bcnu_an antimalarial dug

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B i o c h e m i c a l P h a r m a c o l o g y Vol. 37 No. 5 pp. 855-W0 1988.Printed in Great Britain.

OOOk295@4 $3.W + 0.00@I 1988. Pergamon Journals Ltd.

GLUTATHIONE REDUCTASE INHIBITORS AS POTENTIAL

ANTIMALARIAL DRUGS

EFFECTS OF NITROSOUREAS ON P L A S M O D Z U M FA L C Z PA R U M I NVITRO

YA ZHANG,* ERNST HEMPELMANN and R. HEINER SCHIRMER~Institut fiir Biochemie II, Universitlt Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg,

Federal Republic of Germany

(Recei ved 27 Ap ri l 1987; accept ed 30 A ugust 1987)

Abstract-Malarial parasites are believed to be more susceptible to oxidative stress than their hosts.BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) and HeCNU(l-(2-chloroethyl)-3-(2-hydroxythyl)-l-

nitrosourea), inhibitors of the antioxidant enzyme glutathione reductase, were found to prevent thegrowth of Plasmodium alciparum n all intraerythrocytic stages. When exposing infected red blood ce llsto 38 PM BCNU or 62 PM HeCNU for one life cycle of synchronously growing parasites, the parasitemiadecreased by 90%. During the formation of new ring forms, the parasites are even more susceptible tothese drugs. The treatment with BCNU or HeCNU produced a rapid depletion of GSH in the parasitesand their host cells; in addition, protection against lipid peroxidation was impaired in these cells. Possiblemechanisms for the antimalarial action of the inhibitors are discussed. Our results suggest that erythrocyteglutathione reductase, an enzyme of known structure, might be considered as a target for the design ofantimalarial drugs.

Oxidative stress or oxidant stress in biomedical sys-tems denotes a disturbance of the prooxidant-anti-oxidant balance in favour of the former [l]. Clinicalobservations and experimental evidence suggest thatoxidative stress plays a dominant role in the defenseagainst parasitic infections [2-51. As a point in case,inborn and acquired conditions leading to intra-cellular oxidant stress protect, although not absol-utely, red blood cells from the malaria parasitePlasmodium falciparum [24,6]. This is in keepingwith the observation that parasitized erythrocytesare more susceptible to the toxic effects of H202than normal red cells [7]. A major defense systemfor the detoxification of reactive oxygen species inred blood cells is the glutathione redox cycle con-taining the two enzymes glutathione peroxidase

(ROOH +2

GSH + ROH + H20 + GSSG)and

glutathione reductase (GSSG + NADPH + H+ +2 GSH + NADP+) [8]. Consequently it is to beexpected that inhibitors of these enzymes impair theintraerythrocytic growth of malaria parasites. Herewe describe the antimalarial effects of the glutathionereductase inhibitors BCNUS [&ll] and HeCNU[3,12,13]. BCNU is widely used in tumour therapy[9,10,14]; HeCNU, a less toxic derivative of BCNU,

* Present address: Physiologisch-Chemisches Institut derUniversitlt, D-7400 Tiibingen, Federal Republic ofGermanv.

t To \;hom correspondence should be addressed.$ Abbreviations: BCNU, 1,3-bis(2-chloroethyl)-l-

nitrosourea; HeCNU, I-(2-chloroethyl)-3-(2-hydroxy-ethyl)-1-nitrosourea; GR, glutathione reductase (EC1.6.4.2); I-BHP, t-butyl hydroperoxide; TBA,thiobarbituric acid.

is of interest because it was found to cure rodentmalaria caused by Plasmodium vi nckei [ 51.

MATERIALSANDMETHODS

M aterial s. All reagents (from Boehringer,Mannheim, Merck, Darmstadt, Roth, Serva, Hei-delberg or Sigma, St. Louis, MO) were of the highestpurity available. RPMI-1640 niedium was obtainedfrom Biochrom (Berlin). BCNU and HeCNU werekind gifts of Professor G. Eisenbrand, KaiserslauternUniversity.

Cult i vat i on and i solat i on of the parasi t es. P. fal-ciparum (FCB strain) was cultured according to thecandle jar method [16] in RPMI-1640 medium sup-plemented with 25 mM HEPES, 32 mM NaHC03,

