophage

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Migration Inhibitory Factor and its Pro-inflammatory Responses in Human Peripheral

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Blood Mononuclear Cells

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Souvik Sarkar ‡, Asim A. Siddiqui ‡, Somnath Mazumder ‡, Rudranil De ‡, Shubhra J. Saha ‡,

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Chinmoy Banerjee ‡, Mohd. S. Iqbal ‡, Susanta Adhikari §, Athar Alam ‡, Siddhartha Roy ¶, and

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Uday Bandyopadhyay ‡ *

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‡

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Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4,

10

§

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Bengal, India.

12

¶

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4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.

Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700 009, West

Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology,

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Corresponding author

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Name: Uday Bandyopadhyay

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Address: Division of Infectious Diseases and Immunology,

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CSIR-Indian Institute of Chemical Biology,

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4, Raja S. C. Mullick Road,

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Jadavpur, Kolkata 700032, West Bengal, India.

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Telephone number: + 91-33-24995735

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E-mail address: [email protected]; [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage

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migration inhibitory factor (MIF) non-competitively (Ki = 1.97 ± 0.7 µM). The binding of EA to

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MIF was determined by following the quenching of tryptophan fluorescence. We synthesized

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several EA-derivatives and their structure-activity-relation studies indicated that the planar

29

conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear

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translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to

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promote inflammation. We were interested in evaluating the effect of EA on nuclear

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translocation of NF-κB and chemotactic activity in human PBMCs in presence of MIF. Results

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showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs as evident from

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confocal immuno-fluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of

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PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated pro-

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inflammatory responses in PBMCs by EA.

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KEYWORDS: Ellagic acid, macrophage migration inhibitory factor, tautomerase activity, gallic

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acid, inflammation, chemotaxis.

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INTRODUCTION

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Phenolic compounds or polyphenols comprise one of the most plentiful and widely distributed

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groups of phytochemicals. More than 8,000 different chemical structures have been identified

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and reported 1. Plants produce phenolic compounds as secondary metabolites (i.e. they are not

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essential for growth or reproduction), which exist mostly in their storage tissues. phenolic

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compounds may play roles as antioxidants as well as modulators of several biological

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macromolecules. As a chemical family, polyphenols can be classified into several groups based

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on their basic structures 1. These groups can be distinguished according to the number of

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aromatic rings and the structural moieties that connect them. The phenolics contain at least one

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aromatic ring bearing one hydroxyl (phenol) or more hydroxyl (polyphenol) groups. Thus,

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polyphenolic compounds include a large array of molecules including phenols, phenolic acids,

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tannins, lignans, flavonoids, stilbenes, and lignins. A wide range of biological actions have

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been reported for different polyphenols including antimicrobial, analgesic, antibiotic, anti-

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cancer, and anti-inflammatory properties

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the antioxidant effects of their phenolic groups. Blocking of phenolic group results in decreased

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activity, whereas extended unsaturated bond conjugation, proper ester linkage, extended fused

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ring system increased the activity of these polyphenolics 6-8.

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Ellagic acid (EA), a polyphenolic tannin based dilactone (4,4′,5,5′,6,6′-hexahydroxydiphenic

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acid 2,6,2′,6′-dilactone) is reportedly known to possess a broad range of beneficial human

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health (antiviral 9, antibacterial

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antimutagenic

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properties. Previous studies reveal that aromatic conjugated –OH groups of EA are the main

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scaffold responsible for its antioxidant nature

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activity recent literature suggests several other modes of action of EA, which may not be

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directly linked to its antioxidant property

66

enzymes

67

that plays an important role in both innate and acquired immunity. In certain cancers, pro-

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inflammatory cytokines serve as the major mediators of inter-cellular communication in the

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tumour microenvironment

22-24

13

, antimicrobial

10

14

2-5

. Their biological actions are mostly attributes of

, gastro-protective

, anti-malarial

15

18-19

20-21

11

, and cardio-protective properties

, anti-inflammatory

16

and anti-cancer

12

17

,

)

. Apart from its –OH mediated antioxidant

. EA interacts with receptors, cytokines, and

. Macrophage migration inhibitory factor (MIF) is one of the pivotal cytokines

25

, thus paving the way for developing malignancy; in others,

3



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oncogenic changes drive a tumor-promoting inflammatory microenvironment. With intricate

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and various activities, MIF has been considered to be a missing link between inflammation and

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tumorigenesis

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response, apoptosis, senescence and invasion during carcinogenesis

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MIF, via regulating nuclear translocation of NF-κB, plays an important role in inflammatory

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diseases

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inflammation, mainly due to the role of NF-κB in regulating the expression of several pro-

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inflammatory mediators including cytokines, chemokines, and adhesion molecules

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Inhibition of MIF has been shown to reduce activation of NF- κB and consequent activation of

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other cytokines

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cytokines and suppress the anti-inflammatory cytokines

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cytokines such as MIF further recruit PBMCs to the site of inflammation through chemotactic

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migration

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concentration gradient of MIF is an essential event involved in the inflammatory response.

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Several natural product polyphenolics like curcumin, eugenol, resveratrol, caffeic acid have

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been found to inhibit MIF mainly via blocking the tautomerase activity of the cytozyme36.

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Here we report that EA, a naturally occurring polyphenolic compound, binds to MIF and

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inhibits its tautomerase activity. Spatiotemporal inhibition of EA on MIF tautomerase activity

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is solely dependent on the dimensionally conformed lactone moiety. We further report that EA

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significantly inhibits MIF-induced nuclear translocation of NF-κB and MIF-mediated

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chemotactic migration in human PBMCs, two signature events associated with tissue

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inflammation.

