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Determination of antiplasmodialactivity and binding affinity ofcurcumin and demethoxycurcumintowards PfTrxRRanjith Muniguntia, Symon Gathiakab, Orlando Acevedob, RajnishSahuc, Babu Tekwanic & Angela I. Calderóna

a Department of Pharmacal Sciences, Harrison School ofPharmacy, Auburn University, 4306B Walker Building, Auburn,36849, AL, USAb Departments of Chemistry and Biochemistry, Auburn University,Auburn, AL, USAc School of Pharmacy, National Center for Natural ProductsResearch, University of Mississippi, MS38677, USAPublished online: 21 Jan 2014.

To cite this article: Ranjith Munigunti, Symon Gathiaka, Orlando Acevedo, Rajnish Sahu, BabuTekwani & Angela I. Calderón , Natural Product Research (2014): Determination of antiplasmodialactivity and binding affinity of curcumin and demethoxycurcumin towards PfTrxR, Natural ProductResearch: Formerly Natural Product Letters, DOI: 10.1080/14786419.2013.866112

To link to this article: http://dx.doi.org/10.1080/14786419.2013.866112

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Determination of antiplasmodial activity and binding affinity of curcuminand demethoxycurcumin towards PfTrxR

Ranjith Muniguntia1, Symon Gathiakab1, Orlando Acevedob, Rajnish Sahuc, Babu Tekwanic and

Angela I. Calderona*

aDepartment of Pharmacal Sciences, Harrison School of Pharmacy, Auburn University, 4306B WalkerBuilding, Auburn, 36849 AL, USA; bDepartments of Chemistry and Biochemistry, Auburn University,Auburn, AL, USA; cSchool of Pharmacy, National Center for Natural Products Research, Universityof Mississippi, MS 38677, USA

(Received 15 July 2013; final version received 4 November 2013)

In our study, the inhibitory activity of curcuminoids towards Plasmodium falciparumthioredoxin reductase (PfTrxR) was determined using LC-MS-based functional assayand showed that only demethoxycurcumin (DMC) inhibited PfTrxR (IC50: 2mM).In silicomolecular modelling was used to ascertain and further confirm that the bindingaffinities of curcumin and DMC are towards the dimer interface of PfTrxR. The in vitroantiplasmodial activities of curcumin and DMC were evaluated and shown to be activeagainst chloroquine (CQ)-sensitive (D6 clone) and moderately active against CQ-resistant (W2 clone) strains of Plasmodium falciparum while no cytotoxicity wasobserved against Vero cells.

Keywords: Plasmodium falciparum thioredoxin reductase (PfTrxR); liquid chroma-tography-mass spectrometry-based assays; molecular modelling

Introduction

Malaria disease continues to be a major health problem in most parts of the world, especially in

developing countries where young children and pregnant women are its primary victims. The

majority of deaths due to malaria are caused by Plasmodium falciparum, which causes extreme

oxidative stress in red blood cells. The parasite’s efficient antioxidant system containing

thioredoxin reductase (TrxR) and glutathione reductase enzymes prevents damage caused by

reactive oxygen species (ROS) (Muller 2003). Disruption of these enzymes is a feasible way to

interfere with the erythrocytic development of malaria parasites. As the resistance to known

antiplasmodials is increasing, there is a need to expand the antiplasmodial drug discovery efforts

for new classes of molecules to combat malaria.

Curcuminoids isolated from the rhizomes of Curcuma longa Linn of the Zingiberaceae

family are mostly produced in India and have been used in the food industry as additive,

flavouring, preservative and colouring agent (Purusotam et al. 2011). Commercially available

turmeric may contain essential oils, polyphenols, protein, fat, minerals, carbohydrates and

moisture (Chattopadhyay et al. 2004). The rhizomes of C. longa are used for malaria therapy in

forms of tincture and decoction in Okeigbo, Ondo State, southwest Nigeria (Odugbemi et al.

2007). Curcumin has been shown to regulate a number of biological responses including anti-

tumourigenic, antioxidant and anti-inflammatory effects and anti-microbial activity (Itokowa

et al. 2008). Curcuminoids are obtained as a yellowish pigment and consist primarily of three

q 2014 Taylor & Francis

*Corresponding author. Email: aic0001@auburn.edu

Natural Product Research, 2014

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phenolic compounds: curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (bis-

