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International Journal of Antimicrobial Agents 32 (2008) 349–354

Short communication

Therapeutic effect of a novel anilidoquinoline derivative,2-(2-methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide,in Japanese encephalitis: correlation with in vitro neuroprotection

Joydeep Ghosh a, Vivek Swarup a, Amit Saxena a, Sulagna Das a, Abhijit Hazra b,Priyankar Paira b, Sukdeb Banerjee b, Nirup B. Mondal b, Anirban Basu a,∗

a National Brain Research Centre, Manesar, Haryana 122050, Indiab Steroid and Terpenoid Chemistry Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, India

Received 15 April 2008; accepted 2 May 2008

bstract

2-(2-Methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide, a novel anilidoquinoline derivative, was synthesised and evaluated for its

herapeutic efficacy in treating Japanese encephalitis. The compound showed significant antiviral and antiapoptotic effects in vitro. Significantecreases in viral load (P < 0.01) combined with an increase in survival was observed in Japanese encephalitis virus-infected mice treatedith 2-(2-methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide.2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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eywords: Anilidoquinoline; Japanese encephalitis virus; Neuron; Apoptos

. Introduction

Japanese encephalitis virus (JEV), a neurotropic fla-ivirus, commonly affects children and is a major causef acute encephalopathy [1]. There is no specific therapyor treating patients affected with JEV other than vaccina-ion. The high cost of the vaccine prohibits poor countriesrom affording a routine immunisation programme. Hence,n affordable chemotherapeutic agent is necessary for treat-ng poor patients living in rural areas of underdevelopedountries. The vaccine, containing murine encephalogenicasic protein or gelatin stabiliser, may also cause allergiceactions [2]. Therefore, it is necessary to find compoundshat are cheap, with no or tolerable side effects, combinedith a protective potential when administered several hours

fter infection.

Treatment of mosquito-borne parasitic diseases with

uinoline derivatives has been well known for decades.everal compounds in this class have been screened for

∗ Corresponding author. Tel.: +91 124 233 8921.E-mail address: anirban@nbrc.res.in (A. Basu).

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924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Societyoi:10.1016/j.ijantimicag.2008.05.001

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ntiviral [3–5], bactericidal [6,7], antitumour [8], anti-nflammatory [9,10] and antiprotozoal [11] activity. Thus,

series of anilidoquinoline analogues were synthesisednd evaluated for their antiviral efficacy. It was observedhat the analogue 2-(2-methyl-quinoline-4ylamino)-N-(2-hlorophenyl)-acetamide (PP2) is effective in conferringrotection against JEV. The present study describes theethod of preparation and the process of evaluation of PP2

s an effective anti-JEV agent in vitro and in vivo.

. Materials and methods

.1. Synthesis of anilidoquinoline analogues

Preparation of the compound was carried out in a three-ecked flask. The whole system was placed on a magnetictirrer with a magnetic fly inside the flask. Sodium hydride

as added to the flask and washed with a dry non-polar sol-ent, petroleum ether, to make it free from suspended oil.imethyl sulphoxide was added and the reaction mixture was

tirred until an olive green colour persisted. The substrate,

of Chemotherapy. All rights reserved.

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50 J. Ghosh et al. / International Journal

-aminoquinoline, was added to the reaction mixture withontinuous stirring. Finally, the reactant, mono chloroanilide,as added to the reaction mixture with continuous stirring.he whole operation was carried out in an inert atmosphere,amely nitrogen, argon free from oxygen under an anhy-rous condition. Termination of the reaction was achievedy adding ice-cold water. The whole of the reaction mix-ure was extracted with dichloromethane. The organic layeras washed with water to make it free from alkali (neutral

o litmus). The organic solvent was removed under reducedressure to give PP2 after crystallisation from methanol.