31

26

. Moreover, MIF has been proposed to regulate proliferation, DNA-damage 27-30

. Studies revealed that

. NF-κB signalling has long been known as a significant event regulating

35

33

32

.

. During inflammatory disorders PBMCs enhance the pro-inflammatory 34

. Secreted pro-inflammatory

. Therefore, involvement of PBMCs via chemotactic migration in response to a

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MATERIALS AND METHODS

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Chemicals. Ellagic acid, Pyrene, DMSO, paraformaldehyde, kanamycin, chloramphenicol,

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protease inhibitor cocktail were procured from Sigma (St Louis, MO, USA). PBS (Hyclone),

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Isopropyl-beta-D-thiogalactopyranoside (IPTG), prestained marker and unstained protein

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ladders, rapid DNA ligation kit, NcoI and HindIII were purchased from Fermentas (Thermo

98

Scientific, USA). Luria-Berteni medium was obtained from Hi-Media, India. Gel extraction kit

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and MiniElute PCR purification kit was procured from Qiagen, USA. pET28a was purchased

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from Novagen (Merck KGaA Darmstadt, Germany). Gallic acid and benzyl bromide were

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procured from SRL (New Delhi, India). NF-κB antibodies were obtained from Santa Cruz

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Biotechnology (SantaCruz,CA). AlexaFluor 647 and AlexaFluor 488 conjugated secondary

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antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Mito tracker red CMXRos

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was obtained from Life Technologies. All other chemicals were of analytical grade.

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Over-expression and Purification of Human MIF. Human MIF was cloned by following the

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protocol as described earlier with slight modification

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cloned using pCMV6-XL5 cloning vector obtained from ORIGENE, USA. Complete ORF

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from

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CTAACCATGGCCCCGATGTTCATTGTAAATACCAACGTGCCCC 3’ (NcoI restriction

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site

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TTAAAGCTTCTGCGGTTCTTAGGCGAAGGTAGAGTTGTTCCAGCCC

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restriction site underlined). PCR was performed with initial denaturation at 95 ºC for 3 min

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then 35 cycles of denaturation (95 ºC for 30 sec), annealing (52 ºC for 30 sec) and extension

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(72 ºC for 1 min) with final extension of 72 ºC for 10 min. PCR product was then cloned in

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Escherichia coli expression vector pET28a (Novagen). Ligation mix was transformed in

the

clone

pCMV6-XL5

underlined)

was

PCR

and

37

. In brief, full length MIF cDNA was

amplified

reverse

5


with

forward

primer

primer

5’

5’ 3’

(HindIII


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competent DH5α cells and clones were screened by colony PCR and double digestion release

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of insert from positive colonies.

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Host strain E. coli Rosetta (Novagen) was used for recombinant MIF over-expression and

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purification. Lysogency Broth (LB) (1 liter) was inoculated with 1% (v/v) of overnight (16

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hours) grown culture on the following morning and incubated at 37 ºC with shaking at the rate

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of 200 revolutions per minute. Culture was induced with IPTG (1 mM final concentration) at

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OD600 range 0.6 - 0.8 and was further allowed to grow for 4 hours. Cells were harvested (11000

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×g, 5 min, 4 ºC) and reconstituted in 10 ml of lysis buffer (PBS with 10% (v/v) glycerol)

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containing protease inhibitor cocktail. Cells were incubated on ice for 60 min and subsequently

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lysed by sonication (30 cycles of pulse-rest at 60% amplitude; pulse duration-10 sec and rest

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duration-1 min) after which supernatant was collected by centrifuging the lysate at 40,000 × g.

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Over-expression of MIF in soluble fraction was confirmed by 15% SDS-PAGE of supernatant.

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All further steps were performed at 4 ºC. Obtained supernatant was concentrated to 4 ml by

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Amicon Ultra Centrifugal Filter Ultracel – 3K (Millipore Ireland ltd) and then passed through

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0.2 µm filter. Finally, MIF was purified by performing gel filtration chromatography through

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Sephadex G50 (GE Life Sciences, Uppsala, Sweden) column using PBS as the solvent with a

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flow rate of 6ml/hour.

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MALDI-TOF and Peptide Finger Printing. Purified protein was processed by µ-C18 ZipTip

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tips (Millipore) for removal of salts as per manufacturer’s instructions and subjected to

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MALDI-TOF mass analysis. In short, trifluoroacetic acid (TFA) was added to the purified

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protein solution to 0.1% (pH should be less than 4), then aspirated in pre-equilibrated (with

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0.1%TFA) tips in 10 µl pipette to the maximum volume. Solution was dispensed and aspirated

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again in the tip. This cycle was repeated for 10 times for maximum binding. Washing was

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performed with 0.1% TFA with 5% methanol twice and elution was done in 4 µl of 0.1% TFA

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with 50% acetonitrile. This protein was directly used for MALDI-TOF analysis using an

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Applied Biosystems 4700 Proteomics Analyzer170. For the confirmation of the identity of

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protein, peptide mass fingerprinting was performed. Protein band was excised from gel, de-

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stained followed by reduction, alkylation and digestion into peptides using In-Gel Tryptic

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Digestion Kit (Thermo Scientific) as instructed by the manufacturer. Briefly, gel piece was

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treated with reducing buffer (50 mM TCEP and 25 mM ammonium bicarbonate), then by

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alkylation buffer (100 mM Iodoacetamide and 25 mM ammonium bicarbonate), then shrunk

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using acetonitrile, air-dried and swelled by Activated Trypsin with digestion buffer added after

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15 min (kept overnight at 30 ºC with shaking). Buffer collected back next day without gel. This

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contains peptides released and can directly be used for MALDI MS/MS. 1% trifluoroacetic acid

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was used to extract remaining peptides and deactivate trypsin. This final product was used for

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MALDI MS/MS. Spectrum obtained was searched in NCBI database by GPS explorer using

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MASCOT browser.

