Indian Journal of Biochemistry & Biophysics Vol. 36 , October 1999, pp. 299-304

Inhibition of a protein tyrosine kinase activity in Plasmodium Jalciparum by chloroquine

Arun Sharma* and Neerad C Mishra

Department of Protein Biochemistry, Malaria Research Centre, 22 Sham Nath Marg, Delhi 110054 (Indi a)

Received 14 January 1999; revised 19 April 1999

The aim of the present study was to establish the importance of phosphorylation events for parasi te growth and maturat ion . Investigations into the cytosolic Plasmodium falciparum protein tyrosine kinase (PTK ) activity revealed th at there is a stage specific increase in the activity, in the order ring < trophozoite < schizont in both chloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) st rai ns (p<0.05). Our data also show that in vivo conversion of the schi zont stage to ring stage via release of merozoites is associated with a decrease in PTK activity. Piceatannol , a specific inhibitor o f PTK inhibited the activity in both the CQ-S and CQ-R strains of the parasites. The presence of low levels of chloroq uine (CQ) inhibited the cytosolic PTK activity in a dose dependent manner (ICso = 45 ~moles or 23 ~g/ml ) in CQ-S strain s. The effect of varying concentration of CQ on the kinetics of peptide phosphorylation reveal that CQ was a competiti ve inhibitor of PTK with respect to peptide subst rate and non-competitive with respect to ATP indicating that CQ inhibits PTK activity by binding with protein substrate binding site. These data thus suggests that maturation of malaria parasite may be due to thi s cellul ar PTK and its inhibition by CQ could provide a hypothesis to explain its antimal arial acti vity and e fficacy.

The parasi te protozoan , Plasmodium Jalciparum, is the causative agent of malaria throughout the world and is responsible for an annual death toll of nearly 3 million cases, the majority of which are children and pregnant women I. During the various stages of the life cycle, the parasite undergoes a trophic phase followed by a replicative phase2

. These developmental processes are accompanied by metabolic and morphological changes in the parasite. Since the tools available for control of malaria are rendered inadequate and drug resistant strains are widespread and moreover the immediate prospect of a vaccine is uncertain \ there is an urgent need to obtain fundamental knowledge on the mechanism of parasite development at cellular leve l so that susceptible targets can be identified.

Protein kinases are potentially important drug targets due to their roles in the regulation of cellular replication and differentiation in eukaryotes4 and therefore, their involvement during the maturat ion of Plasmodium spec ies is envi saged). Some preliminary studies on prote in kinases have resulted in the cyclic AMP dependent protein kinase6

, a Ca2+-ac tivated protein kinase7

, a cdc 2 like prote in kinase8 and genes . h . k· 910 F Wit protem mase consensus sequences · . our

*Author for correspondence. Tel. : 9 1 II 3979998; Fax: 9 1 II 2946150

cytosolic phosphoproteins of the paras ite have been identified and monoclonal antibodies have also been genenited ll

. The cDNA for a casein kinase I (CK I) of P. Jalciparum has been cloned, sequenced and expressed in bacteria'.

Since reversible prote in phosphorylation and dephosphorylation constitute a major mechanism of signal transduction 12 catalyzed by specific prote in kinases of which many are phosphoprote ins, one of the immediate goals has been to characterize the various protein kinases in P. Jalciparul17. Protein tyrosine kinases (PTKs) have been found to be inhibited by specific inhibitors. One such inhibitor of plant origin has been identified as piceatannol and has been found to be inhibitory to the PTK in cancerous cells lJ

. Protein tyrosine kinases have been found to be activated by Mg2+ preferred ATP over GTP and was found to be stimulated by polyamines'.

In this article we report PTK express ion during the course of maturation of malaria parasite and its inhibition by CQ and piceatannol. Our studi es show that PTK plays an important rol e in the maturati on / differentiation of the malaria paras ite and may be a potential drug target.

Materials and Methods Reagents used in the study were RPMI 1640

(Gibco Laboratories, Grand Is land , NY): Percol l

300 INDIAN J BIOCHEM BIOPHYS, VOL. , 36, OCTOBER 19lJlJ

(Pharmacia, Uppasa la, Sweden); protease inhibi tors and PTK inhibitors and PTK assay kit (Boehringer Mannheim, GmbH , Germany); CQ and all other reagents were purchased from Sigma Chemical Co. USA and were of analyti cal grade.