.2. Screening of anilidoquinoline analogues by MTTssay

The mouse neuroblastoma cell line N2a was plated at aensity of 5 × 105 cells/well in a six-well plate. After 24 hn Dulbecco’s Modified Eagles Medium (DMEM) with 10%erum, the cells were switched to serum-free medium for2 h. The N2a cell line was then infected with either live JEVGP78 strain) or was mock infected for 1 h. After adsorp-ion, unbound virus was removed by gentle washing withhosphate-buffered saline (PBS). Fresh serum-free mediumas added to each well for further incubation.JEV-infected N2a cells were treated with five different

nilidoquinolones (PP0, PP2, PP4, PP6 and PP8) at a con-entration of 5, 10 and 20 �g/mL in serum-free medium for4 h. The MTT assay, which reflects mitochondrial succinateehydrogenase activity, was performed to assess the levelf cell survival. After treatment with anilidoquinoline ana-ogues, the culture medium was replaced by a solution of

TT (5 mg/mL) in serum-free medium and incubated forh at 37 ◦C. The solution was then removed and the result-

ng blue formazan was solubilised using solubilisation buffer50% dimethylformamide, 20% sodium dodecyl sulphateSDS), with the volume made up to 10 mL using distilledater). Absorbance was measured at 570 nm using a Var-

oskan Flash spectral scanning multimode reader (Thermolectron Corporation, Vantaa, Finland) [12].

.3. Intracellular staining by flow cytometry forEV-specific antigen

N2a cells were plated in six-well plates at a density of× 105 cells/well in 3 mL of medium and were cultured

or 18 h. After 18 h in DMEM with 10% serum, the cellsere switched to serum-free medium for 12 h. The N2a

ell line was then infected with JEV (multiplicity of infec-ion (MoI) = 5) for 1 h. The virus was removed and thelates were then washed with 1× PBS to remove unboundirus. The plates were further incubated with PP2-containingerum-free medium at 37 ◦C. Cells were collected after 24 h.

fter two washes with 1× PBS, cells were first fixed withD CytofixTM solution (BD Biosciences, San Diego, CA)

or 15 min. Cells were then permeabilised by re-suspensionn permeabilisation buffer (BD CytopermTM Plus; BD

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microbial Agents 32 (2008) 349–354

iosciences) and incubated at room temperature for 10 min.ells were washed twice in wash buffer (PBS containing% bovine serum albumin (BSA)) and then re-suspended inash buffer at 1 × 106 cells/100 �L. Primary antibody (JEVakayama strain; Chemicon, Temecula, CA) was added in:100 dilutions and incubated for 30 min at room temper-ture. The cells were washed five times with wash buffer,elleted and then incubated with fluorescein isothiocyanateFITC)-conjugated secondary antibody (Vector Laboratories,urlingame, CA) for 30 min. After three washes with washuffer, the samples were re-suspended in 400 �L of Fluores-ence Activated Cell Sorting (FACS) buffer. Samples werenalysed on a FACSCalibur in the FL1 channel. The per-entage of the population was calculated after gating theopulations on Dot plot in Cell Quest Software [13].

.4. Active caspase-3 assay

Mock-infected, JEV-infected and JEV-infected/PP2-reated N2a cells were sonicated on ice in 100 �L ofysis buffer. The Colorimetric CaspACETM Assay SystemPromega, Madison, WI) was used according to the man-facturer’s instructions for the 96-well plate assay format.uplicate assays were performed for each sample, withlanks and mock-infected cells. Absorbance was measuredt 405 nm using a Varioskan Flash spectral scanning mul-imode reader. Caspase-3 enzyme activity was expressed asicomoles of caspase-3 liberated per milligram of protein perinute [14].

.5. Terminal deoxynucleotide transferase-mediatedUTP nick-end labelling (TUNEL) assay

N2a cells were plated at a density of 5 × 104 cells/welln eight-well chamber slides (Nunc, Roskilde, Denmark) in

edium containing 10% fetal bovine serum (FBS). After4 h cells were grown in serum-free medium for 8 h. Fol-owing JEV infection for 1 h and PP2 treatment for 24 h,poptotic cells were identified using In Situ Cell Death Detec-ion Kit, TMR red (Roche, Mannheim, Germany). Cells werexed with 4% paraformaldehyde in 1× PBS and blockedith 4% BSA containing 0.02% Triton X-100. The fixed

ells were then incubated in the TUNEL mix (terminaleoxynucleotidyl transferase in storage buffer and TMR redabelled-nucleotide mixture in reaction buffer) for 1 h at roomemperature. The slides were mounted with Vectashield®

ounting media containing 4′,6-diamidino-2-phenylindoleDAPI) (Vector Laboratories) [15].