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L-dopachrome Methyl Ester (L-DOPA) Preparation to Follow MIF Tautomerase Activity

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Assay. Tautomerase activity of MIF was followed as described earlier

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determined at 25 ºC by adding L-DOPA (0.25–1.5mM) to a cuvette containing 60 nM MIF in

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10 mM potassium phosphate buffer, pH 6.2 containing 0.5 mM EDTA. The activity of MIF

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was monitored by measuring the conversion of L-DOPA (coloured) to indole carboxylic acid

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methyl ester (colourless) at 475 nm. Inhibitory activity of the synthesized EA derivatives were

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measured by dissolving in dry DMSO, subsequently adding to the cuvette containing MIF at

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different concentrations and finally incubating the mixture for 20 min before addition of L-

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DOPA. The IC50 was obtained with different concentrations of compounds and finally Ki

7


38

. Activity was


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(inhibition constant) was determined using non-linear regression analysis using Prism4

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(GraphPad Prism).

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Fluorescence Spectroscopy. Concentration-dependent response of EA on MIF tryptophan

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fluorescence was monitored as mentioned earlier

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The resulting solution was excited at 295 nm for tryptophan residue and the intrinsic

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fluorescence emission was scanned at 300 to 400 nm. Fluorescence quenching was determined

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by analyzing the data by the classical Stern–Volmer equation

169

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by serial addition of EA to MIF (7.5 µM).

F0/F = 1 + kq τ0 [Q] = 1+ Ksv[Q]

(1)

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where Fo and F are the fluorescence intensities in the absence and presence of the quencher

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respectively, Kq and Ksv are the quenching rate constant and dynamic quenching constant

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respectively, average life time of the protein in absence of the quencher is expressed in terms of

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τo and [Q] is the concentration of the free quencher, respectively as mentioned earlier 40. When

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small molecules interact with several comparable sites in a protein, the equilibrium between

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liberated and bound molecules is expressed by the equation:

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log ((F0- F) / F) = log Kb + n log[Q]

(2)

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where Kb represents binding constant for quencher-protein interaction, n the number of binding

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sites per molecule of MIF. Utilizing Kb, the free energy change (∆G0) value can be estimated

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from the following equation:

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∆G0 = -RT ln Kb

(3)

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The negative sign ∆G0 value validates the spontaneity of quencher-protein interaction.

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Circular Dichroism (CD) Spectroscopy. CD spectroscopy was used to evaluate the structural

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changes in MIF as mentioned before 41. Secondary structure of MIF was evaluated in presence

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of different concentration of EA (10, 25, and 50 µM) in the far-UV range by a J-810 CD

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spectrometer. CD spectra were recorded at a range of 190 nm to 270 nm from 10 µM of MIF in

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optically clear buffer. Spectra represented an average of 5 scans (a step size of 0.2 nm, an

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averaging time of 3 sec) with buffer baseline subtracted; smoothed over 4 data points to

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generate the data reported in units of mean residue ellipticity. Secondary structure analysis of

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the

190

(http://dichroweb.cryst.bbk.ac.uk/html/home.shtml).

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Molecular Docking. Ideal coordinate for EA (Protein Data Bank (PDB) code REF) and MIF

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(PDB code 3L5S; 1.86-Å resolution) were obtained from the PDB. Molecular Docking

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calculations were performed using SwissDock based on the docking software EADock DSS 42.

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A grid (Box size: 40×40×40 Å) was designed in which many binding modes were generated

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and their CHARMM energies were estimated on the grid

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MIF was treated as rigid during docking calculation. Each docking experiment was a composite

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of 250 different consecutive runs. Fast analytical continuum treatment of solvation (FACTS)

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was used to evaluate the modes of binding with most favorable energies and finally clustered.

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Clusters were then ranked according to the average Full Fitness of their elements 44. Results of

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the SwissDock were visualized and rendered by PyMOL molecular viewer.

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General Procedure for Formation of Esters by EDC Coupling. Reactions of 1-ethyl-3-(3-

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dimethylaminopropyl) carbodiimide (EDC) coupling was carried out as described earlier with

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slight modification 45. To a solution of benzoic acid (1 eq), amine (hydrochloride)/alcohol (1.5

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eq), 1-hydroxybenzotriazole (1 eq, for amine), and Et3N (5 eq, for amine)/DMAP (1 eq, for

205

alcohol) in N,N-dimethylformamide, EDC hydrochloride (1.5 eq) was added at 0 ºC. Then the

206

reaction mixture was stirred at room temperature until completion of the reaction (12 hours) as

207

monitored by TLC. Reaction mixture was terminated by the addition of ice and extracted with

CD

spectra

was

performed

9

by

Dichroweb

43

online

server

. Docking type was accurate and



208

ethyl acetate. The ethyl acetate layer was washed with brine and dried over Na2SO4. The

209

solvent was then removed in a rota-vapour under vacuo and the crude mass was

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chromatographed over a silica gel column.

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General Procedure of Debenzylation (Hydrogenolysis). A combination of benzylated

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compound and 10% palladium on carbon (catalytic) in a methanol/chloroform mixture (4:1) (10

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ml) was stirred under hydrogen atmosphere (using hydrogen balloon) until completion of the

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reaction (2 hours) as monitored by TLC. The reaction mixture was passed through a small pad

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of Celite. The filtrate was evaporated in vacuo to dryness to obtain the product.