DMC) (Scheme 1) (Aditya et. al. 2010). It has been reported that curcumin has cytotoxic and

parasiticidal effects in cultures against Leishmania major (Koide et al. 2002), Trypanosoma

brucei (Nose et al. 1998) andGiardia lamblia (Perez-Ariaga et al. 2006). In earlier studies, it was

demonstrated that curcumin has potent activity against P. berghei and P. falciparum when tested

in vitro (Raju et al. 2005; Long et al. 2007). However, the molecular mechanism in the

antiplasmodial activity of curcumin remains to be explored. Curcumin is also considered to be an

ideal molecule for use in combination with other antiplasmodials such as artemisinin because of

high cost, recrudescence and drug resistance of the latter molecule (Raju et al. 2005). In view

of curcumin’s abundance, non-toxic nature and demonstrated therapeutic effects in a variety of

human diseases, it will be useful to further investigate the potential of curcumin in developing

low-cost antiplasmodial therapies. To better evaluate the antiplasmodial activity of

curcuminoids, we tested and reported their binding affinity and inhibitory activity towards

PfTrxR using mass spectrometry and computer-based docking studies. The in vitro

antiplasmodial activity of these compounds was also tested against chloroquine (CQ)-resistant

and CQ-sensitive P. falciparum cultures.

Results and discussion

Based on the report of Mulabagal and Calderon in 2010 (Mulabagal & Calderon 2010) regarding

curcumin and DMC as PfTrxR ligands, we tested these compounds for their ability to inhibit

PfTrxR. Curcumin and DMC displayed good binding affinity for the PfTrxR target enzyme but

curcumin displayed less than 50% inhibition of PfTrxR at 10mM when tested in the functional

assay compared to DMC which displayed more than 50% PfTrxR inhibition at 10mM with an

IC50 value of 2.0mM. The fact that curcumin has been shown to inhibit rat TrxR by alkylating

the cysteine/selenocysteine catalytic active site residues (Fang et al. 2005) supports our data and

prediction that curcumin selectively inhibits mammalian TrxR but not the PfTrxR. The in vitro

antiplasmodial activity of curcumin and DMC was evaluated against both CQ-sensitive

O O

HO OH

OCH3 OCH3O O

HO OH

OCH3O O

HO OH

Curcumin

DMC

bis-DMC

Scheme 1. Chemical structures of curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin(bis-DMC).

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(D6 clone) and CQ-resistant (W2 clone) strains of P. falciparum, while cell cytotoxicity was

determined against Vero cells (Table 1) using the procedures described in the supplementary

information. The two compounds were active against the D6 strain of P. falciparum and

moderately active against the resistant W2 strain. The antiplasmodial activity of curcumin (IC50:

4.21mM) against CQ-resistant strain MP-14 of P. falciparum has been reported by Mishra et al.

(2008). The difference in IC50 values between our results and others could be due to the use of

different strains of the parasite, protocol to measure the parasite growth inhibition and the

detector used for the measurement. Furthermore, the curcuminoids showed cytotoxicity against

Vero cells from 64mM and selectivity index of four-sixfolds and onefold against D6 and W2,

respectively. Curcumin and DMC were tested for their ability to induce signs of oxidative stress

by accelerated generation and accumulation of reactive oxygen intermediates (superoxide

radical, hydroxyl radical and hydrogen peroxide). The intraerythrocytic formation of ROS was

monitored in real time for 120min with 2070-dichlorofluorescein diacetate, a fluorescent ROS

probe. Neither of these two compounds was able to increase the ROS in healthy erythrocytes by

a potential inhibition of mammalian TrxR.

To validate the docking protocols, the bound cofactor, flavin adenine dinucleotide (FAD),

was re-docked as the control (with crystallographically bound water molecules included) as the

structure used does not have a bound ligand and there are no alternative crystal structures of the

target protein. By visual inspection, the flexible docking protocols of AutoDock Vina reasonably

reproduced the experimental binding pose of FAD indicating a good accuracy for the parameters

used in the present docking methodology (Figure S1). Curcumin and DMC were predicted to

bind to the dimer interface of PfTrxR at the intersecting helices between the subunits. An

alternative curcuminoid, bis-DMC, that lacks the methoxy substituents on the phenyl moieties

(Scheme 1) was also found to favour the PfTrxR dimer interface. Intriguingly, all three

curcuminoids were predicted to bind in a nearly identical fashion, regardless of the presence or

lack of methoxy substituents (Figure 1). Examination of the crystal structure (Boumis et al.

2012) reveals that the residues from the intersecting helices that interact with the ligands are

Leu98, Tyr101, Ala102, His104, Met105, Ile108, Asp112, Tyr116 and Pro480 from both

subunits A and B. It is evident that the first phenol moiety (demethoxylated in DMC) interacts

with the residues on subunit A, whereas the other moiety extends towards subunit B (Figure S2).

The results suggest that the methoxy groups may contribute to the difference in inhibition

towards PfTrxR. In curcumin, the ortho-methoxy group can form an intramolecular hydrogen

bond with the phenolic hydrogen, making the H-atom abstraction from the ortho-methoxy

phenols surprisingly easy. However, elucidating how phenyl methoxy groups mediate inhibition

of PfTrxR is more difficult.