Parasite culture CQ-S (FlB-09) and CQ-R (FlB-04) strains of P.

Ja lciparum were cul tured ill vitro in RPM! 1640 medium which was supplemented with 2S mM

HEPESIO.37 mfli! hypoxanthinell 00 mg neomycin basell 0% heat inacti vated fetal calf serum. For culture 10% hematocrit was adjusted with normal 0 +ve red blood cel ls. Paras ites were grown at 37°C in candle jar and monitoring of the paras itaemia was done at 24 hrs '4. The parasitaemia and the stage spec ific parasi ti c development was examined microscopicall y of the JSB stained blood smears.

Isolation oj erythrocytic stages Paras iti zed eryth rocytes were isolated as previ ously

described IS. Briefl y, healthy asynchronous cultures having a paras itaemi a between S-I 0% were taken fo r iso lati on of di ffe rent stages by Percoll density gradient. Different concentrati ons of Percoll (30%, 45 %, 50% and 6S%) were used. The suspended culture was overlaid on 30% Percoll for separation of merozoites. Pellet thus obtained was resuspended in complete media and overlaid on 45 % percoll fo r isolation of sc hi zonts. This process was repeated by overlay ing pellet on 4S % and SO% Percoll gradient to obtain trophozoites (earl y and late stages) and the final pe ll et of 65% Perco ll wit h ring forms. Purity of each form after gradient centrifugation was checked by microscopic examinati on.

Parasite isoiation and cytosol preparation Infected erythrocytes (different stages) were

harves ted by centrifugation (700 g, 10 min, 4°C) and washed twice with phosphate buffer (S mM, pH 8.0). Released paras ites were concentrated by centrifugation ( ISOO g, IS min, 4°C) . Based on the acetyl choline esterase ac tivity I % of the erythrocytes copurified with the paras ites fractions on the Percoll and the overall purity of the parasites recovered was based on the NAOP-glutamate dehydrogenase activity as previously described ' 6

. The isolated paras ites were washed twice and resuspended in lysis buffer (20 mM

Tris, pH 7.S, 10 mM OTT, 2 mM EOTA, 2 mM

EGTA, I rnM PMSF, S /lg/ml leupeptin and 2S0 rnM

sucrose). The paras ites were sonicated fo r I min with alternative cool ing and centri fuged at 150,000 g fo r 30 min to separate cytoso lic frac ti on in a Beckman ultracentrifuge. Cytoso l was further centri fuged at 100,000 g for 60 min and stored fo r the assay of PTK.

PTK assay The PTK acti vity was measured usin g a sli ghtly

modified non rad ioact ive meth od '7 Brie fl y, the standard kinase assay mi xture contains assay buffe r, peptide substrate [111M (biotin EGPWLEEEEEA YGWMOF - ami de)], ATP ( I 111M ), Mg2+ ( 10 mM) ail e! the enzyme sample in appropriate diluti on's . Mini ma l sample vo lume \vas 50 ~ll.

Reactions were initi ated ill tri pli cate by add ition of the paras ite ex trac t. The reacti on mixture was incubated at 37°C fo r varying peri ods of ti me to obtain max imum ac ti vit y. It was observed th at maximum acti vity was obtai ned at 60 min incubati on. Thus in all PTK assays the samples were incubated at 3r C for 60 mi n. After the enzyme was quenched by a specifi c inhibi tor (3 mM piceatann ol, 120 11M

EDT A) phosphorylated and dephosphory lated substrate was immobili zed by binding to a streptavidi n coated microtitre plate and the reaction mi xture was was heci out. The fract ion of the phosphorylated substrate I S determi ned immunochemicall y with ABTS (a registered trade mark of Boehri nger Man nheim) via a highl y spec ific antiphosphotyrosine monoc lonal antibody direc tl y conjugated to perox idase . The absorbance was measured at 405 nm (reference wave length approx. 490 nm) using a microtitre pl ate reader (EUSA reader). The protein concentrati on was measured according to the method of Lowry'~.