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Peripheral Blood Mononuclear Cells (PBMCs) Isolation. Human PBMCs were isolated

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using Histopaque density gradient centrifugation following the instruction of the manufacturer.

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Briefly, 10 ml of blood was collected from median cubital vein of a healthy volunteer by sterile

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disposable syringe and dispensed in tubes containing heparin. Next 6 mL of blood was

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carefully layered on a preformed Histopaque gradient (6 ml) prepared by layering Histopaque

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1077 over Histopaque 11191 at 1:1 ratio. The tubes were next centrifuged at 700 × g for 30 min

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at room temperature. The mononuclear cell fraction was carefully aspirated out and collected in

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a fresh tube. The cells were washed thrice in phosphate buffered saline (PBS) and checked for

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cell viability by Trypan blue. 90-95% of the isolated cells were alive. Finally an aliquot of the

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isolated cells were stained by crystal violet and observed under microscope to check the purity

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of the isolated PBMCs. Isolated PBMCs were resuspended in RPMI-1640 containing 5% FBS

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(Gibco) and maintained at 37 ºC, 5% CO2 environment for all the subsequent experiments.

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Confocal Microscopy. Isolated PBMCs were used for immune-fluorescent microscopy as

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mentioned earlier

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RPMI-1640 containing 10% FBS and treated in 4 sets: Control, MIF (1µg/ml), EA (50 µM) and

46-47

with minor modifications. Briefly, about 1×106 cells/ml were plated in

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EA+MIF for 4hours at 37 ºC and 5% CO2. Cells after treatment were washed with pre-warmed

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PBS (HyClone) and fixed with 4% paraformaldehyde at room temperature for 10 min. Cells

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were next washed with PBS by swirling motion and permeabilized with 0.1% Triton X-100 at

234

room temperature for 10 min. After washing, the cells were blocked in a solution containing

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2% BSA in PBS for 2 hours at room temperature. Immunostaining of the fixed cells was

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performed by incubating the cells in the blocking solution containing rabbit anti-NF-κB (p65)

237

antibody overnight. Next day the cells were stained with anti-rabbit Alexa Fluor 647-

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conjugated secondary antibody (1:2000; Invitrogen). Nucleus was stained with DAPI. Cells

239

were imaged at 512 × 512 resolutions with Leica TCS-SP8-Confocal microscope. Samples

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were viewed under 63X oil immersion lens and digital zooming was performed according to

241

the requirement. Overlap of red (NF-κB) and blue (nucleus) signals was quantified using the

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software provided with the microscope. Pearson’s correlation coefficient was considered to

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present the intranuclear NF-κB. The data presented are a representation of one of four

244

experiments showing similar values.

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Chemotaxis Assay. Chemotactic assay was performed with human PBMCs as mentioned

246

before

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loaded into the inserts (having polycarbonate membrane with 5-µm pore size) of a Transwell

248

cell migration chamber (Corning) in a 24-well plate format. The chemo-attractant MIF (1µg ml-

249

1

250

of EA (10-50 µM). Cells without any treatment were used as a control and macrophage

251

inflammatory protein-1 alpha (MIP-1α), a chemo-attractant cytokine, was used as positive

252

control. Heat-inactivated MIF (MIF heated to 65 ºC for 30 min) was also used as a negative

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control. Transwell plates were incubated for 4 hours at 37 ºC in 5% CO2. PBMCs that had

47-48

with minor modifications. Briefly, isolated PBMCs (2.5 × 105 cells/well) were

) was added into the lower chambers in presence and/or absence of increasing concentrations

11



254

migrated through the membrane were dislodged using 2 mM EDTA and fixed with methanol.

255

The number of cells that migrated from the upper chamber into the filter and reached its lower

256

side was determined by staining with crystal violet and counting under microscope (Leica). To

257

compare the results from different experiments, a chemotaxis index (CI) (CI = number of

258

PBMCs migrating toward MIF or MIF plus EA-loaded wells/number of PBMCs migrating

259

toward control medium) was calculated. The counts provided were the results observed in five

260

randomly selected fields under microscope. The results were reported as the mean CI ± S.D.

261

change with respect to control of at least three independent experiments.

262

Statistical Analysis. For IC50 determination, respective experiments were repeated thrice in

263

triplicates. Data were presented as mean ± SD. Student’s t test was used to analyse the data in

264

GraphPadPrism 4. Values of p < 0.05 were considered significant.

265 266 267

RESULTS

268

Purification of MIF. MIF was cloned in pET28a vector, over-expressed in E. coli and purified

269

using gel permeation chromatography. Purity of the protein was checked by 15% SDS-PAGE

270

and molecular weight (12.556 kDa) was confirmed by MALDI-TOF mass spectrometry (Figure

271

1, inset a). Peptide fingerprinting (Figure 1, inset b) finally confirmed the identity of MIF by

272

MALDI MS/MS with perfect match from NCBI database.

273

Ellagic Acid (EA) Inhibits MIF Tautomerase: Kinetics and Mode of Inhibition. The

274

tautomerase activity of recombinant MIF was measured by following the tautomerization of L-

275

DOPA in absence or presence of increasing concentrations of EA. Data indicated that EA

276

significantly inhibited the tautomerase activity of MIF (IC50 = 4.77 ± 0.52 µM) (Figure 2).