Earlier theoretical studies have shown that hydrogen bonding between ortho-methoxy

oxygen and phenolic hydrogen in curcumin influences the planarity, conformation and ability to

undergo oxidation (Sandur et al. 2007). From the current docking simulations, the phenyl moiety

Table 1. Inhibition of PfTrxR, predicted binding affinities and antiplasmodial activity of curcuminoids.

Testcompounds

PfTrxRIC50 (mM)

Computed dimerinterface affinity

(kcal/mol)

Pf (D6)CQ-sensitiveIC50 (mM)

SID6

Pf (W2)CQ-resistantIC50 (mM)

SIW2

Vero IC50

(mM)

Curcumin NA 29.4 15.9 ^ 2.1 4.0 41.2 ^ 6.2 1.6 64.6 ^ 1.73DMC 2.03 ^ 1.05 29.6 17.7 ^ 3.1 5.0 82.7 ^ 10.3 1.1 89.7 ^ 4.51bis-DMC NA 29.8 ND ND ND ND NDCQ 0.055 ^ 0.006 0.440 ^ 0.045 NC

Notes: NA, not active i.e, PfTrxR inhibition was , 50% at 10mM; ND, not determined; NC, no cytotoxicity up toconcentration much higher than the concentration responsible for its antiplasmodial activity.

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formed a pi–pi stacking interaction with the Tyr101 from each respective subunit. The hydroxyl

group in DMC formed a hydrogen bond with a distance of 2.5 A to the oxygen of Tyr116 in

subunit A, albeit at an angle of 1108. Curcumin’s OH group is bent in the opposite direction

because of the conformational change, and thus, the H bond is not realised. For subunit B, the

opposite trend occurs. Curcumin forms a 2.4 A H bond with the Tyr 1010 at an angle of 1388 andDMC’s OH group bends to avoid the interaction (Figure 1). The hydrophobic a, b-unsaturatedchain interacts with the side chain of Ile 108 from subunit A. The experimental data show a

marked difference between the two compounds with respect to PfTrxR inhibitory activity,

whereas docking analysis predicted indistinguishable binding affinities despite the subtle

differences in their binding poses (Table 1).

The methoxyphenyl moiety of the curcuminoids is shown to lay in a narrow groove in the

dimer inter-subunit interface, sandwiched by Tyr101 and Tyr116, thereby locking the rings into

position (Figure 2). The structural differences in the curcuminoids could point to the differences

in experimental values. For example, the pi–pi interactions between the phenyl moieties of the

curcuminoids and the Tyr residues (Figure 2) suggest that the methoxy groups on curcumin and

DMC are parallel to the hydroxyl group of Tyr 101 and may be less favourable due to poorer

Figure 1. (Colour online) The interactions between DMC (cyan), curcumin (tan) and bis-DMC (purple)with PfTrxR.

Figure 2. (Colour online) Hydrophobicity surface for curcumin-docked PfTrxR. Colour scale: white to bluefor the most hydrophilic residues and orange to red for the most hydrophobic residues.

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sterics (Figure 1). The lack of a methoxy group in bis-DMC would not incur the same energetic

penalty, and therefore, the binding affinity is slightly enhanced according to the calculations.

Comparisons to the mammalian TrxR are difficult to make as curcumin binds covalently (Fang

et al. 2005), precluding the use of docking calculations. It may be reasonable to assume that

curcuminoid derivatives DMC and bis-DMC would also bind covalently to TrxR.

Based on the little correlation between the results from phenotypic screening and docking

studies of curcumin and DMC to support the activity through PfTrxR inhibition, the

antiplasmodial activity might be due to other mechanism of actions such as inhibition of Ca(2þ)-

ATPase (PfATP6) (Shukla et al. 2012), S-adenosyl-L-homocysteine hydrolase (PfSAHH) (Singh

et al. 2013) or any other target.

Conclusions

In this study, phenotypic screening and docking studies have been applied as tools for the

assessment of the potential of curcuminoids as antiplasmodials through PfTrxR inhibition. Even

though the docking studies predicted close proximity in binding affinities of curcumin and DMC

to PfTrxR at the dimer interface, only DMC was found to inhibit PfTrxR with an IC50 value of

2mMwhen tested in the functional assay. Curcumin did not inhibit PfTrxR, and therefore, it was

evident from earlier studies that curcumin selectively inhibits mammalian TrxR and not PfTrxR

owing to the differences in active sites of these two enzymes. However, these two compounds

showed antiplasmodial activities when tested in vitro against P. falciparum.

Supplementary material

Experimental details relating to this article are available online, alongside Figures S1 and S2.

Acknowledgements

The authors are deeply indebted to Dr Katja Becker from the Justus-Liebig University, Germany forsupplying the enzyme PfTrxR.

Note

1. These authors contributed equally to this work.

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