PTK activity ill injected all d 1Il/.i/ ~lecred eryllJ m cyle.\' In experiments comparing the in fected and

uninfected erythrocytes, cultures were enriched fo r paras itized cell s by load ing 2 ml of infec ted ce lls at 10 % hematocrit of 6S% Percoll ill RPM1 1640. The tubes were centri fuged in a table top centri fuge at 2000 rpm for 20 min . The layer of the infected erythrocytes at the top of Perco ll was washed twice with RPMI 1640 and subj ected to lys is procedure as described for the isolated parasites.

Inhibitor studies PTK activity was inhibited with piceatannol, a

specific inhibitor of PTK. Cytosol of the trophozo ites

SHARMA & MISHRA: INHIBITION OF PROTEIN TYROSINE KINASE BY CHLOROQ UINE 301

of CQ-S strain was used to assay PTK activity . CQ was dissolved in water and appropriate dilutions

(0-300 flM) were used for inhibition of PTK activity .

Data analysis Analysis of data was performed using one way

analysis of variance. Two variables were compared by the student's t-test.

Results PTK activity was found to be distributed in cytosol

and membrane fractions of the parasite l9. As the

activity was predominantly cytosolic in different stages of parasite maturation, cytosol of the trophozoite stage was used as a source of the enzyme for subsequent experiment. The non-radioactive PTK assay provides a fast, specific and reproducible method for the quantitative and comparative determination of the enzyme activity from various biological samples. This assay of PTK has been found to be specific since there is no interference with serine and threonine kinases because the antiphosphotyrosine monoclonal antibody does not cross react with phosphoserine and phosphothreonine.

Expression oj PTK activity The relative PTK activity in cytosol of the CQ-S

and CQ-R strains varied from one stage to another in P.Jalciparum. As the parasite matures from ring stage to the schizont, PTK activity was found to be increased (p < 0.p5), however, the activity was found to be lower in the merozoite stage (Table I ). Our results show that piceatannol and CQ both inhibit the activity of these kinases in the later stages of parasite maturation, i.e trophozoite and schizont. In CQ-R strains, however, piceatannol was found to be

significantly inhibitory to the actI vIt y of PTK whereas CQ does not have a significant impact on the status of PTK in all the stages of maturati on. To determine any contribution from the contaminating erythrocytes and the leukocytes, we have al so assayed the un'infected and infected e rythrocyte preparations in which the parasites were grown for the PTK activity. Based on the number of contaminating erythrocytes in the free paras ites fracti ons (determined microscopically) only about 0.02% of the total PTK activity in the free parasites could be accounted for the contaminating erythrocytes and leukocytes. To also assess the amount of the paras ite kinase activity relative to the host PTK actvity, total PTK activity of the infected and uninfec ted erythrocytes were compared. Of the total PTK activity about 90% of the activity was of the parasite origin and only about 10 % could be attributed to the host enzyme activity (Data not shown) .

Inhibition oj PTK activity ill vitro The synthetic samples of piceatannol and CQ were

tested for their ability to inhibit the activity of cytosolic PTK of trophozoite stage of P. falciparwl1. Piceatannol inhibited the PTK activity in a dose depenc:Ient manner in both the CQ-S strains of P. Jalciparum (Fig. I). The phosphory lation of peptide substrate on the tyrosine residue was a lso found to be inhibited in a dose dependent manner by increasing concentration of CQ in CQ-S strain (Fig 2) . However, in CQ-R strain the activity was on ly marginally inhibited even by the maximum concentration of CQ used (Fig. 2).

Kinetics oj CQ inhibition In order to examine the mechanism of interaction

Table I-Distribution and inhibition of cytosolic protein tyrosine kinase activity in developmental stages of Pla.l'/l w tiilllll /a ici/w/'I II II

Protein tyrosine kinase activity

(p moles phosphate incorporatedlmg protein/min)

Chloroquine Sensitive strain (FlB-D9) Chloroquine Resistant strain (FJB-D4)

Stages Status Chloroquine Piceatanno! Status Chloroqlline

Ring

Tropho

Schizont

Mero

4.4 ± 0.4

6.1 ±0.5*

6.6 ± 0.5*

4.3 ± 0. 3

(0. I mM)

3.6 ± 0.3

3.7 ± 0.4

4.0 ±0.3

2.7 ± 0.2

(0.25mM)

3.9 ± 0.3 4.3 ± 0.4

3.8 ± 0.2 5.6 ± 0.4*

4.1 ±0.3 7.2 ± 0.5*

2.7 ±0.2 5.0 ± 0.4

Values are for triplicate assays for 5 independent culture experiments and are expressed as mean ± SEM *p < 0.05