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277

Next, we were interested in analyzing the type of inhibition offered by EA. Concentration

278

response curve and Lineweaver-Burk plot were plotted (Figure 3A and 3B). Lineweaver-Burk

279

plot of concentration-response curves revealed a non-competitive mode of inhibition of MIF by

280

EA. The inhibition constant (Ki) was determined from concentration response curve and found

281

to be 1.97 ± 0.7 µM. Binding of EA to MIF was assessed by following the quenching of

282

tryptophan (Trp-108) fluorescence in presence of increasing concentrations of EA. The only

283

tryptophan in human MIF is distantly located from the catalytic Pro-1 and is exposed to the

284

solvent. MIF in optically clear assay buffer was excited at 295 nm for recoding the fluorescence

285

spectra which was mainly attributed to the only tryptophan residue. MIF showed emission

286

maxima at 348 nm. Protein solution was titrated by consecutive additions of EA (1-20 µM) and

287

its fluorescence intensity was gradually found to decrease with increasing concentrations of EA

288

(Figure 4A). Small blue shift of the λmax was also observed with increasing concentrations of

289

EA. The nature of fluorescence quenching of MIF-EA (0-20 µM) interaction was analyzed

290

using the Stern-Volmer equation (Figure 4B). Subsequently KSV was calculated from the linear

291

regression curve of F0/F versus [Q]. The binding constant (Kb), number of binding sites (n), and

292

free energy change (∆G0) of EA-MIF interaction were determined from the equations (2 and 3),

293

respectively (Figure 4C). Binding affinity of EA was found to be quite strong and its value was

294

found to be ∆G0 = -33.53 ± 0.729 kJ.mol-1. We analysed the conformation of MIF in presence

295

of 10, 25, 50 µM EA by CD spectroscopy to confirm any impact of EA on MIF secondary

296

structure (manifested as gradual decrease of MIF fluorescence). Secondary structural elements

297

like α-helices, β-sheets, and random coils absorb specific components of the circularly

298

polarised light of a CD spectra thereby providing information about specific structural elements

299

within a protein. The addition of EA did not alter much of the basic secondary structure of MIF

13



300

(Figure 4D). For further confirmation of minimum change in structure we analysed the spectral

301

data in DICHROWEB web server by four widely used algorithms. Outcome of these

302

algorithms established (data not shown) that EA did not significantly alter the secondary

303

structure of MIF.

304

To get the exact binding location of EA with MIF we tried to co-crystallize EA with MIF but

305

failed to get the co-crystallized product. Hence molecular docking was performed to get a

306

preliminary idea about the possible interaction between MIF and EA. Structural details for

307

human MIF (Protein Data Bank code 3L5S; 1.86-Å resolution) and EA (PubChem CID:

308

5281855) were obtained from the respective databases. For molecular docking studies, the

309

homotrimer of MIF was docked with EA. Each docking experiment was a composite of 250

310

independent runs. Molecular docking outputs showed that EA interacted at or close to the

311

active site of the N-terminal of MIF. EA binds in a pocket formed by the N-terminal (Pro-1,

312

Lys-32, Tyr-36) and C-terminal (Ile-64, Trp-108, Phe-113) residues of chain A and Tyr-95 of

313

chain B (Figure 5A). In this orientation EA formed hydrogen bond interactions with MIF

314

through amino acids Pro-1, Lys-32, Tyr-36, Ile-64, Tyr-95, Trp-108 and Tyr-95 (Figure 5B).

315

The ligand was also stabilized by hydrophobic interactions with the aromatic side chains of

316

Tyr-36, Ile64, Tyr-95, Trp-108 and Phe-113. In addition, aromatic side chain of Tyr-36 makes a

317

pi-pi stacking interaction with EA. These hydrogen bonds and hydrophobic interactions were

318

thought to play a role in the binding of EA with MIF.

319

Functional Group Pattern Recognition Study: Orientation and Role of Lactone Moiety.

320

Following the identification of EA as a new MIF inhibitor, we were now interested to identify

321

the active pharmacophore. We, therefore synthesised several molecules as structural

322

dissects/parts of EA and screened for MIF tautomerase activity. Gallic acid a unit structural

14


Page 14 of 38

Page 15 of 38


323

scaffold of EA did not show any activity. Synthesized EA metabolites urolithin B and urolithin

324

A (Figure 6A) contained only three fused rings with one lactone group and showed inhibitory

325

activity with higher IC50 values (Table 1). To check the specific involvement of lactone moiety

326

on tautomerase inhibition we synthesised distant dimeric gallic acid derivative (compound 2d).

327

Compound 2d, a dimer of GA comprising two labile ester groups and six free hydroxyl groups

328

(like EA) was synthesised using distant linker ethylene glycol (Figure 6B). Compound 2d

329

showed very low activity against MIF (IC50 = 35.9 ± 0.72 µM) (Table 1).To explore the role of

330

planar conjugated fused system we took pyrene, a planar polycyclic, fused aromatic

331

hydrocarbon without any functional group. Pyrene did not show any tautomerase inhibitory

332

activity. Thus, compounds having lactone or ester groups were found to be active and hydroxyl

333

groups had no effect on MIF tautomerase inhibition.