50 mg/ml hypoxanthine, 100 mg/l gentamycine and10% human serum from A(+) blood. The pH of theculture medium was 7.4 at 37”. In this paper thesupplemented medium is designated RPM1 medium;the supplemented medium plus serum is called cul-ture medium. At about 15% parasitemia with theparasites being present as trophozoites and schizonts,the erythrocytes were collected by centrifugation andwashed with isotonic medium (68 mM NaCl, 4.8 mMKCl, 1.2mM MgS04, 5 mM glucose in 50 mMsodium phosphate, pH 7.4). The cells were lysedby incubation at 37” for 10 min in 20 vol. of 150 mg/lsaponin in isotonic medium [17]. The parasites werepelletted by. centrifugation at 5OOg, washed twice

with isotonic medium and used immediately.Concentr ati on effect of BCNU and HeCNU on the

growth of P. falciparum in vitro. Sorbitol-synchron-ized parasites [18] were used in all experiments;the initial parasitemia was 1.5% with the parasitesbeing in the ring stage. Infected erythrocytes (25 ,ul

855


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856 Y ZHANG E. HEMPELMANN and R. H. SCHIRMER

in 500 ~1 medium) were cultured in 24-well plasticplates (Greiner, F.R.G.) and exposed to variousconcentrations of BCNU or HeCNU. Final para-sitemia was determined after 48 hr of culture bycounting the number of parasites per 2000 erythro-cytes in Giemsa-stained smears; the average wascalculated from duplicate culture dishes [19].

Effect of BCNU on eryt hrocyte i nvasion and theformat ion of new ri ngs. Highly synchronous parasitesobtained by treatment with 5% sorbitol with a cul-tural interval of 30 hr were grown in 24-well plasticplates. The cultures were divided into three groups,each group comprising 5 wells.

Group 1.

Infected erythrocytes (10% parasitemia) wereexposed to BCNU from 14 to 22 hr after the secondsynchronization. Then, the erythrocytes werewashed three times and added to washed fresh

erythrocytes to obtain 1.2% parasitemia. The culturewas then continued in BCNU-free medium until52 hr.

4 48 nAge of culture (hr)

Fig. 1. Inhibition of parasite growth by BCNU and theeffect of glutathione. P. fulciparum was cultured in mediumwithout additions (O), in medium with 20 mM GSH (m),with 20 mM GSSG (O), with 100 pM BCNU (A) and with

Group 2. 100PM BCNU plus 20 mM GSH (A).

Infected erythrocytes with 1.2% parasitemia werecultured in BCNU-free medium until 44 hr and thenexposed to BCNU for 8 hr.

Group 3.

The parasites in the control group were culturedwithout inhibitor. Final parasitemia was monitoredfor the three groups by microscopic examination at52 hr. Statistical analyses were performed byStudent’s two-tailed test for unpaired data. Theresults are expressed as means + standard error offive test samples.

tungstic acid was added, followed by 3 ml chloroformin order to remove dispersed lipids. After shakingthe sample for 3 min, the two phases were separatedby centrifugation. The water layer was taken forfluorometric measurement at 553 nm with excitationat 515 nm. The results are expressed as equivalentsof lipid peroxidation (nmol TBA/g Hb).

Gl ut at hi one reduct ase assay and prot ei n deter-minat ion. Isolated P. falciparum and washederythrocytes were dissolved with 4 vol. of solution T(10 mM MgC12, 10 mM mercaptoethanol, 4 mMEDTA, 4% Triton X-100, 50% glycerol in 10mMpotassium phosphate, pH 6). Glutathionereductase was assayed according to Krohne-Ehrichet al . [ 20]. The enzyme activity is given in U/ml cellsor in mU/mg protein. Protein was determined asdescribed by Heinzel et al. [21].

RESULTS

General observations

Effect of BCNU and H eCNU on the glutat hionelevels of host cells and parasites. Five percent sus-pensions of washed fresh erythrocytes and infectederythrocytes in RPMI-medium were exposed at 37”to 500 PM BCNU or 500 PM HeCNU. Aliquots con-taining 20~1 erythrocytes were taken at differenttimes and prepared for the determination ofglutathione according to the method of Anderson[ =

The capacity of glutathione reductase in humanerythrocytes is 1.0-l .4 U/ml cells corresponding to3.5-5 U per g haemoglobin; a similar activity of theenzyme was determined for P. fal ciparu m (44.8 U/g protein). The activity of red blood cell GR wasfound to decrease by less than 20% in 3 days underconditions used for culturing the intraerythrocyticforms of P. falciparum.

BCNU and HeCNU led to complete (298%) andpractically irreversible inhibition of intracellularglutathione reductase. Haemolysis was not observedwhich is consistent with the finding of Loos et al . [24]that erythrocytes without detectable GR activity canfulfil their functions in vi vo.