.6. Immunoblot analysis

Western blot analysis was performed with protein isolated

rom mock-infected, JEV-infected and JEV-infected/PP2-reated N2a cells. Each sample (20 �g) was electrophorescednd transferred onto a nitrocellulose membrane. The mem-ranes were then blocked and incubated with several primary

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J. Ghosh et al. / International Journal

ntibodies, including Bax and Bcl-2 (1:1000; Santa Cruziotechnology Inc., Santa Cruz, CA) and �-tubulin (1:2500;igma, St. Louis, MO). Appropriate horseradish peroxidase-onjugated secondary antibodies were used and blots wereeveloped using enhanced chemiluminescence Western blot-ing detection reagents (Amersham Pharmacia Biotech, Littlehalfont, UK).

.7. PP2 administration in an animal model of Japanesencephalitis

BALB/c mice (age 4–5 weeks) were infected intra-enously with a lethal dose of 3 × 105 plaque-forming unitsPFU) of JEV (GP78 strain). One day following virus inocu-ation, animals started receiving PP2 intraperitoneally twiceaily (40 mg/kg body weight) for the next 7 days. Observationf animal survival experiments was performed in a maskedanner to avoid bias towards any one group of animals.

.8. Plaque assay

For analysis of viral load in the central nervous system ofnfected mice, brains were recovered after extensive cardiacerfusion with PBS, dissected, cooled on ice, homogenisednd titrated for virus by plaque formation on porcine sta-le (PS) kidney cell monolayers. PS cells were seeded in5-mm dishes and formed semiconfluent monolayers aftera. 18 h. Monolayers were inoculated with 10-fold dilutionsf virus sample made in minimal essential medium (MEM)ontaining 1% FBS and incubated for 1 h at 37 ◦C with occa-ional shaking. The inoculum was removed by aspirationnd the monolayers were overlaid with MEM containing% FBS, 1% low-melting-point agarose and a cocktail ofntibiotic/Antimycotic solution (Gibco–Invitrogen Corp.,ew York, NY) containing penicillin, streptomycin and

mphotericin B. Plates were incubated at 37 ◦C for 4 daysntil plaques became visible. The cells were fixed with 10%ormaldehyde and stained with crystal violet and the plaquesere counted [13].

.9. Statistics

Data are expressed as mean ± standard error of the mean.omparisons among groups were performed by one-waynalysis of variance (ANOVA) followed by Bonferroni’s mul-iple comparisons post-test.

. Results

.1. PP2 has potent antiviral activity in vitro

N2a cells were used to assess the efficacy of variousnilidoquinolines towards preventing JEV-induced neuronaleath. The results (Fig. 1A) showed that at all the MoIs, PP2Fig. 1B) was the most potent anilidoquinoline in conferring

itis

microbial Agents 32 (2008) 349–354 351

rotection from JEV-induced neurotoxicity. PP2 showedaximum antiviral action along with minimal cytotoxic

ffect at a concentration of 10 �g/mL. At a MoI of 0.5, the sur-ival rate of JEV-infected/PP2-treated cells was found to be.8–2.5-fold greater (P < 0.01) than JEV-infected cells treatedith other anilidoquinolines. At a higher MoI (5 and 50),

he survival rate of JEV-infected/PP2-treated cells was foundo be 1.2–2.2-fold greater (P < 0.001) compared with JEV-nfected cells treated with other anilidoquinolines (Fig. 1A).o determine whether PP2 affects neuronal morphology,ock-infected, JEV-infected and JEV-infected/PP2-treated

ells were observed under a light microscope. In JEV-infectedells (Fig. 1D), the morphology was significantly altered dueo JEV-induced neurotoxicity compared with mock-infectedells (Fig. 1C) or JEV-infected/PP2-treated cells (Fig. 1E).

.2. PP2 treatment reduces intracellular viral load initro

Intracellular staining of JEV-specific antigen in N2a cellsas performed to assess whether PP2 could decrease the

ntracellular viral load. It was observed that after 24 h theEV-infected cells comprised 90% of the total cell popula-ion, whereas after PP2 treatment the level came down to5% (P < 0.01) (Fig. 1F and G).