334

EA Prevents MIF-mediated Nuclear Translocation of NF-κB: a Molecular Event

335

Essential for Pro-inflammatory Signalling. MIF promotes inflammation by activating

336

signalling cascades involving NF-kB. MIF is known to cause translocation of NF-kB into the

337

nucleus. Therefore nuclear translocation of NF-κB was followed by confocal microscopy in

338

PBMCs. Blue fluorescence represented nucleus (Figure 7, first column) while red fluorescence

339

represented cells immuno-stained with NF-κB primary antibody and Alexa Fluor 647

340

conjugated secondary antibody (Figure 7, second column). The third and fourth column

341

represented the merged image of the red and blue fluorescence while area of intranuclear NF-

342

κB was depicted as white dots in the adjacent panels. In the fifth column, intranuclear versus

343

non-intranuclear NF-κB was presented as bar diagram. In the control set (Figure 7, first row),

344

there was a basal level of NF-κB (red fluorescence) in the nucleus; however the red signal was

345

confined mostly in the cytosol. PBMCs with exogenously treated MIF (Figure 7, second row)

15



346

showed a significantly higher level of NF-κB in the nucleus depicting the role of MIF on

347

nuclear translocation of NF-κB. On the contrary, PBMCs treated with MIF pre-incubated with

348

EA (50 µM) showed a much lower intranuclear NF-κB as evident from reduced overlap of the

349

red and blue signals and the white dots. Because PBMCs normally secrete a basal level of MIF

350

34

351

control. Thus, the data showed that stimulatory effect of MIF on the nuclear translocation of

352

NF-κB was significantly compromised upon treatment with EA.

353

EA Prevents MIF-mediated Chemotaxis of Human Peripheral Blood Mononuclear Cells

354

(PBMCs): a Cellular Event Necessary for Inflammation. MIF promotes macrophage

355

chemotactic migration and also plays an important role in neutrophil infiltration during

356

inflammatory diseases. To check any probable effect of EA on MIF-mediated chemotactic

357

activity, we performed a chemotactic migration assay using PBMCs in presence or absence of

358

increasing concentrations of EA. EA showed a potent concentration-dependent inhibitory effect

359

on MIF-induced chemotactic migration of PBMCs (CI = 2.31, 1.93 and 1.94 for EA 10 µM, 25

360

µM and 50 µM respectively compared with CI = 3.38 for MIF treated cells where control set CI

361

was fixed at 0.0) (Figure 8). We validated the chemotactic migration assay using heat-

362

inactivated MIF which further showed negligible chemotactic migration of PBMCs compared

363

to control (Figure 8). MIP-1α, a classical chemo-attractant, used in this assay also resulted in

364

significant chemotactic migration (CI = 2.96) of the PBMCs. However, it is interesting to note

365

that EA even at a high concentration (50 µM) failed to inhibit the MIP-1α -induced chemotactic

366

migration of PBMCs (Figure 8) suggesting the specificity of EA for MIF-mediated chemotaxis.

, treatment of PBMCs with EA alone also resulted in lowering of nuclear NF-κB compared to

367 368

16


Page 16 of 38

Page 17 of 38


369

DISCUSSION

370

EA is long since known for its antioxidant and anti-inflammatory activity. But, here we report

371

that EA inhibits tautomerase activity of human MIF, a pro-inflammatory protein. Further we

372

identified that the planar conjugated lactone moiety is responsible for tautomerase inhibition

373

after performing extensive structure-function analysis. EA also inhibited MIF-mediated

374

proinflammatory responses in PBMCs.

375

The inhibitory action of EA against MIF followed a concentration gradient. Moreover, EA non-

376

competitively inhibited MIF as there was no structural similarity between EA and MIF’s assay

377

substrate L-DOPA. This suggested that EA might bind to the allosteric site (away from active

378

site) or close to the active site with slack binding of MIF, which did not block it from binding

379

to the substrate but inhibited the tautomerase activity of MIF. Binding of EA to MIF was

380

evaluated by fluorescence spectroscopy. Structural conformations of a globular protein may

381

change upon binding of small molecule due to alterations in the intra-molecular forces

382

responsible for maintaining the secondary and tertiary structures.

383

fluorescence

384

microenvironment of Trp. Emission spectra of MIF were measured using excitation wavelength

385

at 295 nm to ensure that light caused excitation of Trp residues only. Blue shift of the λmax of

386

Trp emission spectra indicated a decrease in hydrophilicity in its surrounding region within the

387

globular protein indicating that Trp residue might have shifted to a more hydrophobic

388

environment due to a presumptive change in the secondary structure of the protein. Moreover,

389

the free energy change having negative sign indicated the spontaneity and stability of EA-MIF

390

interaction. Since human MIF has only one Trp, which is distant from the catalytic Pro-1 and

391

exposed to the solvent, therefore the results of the fluorimetric quenching of Trp λmax may

λmax

therefore

suggested

that

17

EA-MIF

interaction


Decrease inTrp-108 might

alter

the


392

result in either a change in microenvironment around Trp or a gross alteration in the secondary

393

structure of MIF upon addition of EA

394

reflected in circular dichroism (CD) spectra. Our CD data however indicated almost no changes

395

in the secondary structure of MIF even in presence of high concentrations of EA (50 µM),

396

therefore concluding that quenching of MIF fluorescence was probably due to alteration in the

397

microenvironment around Trp-108 and not due to the alteration in the secondary structure of

398

the whole protein. Molecular docking of EA to MIF revealed that NH backbone of Pro-1, Lys-

399

32, Tyr-36, CO terminal of Ile-64, Trp-108, Phe-113, Tyr-95 and pi-pi interactions with Tyr-

400

36, Ile64, Tyr-95, Trp-108 and Phe-113 allowed a favourable interaction of EA with MIF.

401

These interactions shown in docking study were consistent with our previous kinetics of

402

tautomerase inhibition and fluorimetric binding.