Effect of BCNU on t- buty l hydroperoxide-i nducedli pid peroxidat ion. A 5 suspension of erythrocytesin RPMI-medium was incubated with 1 mM t-BHPand 0, 100 or SOOpM BCNU, respectively. Lipidperoxidation was measured according to Yagi’s

method [23] with modifications: 30 ~1 erythrocyteswas pipetted into 2.5 ml distilled water and 0.5 mlTBA reagent (equal volumes of 0.67% thiobarbituricacid and acetic acid) was added to the solution. Thereaction mixture was heated for 60 min at 95” in awater bath. After cooling, 0.5 ml 10% phospho-

Inhibi t i on of parasi t e grow th by BCNU and HeCNUin vitro

As shown in Figs 1 and 2 the growth of P. fal-ciparum is completely inhibited by 100pM BCNUor 100 PM HeCNU, even in the presence of a highGSH level in the culture medium. The concentration-

dependence of the inhibitory effects is shown inFig. 3. When exposing the parasites for one life cycleto the nitrosourea compounds, IQ and ICY werefound to be 13 PM and 38 PM for BCNU; the cor-responding values for HeCNU were 18 ,uM and62 PM.


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Antimalarial action of BCNU and HeCNU 857

Fig. 2. Inhibition of parasite growth by HeCNU. P. fa l -ciparum was cultured in medium without additions (a),medium with 100 FM HeCNU (0)) with 100 PM HeCNUplus 5 mM GSH (Cl) and with 100 PM HeCNU plus 20 mMGSH (m). Each point represents the mean of three

experiments.

Sensi t ivi ty o the parasit es to BCNU and HeCNU atdif ferent stages of t heir l if e cycle

The inhibitory effects of BCNU and HeCNU onparasite growth are stage-independent. This wasshown by exposing synchronous cultures of P. fal-ciparum to the inhibitors either during the ring stage(from 0 to 24 hr of the intraerythrocytic life cycle)or during the trophozoite/schizont stage lasting from28 to 48 hr (Fig. 3). The transition from one life cycleto another which includes merozoite release from aparasitized red blood cell, invasion of new erythro-

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BCNU-incubated

BCNU B6NU BCNU B&NU

Fig. 4. BCNU effect on parasite reinvasion and the for-mation of new rings. Highly synchronous parasites were

exposed to BCNU for 8 hr during the late ring stage (14-22 hr) and during the formation of new rings (44-52 hr).Final parasitemia was determined at 52 hr. The results are

expressed as means + standard error of five samples.

cytes and ring formation of the parasites, comprisesthe time interval from about 44 to 52 hr. Duringthese 8 hours the parasites were found to be moresusceptible to BCNU than at other stages (Fig. 4).At a concentration of 3 PM, BCNU inhibits theformation of new rings (P < 0.001) but has no stat-istically significant effect (P > 0.05) on the growthand differentiation of the ring forms present at 14-22 hr of a cycle.

Depleti on of glut athi one in heparasit es and their hostcells

In many cells the inhibition of glutathionereductase leads to a decrease of the GSH level[lo, 11,25,26]. As shown in Fig. 5 this is also thecase for erythrocytes and intraerythrocytic P. fal-ciparum. In the presence of 500 PM BCNU the intra-

i ; ‘0 30 100 300

Grantration d inhibitors (qhl )

Fig. 3. Concentration effect of BCNU and HeCNU on the inhibition of P. fulciparum. Synchronous P.falciparum was cultured in the medium with various concentrations of BCNU (-) or HeCNU (---)for a whole life cycle (from 0 to 48 hr) (O), during the ring stage (O-24 hr) (W), or the trophozoite/

schizont stage (28-48 hr) (0).


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858 Y. ZHANG. E. HEMPELMANN and R. H. SCHIRMER

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i5 6 5)2 5kld

“0

inhitda BcNU ““yc,

Fig. 5. Depletion of glutathione by BCNU and HeCNU. The cells were incubated in RPMI mediumwith 500 PM BCNU or 500 PM HeCNU. GSH was determined at 45 min, 2 and 5 hr. Values aremeans f standard error. Numbers within the bars indicate the numbers of samples. * P < 0.001. (a)erythrocytes; (b) infected erythrocytes with 15% parasitemia; (c) P. alciparum solated from parasitized

red cells.

cellular GSH concentration drops to 10% of theinitial value within 45 min; HeCNU has a similareffect but it acts more slowly than BCNU (Fig. 5).The GSH level in the erythrocyte and the GSHlevel in the parasite are affected by the glutathionereductase inhibitors in a similar fashion, the timecourses and the resulting GSH levels being almostidentical for the two cells (data not shown).

Enhanced li pid peroxidat ion of host eryt hrocytes int he pr esence of BCNU

Glutathione and glutathione reductase are partsof the glutathione redox cycle which protects cellsagainst damage by oxidants [ 1,8, lo]. Consequentlyit is to be expected for situations of oxidative stressthat the presence of glutathione reductase inhibitorsleads to increased peroxidation of membrane lipidsand other cell constituents. Figure 6 shows that redcells are susceptible to 1 mM t-butyl hydroperoxideand that the effect of this oxidative stressor is clearly

i s 12 16 20 24Incubation time hr 1

Fig. 6. Lipid peroxidation of erythrocytes in the presenceof BCNU. Erythrocytes were incubated in RPMI-mediumwithout additions (O), in medium with 1 mM t-BHP (M),with 1 mM f-BHP plus 100 /*M BCNU (0) and with 1 mM

f-BHP plus 500 pM BCNU (0).

enhanced by BCNU in a concentration-dependentfashion.