(0. 11llM)

3.9 ± 0.4

5.6 ± 0.5

5.9 ± 0.4

4 .7 ± 0.5

Piceatw/lw/

(0.25 mMJ

3.4 ± 0.2

3.g ± 0.2

4 .0 ± 0.3

2.5 ±O.I

302 INDIAN] BIOCHEM BIOPHYS, VOL. , 36, OCTOBER 1999

::.:: l-

100

n. 20

PICEATANNOL( ~g Iml }

Fig. I-Inhibition of cytosolic PTK" ac tivity by increasing concentrations of piceatanno l in CQ-S strain o f Plasmodium Jalciparum

-l

.?l I-z 8 l) CQ(R}

'If.

>-l-S; i= 40 ':;i ::.:: I-

20 n.

O~--~---L--~ __ ~ ____ L-__ ~ ___ J

~ 100 1~ ~OO 2~ 300

CHLOROQUiNE (~M) 0-0

Fig. 2-Effect of CQ on cytosolic PTK activity of trophozoites in chloroquine sensitive (CQ-S) and resistant (CQ-R) strains of Plasmodium JalciparulIl

of CQ with tyrosine kinases, effect of varying concentrations of CQ on the kinetics of peptide phosphorylation were examined both in presence of peptide substrate and A TP using Lineweaver Burk double reciprocal plot. As shown in Fig. 3A, CQ was found to be a competitive inhibitor of PTK with respect to peptide (K; = 21 f.lM) and a non-competitive inhibitor with respect to A TP (Fig. 3 B) respectively.

Discussion The results of this study indicate that the PTK

activities associated with the CQ-R and CQ-S strains

® 100

8 .0.

6 .0 "> :::::

4 .0

0

I 10

® 8

6 :;

"

0 .2 0.4 0.6 0 .8

I/(PEPTIDE); lItmMI

~ 0.05 0..10. 0..15 0.20

lI (ATP); 1/(}JM)

.0

'0

1.0

Fi g. 3-Kinetic analysis o r the interactions or CQ with thc cytosolie PTK acti vity in trophozoites or chl oroquin e sensiti ve (CQ-S) strains. [Lineweaver Burk doubl e reciprocal plot of (A): increasing concentrations of peptide substrate on the in hibiti on of PTK by CQ. (B): increasing concentrations o r ATP on the inhibition of PTK by CQ. The final concentrati ons o r CQ in the assay mixture were 0 (0----0), 25 (0·--- 0). 50 (e ----e ) and 100 11M (6----6) respective ly. Each point is average (l r tri plicat e determinations.]

of parasites are different in terms of the ir re lation to antimalarial drug CQ. Plasmodium PTK activity was found to be increased « 0 .05) as the parasite matures from ring fonn to schizont, both in CQ-R and CQ-S strains (Table I). Our results further show that piceatannol and CQ both inhibi t the activity of these kinases in the later stages of maturation. Ring stage parasites were not found to be s ignificantl y inhibited by both the inhibitors (Table I) . The occurrence of PTK acti vity among P. falciparulIl strains (CQ-S and CQ-R) and their inhibition by piceatannol and CQ suggest a plausible role and metaboli sm of second

..

SHARMA & MISHRA: INHIBITION OF PROTEIN TYROSINE KINASE BY CHLOROQU INE 303

messengers in the regulation of cell growth and differentiation2

. Red cells infected with malaria parasite can accumulate chloroquine and the intravacuolar concentration can reach upto I mM when the extracellular concentrations were at a level known to inhibit parasite development. It is thus quite likely that parasites have an additional chloroquine concentrating mechanism. Resistant parasites do not have the ability to concentrate drugs to such high levels found in the CQ-S strains presumably keeping the concentration of CQ below those that would ultimately inhibit the enzymes20

. An efflux mechanism thus releases chl oroquine from the resistant parasites 40-50 times faster than that of the sensitive paras ites21

. Protein kinases are good chemotherapeutic targets in that they are often involved in the regulatory aspects of cellular physiology. The cdk 2 kinase, a p34 like protein kinase involved in the control of Gi to S phase transition of eucaryotic cell have been recently cloned and sequenced22

. Another protein kinase cDNA (casein kinase 1) of P. Jalciparum have been cloned, sequenced and expressed in bacteria. The activity showed a stage specific expression in the parasite, suggesting transcriptional as well as post transcriptional regulations3. Recently tyrosine kinase activity has been shown to be strongly upregulated by IFN-y and shown to be involved in the growth process23 . Our results on the inhibition of protein tyrosine kinase by antimalarial drug chloroquine opens up new drug targets for the killing of the malarial parasite.