403

Next, we were interested to identify the active functional scaffold of EA responsible for MIF

404

tautomerase inhibition. Gallic acid (GA), a unit structural motif of EA previously shown to

405

exhibit anti-inflammatory and antioxidant activity

406

activity. To test this hypothesis we performed dopachrome tautomerization assay to screen the

407

inhibitory activity of GA and all synthetic GA-related polyphenolic compounds. We found EA

408

as the most potent inhibitor among all the compounds followed by compound 2d, urolithin A,

409

urolithin B. GA however exhibited no inhibitory activity at all. Therefore, it appears that the

410

difference in activity between EA and GA probably arises from the two lactone groups present

411

in EA, although both have free hydroxyl groups. Compound 2d having ester group was also

412

found to be active. Conjugated aromatic system was also not responsible for the inhibition of

413

tautomerase activity because pyrene did not exhibit any inhibitory activity. Therefore, ester

414

groups play an important role whereas hydroxyl groups do not have any impact on inhibition of

49

. This change in protein structure is expected to get

18

50-51

, may also inhibit MIF tautomerase


Page 18 of 38

Page 19 of 38


415

MIF tautomerase activity. Although free ester groups are capable of inhibiting MIF, their

416

activity is much less than cyclic ester group i.e. lactone as evident from the IC50 values. The

417

presence of higher inhibitory activity of EA (having two lactone groups) in comparison to

418

urolithin A and urolithin B (both having single lactone group) indicates the importance of

419

conformationally restricted lactones as well as the role of lactones in MIF inhibition. Thus,

420

structurally restricted lactone group of EA is probably the most essential scaffold responsible

421

for MIF inhibition.

422

MIF is an inflammatory cytokine

423

mediated inflammatory response such as nuclear translocation of NF-κB in PBMCs. It is

424

reported that binding of MIF to CD74 complex involving CD44 leads to the release of the

425

intracellular domain of CD74 (CD74 ICD), which triggers the downstream signalling pathway

426

leading to the activation of NF-κB

427

inflammation by switching on the transcription of a plethora of pro-inflammatory target genes.

428

As NF-κB is a transcription factor, that enters the nucleus and binds to the DNA of its targets to

429

mediate its actions

430

translocation of NF-κB into the nucleus. Data clearly indicated that MIF greatly induced NF-κB

431

translocation to the nucleus in PBMCs, whereas the control (without MIF) and EA (50 µM)

432

treated cells did not show such NF-kB translocation as evident from confocal images.

433

Interestingly ISO-1, which is a well established MIF inhibitor, reported to bind the tautomerase

434

active site of MIF, inhibits its pro-inflammatory activity and also significantly reduces the

435

nuclear translocation of NF-kB 55. Inhibition of MIF tautomerase activity or interference in the

436

tautomerase active site might therefore be a restraining factor behind the inhibition of MIF-

437

induced nuclear translocation of NF- κB. However, we cannot exclude other possibilities of

54

52

. Therefore, we investigated the effect of EA on MIF-

53

. Activation of the NF-κB inturn plays a central role in

, therefore we investigated the inhibitory effect of EA on MIF-induced

19



438

anti-inflammatory property of EA that can mediate the inhibition of nuclear translocation of

439

NF-κB.

440

Chemotactic migration plays a major role during inflammation by leucocyte recruitment to the

441

site of inflammation. MIF promotes macrophage chemotactic migration and also plays an

442

important role in leukocyte recruitment during inflammation

443

chemotaxis of PBMCs and this inhibition was specific as no inhibition was found when MIP-1α

444

was used as the chemoattractant. However, knowledge about the exact mechanism of inhibition

445

of MIF mediated chemotaxis by EA needs further detailed investigation. Blocking of

446

tautomerase activity or tautomerase active site of MIF by EA might be a reason behind this

447

inhibition. This study therefore reports that naturally occurring antioxidant EA inhibits the

448

tautomerase activity of MIF and MIF-mediated pro-inflammatory responses in PBMCs.

56

. EA inhibited MIF-induced

449 450

ACKNOWLEDGMENTS

451

We thank the Council of Scientific and Industrial Research, New Delhi, for offering fellowship

452

to Souvik Sarkar to carry out this work. This work was supported by Council of Scientific and

453

Industrial Research, New Delhi [Grant number BEnD, BSC 0206].

454 455

Supporting Information:

456

Supporting Information include experimental procedures for the synthesis of compounds, 1H,

457

and 13C NMR spectra.

458 459 460

20


Page 20 of 38

Page 21 of 38


461

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Med Cell Longev 2014, 2014, 901315.

605

51.

606

Bandyopadhyay, U., Gallic acid prevents nonsteroidal anti-inflammatory drug-induced gastropathy in rat

607

by blocking oxidative stress and apoptosis. Free Radic Biol Med 2010, 49, 258-67.

608

52.

609

Chugh, R.; Davies, P.; Bloom, O., Elevated circulating levels of the pro-inflammatory cytokine

610

macrophage migration inhibitory factor in individuals with acute spinal cord injury. Arch Phys Med

611

Rehabil 2015, 96, 633-44.

612

53.

613

Lolis, E.; Noble, P.; Knudson, W.; Bucala, R., CD44 is the signaling component of the macrophage

614

migration inhibitory factor-CD74 receptor complex. Immunity 2006, 25, 595-606.

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54.

616

D., Anti-inflammatory function of arctiin by inhibiting COX-2 expression via NF-kappaB pathways. J

617

Inflamm (Lond) 2011, 8, 16.

618

55.

619

Ochani, K.; Bacher, M.; Nicoletti, F.; Metz, C.; Pavlov, V. A.; Miller, E. J.; Tracey, K. J., ISO-1 binding

620

to the tautomerase active site of MIF inhibits its pro-inflammatory activity and increases survival in

621

severe sepsis. J Biol Chem 2005, 280, 36541-4.

622

56.

623

Hickey, M. J., Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic

624

responses via MAPK and Rho GTPase. J Immunol 2011, 186, 4915-24.