DISCUSSION

The results reported here indicate that inhibitorsof glutathione reductase are active as antimalarialagents i n v i t r o (Figs 1 and 2). The different intra-erythrocytic stages are equally sensitive to thesedrugs (Fig. 3), but in the events leading to merozoiteinvasion and ring formation the parasite is moresusceptible to BCNU (Fig. 4). This is in keeping withthe observation of Wozencraft et al . [27,28], thatmerozoite invasion is precluded when the parasitesare exposed to an oxidant-generating system.

BCNU and HeCNU cause a drop of the gluta-thione levels to 10% in the parasites and their hostcells within 45 min (Fig. 5). The most likely expla-nation for the loss of glutathione is as follows. Thenormal cell metabolism leads to the oxidation ofGSH to GSSG. In the absence of glutathionereductase activity GSSG is not re-reduced butexported from the cells instead. Such an oxidation-excretion mechanism for glutathione has indeed beendemonstrated for human erythrocytes [29]. In ourexperiment the de novo synthesis of reducedglutathione (GSH) apparently did not keep up withthe loss, although the RPM1 medium contains theconstituent amino acids of GSH.

An obvious question is whether it is the loss ofglutathione or the inactivation of glutathionereductase which is the more important for the inhi-bition of parasite growth. The two mechanisms can-not be separated sharply from each other becausethe depletion of glutathione is accompanied by a

low [GSH]/[GSSG] ratio which probably leads toinhibition of protein synthesis and DNA synthesis inthe parasite [8].

Depleti on of glutat hione

According to literature reports a low level of GSH


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Antimalarial action of BCNU and HeCNU 8.59

in host erythrocytes interferes with the growth of P.falciparum [30,31]. This is consistent with studies onred blood cells whose glutathione reductase had beeninactivated by BCNU with a remaining activity ofless than 2%. In a BCNU-free culture medium thesecells did not serve as host cells for P. falciparumbut when GSH (20 mM) was added to the mediumparasite growth was found to be normal [ll]. Asshown in Fig. 1, parasites do not grow when othGSH and BCNU are present in the culture medium.These findings together suggest that a high GSHlevel is essential for the invasion process and/or forthe early development of P. falciparum. As soon asthe parasite contains sufficient glutathione reductaseof its own [32-341 it can grow unless the newly-synthesized enzyme is inhibited by BCNU.

Inacti vat ion of glutat hione reductase

The inactivation of the enzyme could play the

predominant role when parasitized red blood cellsare exposed to oxidative stress. Oxidative stress isbelieved to be a major defense mechanism againstmalarial parasites [2,3,7,35]. As shown in Fig. 6,BCNU impairs the protection of host erythrocytesagainst oxidative damage caused experimentally byt-butyl hydroperoxide. This is to be expected becauseperoxides are detoxified by the glutathione redoxcycle containing glutathione peroxidase andglutathione reductase as enzymes [8,33,36].

Glutathione reductase is not essential for the nor-mal function of erythrocytes [9,24]. This fact isimportant when considering glutathione reductaseinhibitors for the prevention and treatment ofmalaria. In addition, a single dose of the inhibitor isexpected to have a longer-lasting effect in erythro-cytes than in cells which are able to synthesize theenzyme de nova. Lens fibres which like erythrocytescannot synthesize proteins are not expected to bereached by lipophilic compounds such as BCNU.BCNU and even HeCNU which is curative againstPlasmodium vinckei infections in mice [15] might betoo toxic to be considered as antimalarial agents. Itremains to be studied whether the toxic side-effectsare due to the inhibition of glutathione reductase. Ifnot, glutathione reductase, an enzyme of knownthree-dimensional structure [37] and known ster-

eochemistry of catalysis [38], might be considered asa target for drug design. A number of compoundswith very different structures, such as HeCNU, para-quat and triiodothyronine are known as ligands ofthe enzyme [8] and can serve as starting points fortailoring drugs. As a first step towards designingbetter inhibitors, crystals of BCNU and HeCNU-inactivated glutathione reductase have recently beenanalyzed by X-ray crystallography [39].

Acknow ledgemenrs-We are supported by the DeutscheForschungsgemeinschaft (Schi 102/7-4). Ya Zhang is ascholar of the Deutscher Akademischer Austauschdienst(DAAD). This work is part of his PhD thesis at TiibingenUniversity.

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