.3. PP2 treatment confers protection from JEV-inducedpoptotic neuronal death

Mock-infected, JEV-infected and JEV-infected/PP2-reated N2a cells were subjected to the TUNEL assay. Whilstegligible cell death was observed in mock-infected N2aells (Fig. 2A), JEV-infected samples (Fig. 2B) had signif-cant TUNEL-positive cells (marker for apoptotic nuclei),nd the number of TUNEL-positive cells reduced drasticallyn JEV-infected/PP2-treated samples (Fig. 2C). To corrob-rate this finding, immunoblot analysis of protein lysatesrom mock-infected, JEV-infected and JEV-infected/PP2-reated N2a cells was performed for Bcl-2 and Bax (Fig. 2D).

hilst in JEV-infected/PP2-treated samples the Bcl-2 levelas significantly increased (3.8-fold increase compared with

EV-infected sample; P < 0.001), the Bax level was signifi-antly increased in only JEV-infected cells (1.2-fold increaseompared with JEV-infected/PP2-treated sample; P < 0.001),hus confirming that PP2 abrogates neuronal death by itsntiapoptotic properties.

As activation of caspase-3 is a hallmark of apoptosis,he amount of active caspase-3 in N2a cells following viralnfection was evaluated. In JEV-infected samples, a 56-old increase (compared with mock-infected cells; P < 0.001)

n caspase-3 activity was observed. On the other hand,here was a 5-fold decrease in caspase-3 activity in JEV-nfected/PP2-treated samples compared with JEV-infectedamples (Fig. 2E; P < 0.001).

352 J. Ghosh et al. / International Journal of Antimicrobial Agents 32 (2008) 349–354

Fig. 1. 2-(2-Methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide (PP2) is a potent anilidoquinoline analogue in preventing Japanese encephalitis virus(JEV)-induced neurotoxicity and decreases intracellular viral load. (A) N2a cells were infected with JEV and then treated with five different anilidoquinolones at aconcentration of 10 �g/mL. PP2 had the most potent antiviral activity compared with other anilidoquinoline analogues. (B) Structure of the anilidoquinoline PP2.(C–E) Mock-infected, JEV-infected and JEV-infected/PP2-treated cells were processed for microscopic observation. In JEV-infected cells (D), the morphologywas significantly altered due to JEV-induced neurotoxicity compared with mock-infected cells (C). The morphology of JEV-infected/PP2-treated cells (E)s tment sH esentata

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howed no visible change compared with mock-infected cells. (F) PP2 treaistogram from Fluorescence Activated Cell Sorting (FACS) analysis is repr

nalysis for intracellular JEV-specific antigen.

.4. PP2 confers complete protection to animals fromEV

PP2 treatment following JEV infection completelyeduced the mortality rate (15 of 15 animals survived fol-owing PP2 treatment in the JEV-infected group) (Fig. 3A).

hilst all infected animals that did not receive any PP2 treat-ent succumbed to infection, PP2 alone (drug control) had

o effect on mortality or on the behavioural outcome of ani-als (data not shown). Infection with JEV was accompanied

y distinct symptoms, and treatment with PP2 followingirus infection significantly rescued animals from suffer-ng.

.5. PP2 treatment reduces the viral titre of JEV in vivo

Virus isolated from JEV-infected animal brains wasitrated by plaque formation on PS monolayers. Treatmentith PP2 significantly reduced the virus titre in JEV-infected

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ignificantly reduced the JEV-specific viral antigen in N2a cells (P < 0.01).ive of three independent experiments. (G) Graphical interpretation of FACS

nimals (3 × 105 PFU/mL in JEV-infected mice, which afterreatment reduced to 5 × 103 PFU/mL; P < 0.01) (Fig. 3B).

. Discussion

Despite the significant disease burden caused by JEV,o specific antiviral therapy is currently licensed for treat-ent. Previous studies have reported that anilidoquinoline

nalogues have potent antiviral and anti-inflammatory activ-ties. In this study, we synthesised a series of anilidoquinolinenalogues and screened them for potential anti-JEV activity.ne of the analogues, PP2, showed significantly increased

evels of anti-JEV activity. In addition, PP2 also showedntiapoptotic effects in vitro. PP2 also conferred complete

rotection to JEV-infected animals when administered 24 hfter infection.