Swope, M. D.; Sun, H. W.; Klockow, B.; Blake, P.; Lolis, E., Macrophage migration inhibitory

Yang, H. L.; Huang, P. J.; Liu, Y. R.; Kumar, K. J.; Hsu, L. S.; Lu, T. L.; Chia, Y. C.; Takajo, T.;

Pal, C.; Bindu, S.; Dey, S.; Alam, A.; Goyal, M.; Iqbal, M. S.; Maity, P.; Adhikari, S. S.;

Bank, M.; Stein, A.; Sison, C.; Glazer, A.; Jassal, N.; McCarthy, D.; Shatzer, M.; Hahn, B.;

Shi, X.; Leng, L.; Wang, T.; Wang, W.; Du, X.; Li, J.; McDonald, C.; Chen, Z.; Murphy, J. W.;

Lee, S.; Shin, S.; Kim, H.; Han, S.; Kim, K.; Kwon, J.; Kwak, J. H.; Lee, C. K.; Ha, N. J.; Yim,

Al-Abed, Y.; Dabideen, D.; Aljabari, B.; Valster, A.; Messmer, D.; Ochani, M.; Tanovic, M.;

Fan, H.; Hall, P.; Santos, L. L.; Gregory, J. L.; Fingerle-Rowson, G.; Bucala, R.; Morand, E. F.;

625 626 627 26


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628

FIGURE CAPTIONS

629

Figure 1. Purification of recombinant MIF. MALDI spectrum showing molecular mass of MIF. Inset a;

630

15% SDS-PAGE . Lane 1-pre-stained molecular weight marker, Lane 2- MIF. Inset b; MALDI MS/MS

631

for confirmation of protein identity by peptide finger printing and matching fragmented sequences with

632

data base.

633

Figure 2. Inhibition of MIF tautomerase activity by EA. MIF (60 nM) is treated with different

634

concentrations of EA for 30 min to measure the tautomerase activity at 474 nm. Data are presented as

635

mean ± S.D.

636

Figure 3. Kinetics of inhibition of MIF by EA. (A) Concentration-response curve of EA versus substrate

637

L-DOPA. (B) Inhibition constant (Ki) of EA against L-DOPA. Non-competitive inhibition pattern of EA

638

with MIF is reveal by Lineweaver-Burk plot. The details of the methodologies are described under

639

‘Materials and methods’. Data are presented as mean ± S.D. of three independent experiments.

640

Figure 4. Binding of EA to MIF. (A) Fluorescence quenching spectra of MIF. Protein solution is excited

641

and quenching of fluorescence is recorded in the presence of varying concentrations of EA (1-20 µM). (B)

642

Stern-Volmer plot of decrease in fluorescence of MIF in the presence of varying concentrations of EA.

643

Dynamic quenching rate constant Ksv is evaluated from the slope of the line. (C) Logarithmic plots of

644

relative fluorescence quenching of MIF against logarithmic concentrations of EA to calculate Kb. Kb is

645

calculated from the intersection of the line with y-axes and number of binding sites (n) from the slope of

646

the line. (D) CD spectra of MIF in presence or absence of EA.

647

Figure 5. Molecular docking of EA with MIF. (A) Surface diagram of MIF homotrimers docked with

648

EA. Each monomer is indicated by a different color: chain A, orange; chain B, cyan; chain C, magenta.

649

EA is shown in green color. The docked conformation produced by SwissDock indicates that EA interacts

650

with MIF in a pocket formed at the interface of chains A and B. (B) Interacting side chains of amino acid

651

residues are shown in sticks, and the ligand in ball and stick model. Hydrogen bonds are depicted as blue

27



Page 28 of 38

652

dashed lines. Binding pocket is formed by the side chains of Pro-1, Lys-32, Tyr-36, Ile-64, Trp-108, Phe-

653

113 and Tyr-95 from two neighbouring subunits. The Figures are generated using PyMOL.

654

Figure 6. Reagents and condition. (A)(i)(a) aq.NaOH, 30 min, reflux; (b) CuSO4 , 10 min, reflux; (ii)

655

AlCl3, chlorobenzene, 2.5 hours, reflux. (B)(i) BnCl, K2CO3, (n-Bu)4N+I-, acetone, 6 hours, reflux; (ii) 20

656

% KOH , ethanol, 2 hours, reflux; (iii) EDC-HCl, Et3N, DMF, overnight rt; (iv) 10% Pd-C, methanol-

657

chloroform, rt, 30 min.

658

Figure 7. EA inhibits MIF-mediated nuclear translocation NF -κB in PBMCs. Localization of NF-κB

659

(red) into the nucleus (blue) is calculated from Pearson’s correlation coefficient (5th column). The white

660

spots in the 4th column indicate area of maximum intranuclear NF-κB. Scale bars indicate 10 µm. Data

661

are presented as mean ± S.D. of three independent experiments.

662

Figure 8. EA inhibits MIF-mediated chemotactic migration of PBMCs. Chemotactic index is plotted with

663

the number of cells migrated towards the lower chamber and expressed as change over control keeping

664

the control value at zero. Data are presented as mean ± S.D. (##, p < 0.001 versus control; *, p< 0.01; **,

665

p < 0.001 versus MIF).

28


Page 29 of 38


Table 1 Chemical structure and activities of EA derivatives against MIF tautomerase Inhibition of tautomerase activity of MIF (IC50) (µM) Mean ± S.D

Compounds

4.77 ± 0.52 EA

Not active GA

35.9 ± 0.72

Compound 2d

50.03 ± 4.37 Urolithin A

57.42 ± 3.93 Urolithin B

Not active Pyrene

29



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60x47mm (300 x 300 DPI)


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Ellagic Acid, a Dietary Polyphenol, Inhibits ... - ACS Publications

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