A protein kinase activity and major cytoplasmic phosphoproteins of the malarial parasite have been described previousl/·24

. Evidences have also been presented that a 46 kDa phosphoprotein is a protein kinase ll . Moreover, the protein designated as ppg is 36, 34 and 32 kDa in P. Jalciparul17, P. berghei and P. chabaudi respectively. These results imply that 34 kDa phosphoprotein may be a protein kinase capable of phosphorylating proteins or may act as an activator of a protein kinase. It is therefore, suggested that during the course of maturation thi s 34 kDa protein might be acting as an activator of PTK activity. As the parasite schizont gets ready to release fresh merozoites it is quite probable that thi s protein factor may undergo proteolysis leading to the production of smaller fragments of 19 kDa and 15 kDa. Both these fractions have been demonstrated not to be an activator of protein kinase activity2. We may

therefore speculate that our observati on of a lower activity in merozoites may be having a co relati on with this proteolytic ac ti vity. The proteo lyti c processing of MSP-l and its potential role in invasion25

.26 is an example of developmentall y

regulated proteolys is. In addition chymotrypsin like protease acti vity has been implicated in the release of merozoites from schizonts and proteases ex pressed specifically during the schizont stage and merozo ite

. h b d 'b d ry7.ry? D' I stage ave een escn e - - . UrIng t le same period the protein kinase act ivity decreases suggesting that the proteolytic processing of thi s 34 kDa protein may be a phys iologica l regulator of

. • . ry kll1ase actlvlty-.

The kinetic, catalytic and physical properties of the membrane bound and cytosolic PTK ha ve been found to be indistinguishable ; hence trophozoite cytosol of the CQ-S P. Jalciparum strain have been taken as a source of PTK for characterization of the enzyme and studying its kinetics. To examine the mechani sm of interaction of CQ with PTK, the effect of varyin g concentrations of inhibitor on the kineti cs of peptide phosphorylation was studied. As shown in Fig. 3A and B, CQ has been found to be a competit ive inhibitor of PTK with respect to peptide substrate used. (K j = 21 11M) and non-compet iti ve with respect to ATP. Our data thus indicate that CQ inhibits the activity by binding to the protein substrate binding sites. The mode of action is similar to that observed for synthetic analogue (tryphostins/o, which share a structural similarity to tyrosine. Our results are in agreement with those of Geahlen and' McLaughlin " who showed that piceatannol can inhibit PTK in intact cancerous cells. The identification of CQ as an inhibitor of PTK provides important informati on regarding the type of compounds that can interact at the protein substrate binding sites of PTKs.

Protein kinases are the principal signal enzymes enabling cell to cell communication , growth regulation and differentiation ' l. Although the exact function of these protein kinase:, durin g the matunition of the paras ite is not known at thi s time. it is apparent from our study tha t PTK may play important roles in the maturati on and deve lopment of the parasite through phosphorylat ion of spec ific proteins. Growing resistance of P. /a/ciparuJI1 to CQ32.33 and other antimalarials has led to the search of other antimalarials including natural plant

d 14 15 I . d . I h pro ucts' .. . n our prevIOus stu les we lave sown that piceatannol, an antileukemic principle in the seed

304 INDIAN J BIOCHEM BIOPHYS, VOL., 36, OCTOBER 1999

of Euphoria lagascae is inhibitory to P. Jalciparum PTKI9

. Determination and expression of this developmentally regulated PTK in CQ-R strains and its inhibition by piceatannol would allow us to co­ordinate studies on various aspects of development of new drugs for CQ-R malaria.

Acknowledgement We are grateful to Dr V P Sharma, former Director,

Malaria Research Centre, for the encouragement and advice. We thank Dr (Mrs) Sukla Biswas for supplying Plasmodium Jalciparum strains. Authors are also thankful to Mr Bhanu Arya and Mrs Poonam Gupta for their technical assistance.

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