In summary, the anilidoquinoline analogue PP2 is aromising antiviral agent with clear evidence of in vivo and

J. Ghosh et al. / International Journal of Antimicrobial Agents 32 (2008) 349–354 353

Fig. 2. 2-(2-Methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide (PP2) prevents Japanese encephalitis virus (JEV)-induced neuronal apoptosis. (A–C)Apoptotic cell death assay of N2a cells showing TUNEL-positive cells (pink) co-localised with 4′,6-diamidino-2-phenylindole (DAPI). Negligible cell deathwas observed in mock-infected N2a cells (A), whereas a profound increase in the number of TUNEL-positive cells was observed when N2a was culturedin the presence of JEV (B). N2a cells cultured in the presence of JEV and treated with PP2 showed a drastic decrease in TUNEL-positive cells (C). Scalebars, 50 �M (magnification 20×). (D) Proteins from mock-infected and JEV-infected N2a samples were subjected to immunoblot analysis. Whilst the JEV-infected/PP2-treated sample showed a significantly increased level of Bcl-2, the Bax level was significantly increased in only JEV-infected cells compared withJEV-infected/PP2-treated cells. The blot shown here is representative of three independent experiments. (E) Protein lysates were analysed for caspase-3-specificactivity. Caspase-3 enzyme activity was expressed as picomoles of caspase-3 liberated per milligram of protein per minute. In JEV-infected samples, there wasa V-infeo refereno

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significant increase in caspase-3 level compared with mock-infected and JEf the mean from three independent experiments. (For interpretation of thef the article.)

n vitro activity against JEV infection. The anilidoquinolinenalogue (i) reduces the intracellular viral load, (ii) preventspoptotic cellular death in vitro, (iii) upregulates the expres-ion level of Bcl-2 and downregulates the expression level of

ax in vitro, (iv) reduces active caspase-3 activity in vitro, (v)

ncreases the lifespan of JEV-infected mice and (vi) causessignificant reduction in viral load in JEV-infected mouse

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ig. 3. (A) 2-(2-Methyl-quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide (PP2)as significantly increased in groups that received PP2 treatment (n = 15 for each gr

nimal. (B) Virus isolated from control, JEV-infected and JEV-infected/PP2-treatehe viral titre obtained after infection with JEV was 3 × 105 plaque-forming units× 103 PFU/mL (*P < 0.01). All the tissue samples were collected on the 8th day pean from six individual animals from each group.

cted/PP2-treated samples (P < 0.001). Data represent mean ± standard errorces to colour in this figure legend, the reader is referred to the web version

rain. As PP2 can be synthesised quite readily, it is intrigu-ng to consider this anilidoquinoline analogue as a possibleffordable therapeutic agent in regions of the globe with aigh level of JEV-induced mortality and yet hard-pressed for

ealthcare resources. Based on the data presented here, weelieve that further study of PP2 is warranted as a possiblereatment for JEV infection.

treatment confers complete protection to mice infected with JEV. Survivaloup). Treatment with PP2 alone behaves in a similar fashion as in a controld BALB/c mice was subjected to plaque assay to determine the viral load.(PFU)/mL, whilst PP2 treatment significantly decreases the viral titre up toost infection. Data shown are representative of mean ± standard error of the

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Japanese encephalitis. J Neurochem 2007;103:771–83.[15] Swarup V, Ghosh J, Duseja R, Ghosh S, Basu A. Japanese encephalitis

54 J. Ghosh et al. / International Journal

cknowledgments

The authors thank Kanhaiya Lal Kumawat for tech-ical assistance as well as Vijayalakshmi Ravindranath,irector of the National Brain Research Centre, for her

ncouragement.Funding: This work was supported by grant nos.

T/PR/5799/MED/14/698/2005 and BT/PR/8682/MED/4/1275/2007 from the Department of Biotechnology, Gov-rnment of India, to AB.

Competing interests: None declared.Ethical approval: All experiments were performed

ccording to the protocol approved by the Institutional Ani-al Ethics Committee of the National Brain Research Centre,aryana, India.

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