Molecular and Biochemical Parasitology 106 (2000) 1–10

Mini Review

Metabolic control analysis of glycolysis in trypanosomes asan approach to improve selectivity and effectiveness of

drugs

Barbara M. Bakker a, Hans V. Westerhoff b,c, Fred R. Opperdoes d,e,Paul A.M. Michels d,e,*

a Kluy6er Institute of Biotechnology, Delft Uni6ersity of Technology, Delft, The Netherlandsb Molecular Cell Physiology, BioCentrum Amsterdam, Vrije Uni6ersiteit, Amsterdam, The Netherlands

c E.C. Slater Institute, BioCentrum Amsterdam, Uni6ersity of Amsterdam, Amsterdam, The Netherlandsd Research Unit for Tropical Diseases, Christian de Du6e Institute of Cellular Pathology, Uni6ersite Catholique de Lou6ain,

ICP-TROP 74.39, A6enue Hippocrate 74, B-1200 Brussels, Belgiume Laboratory of Biochemistry, Uni6ersite Catholique de Lou6ain, ICP-TROP 74.39, A6enue Hippocrate 74,

B-1200 Brussels, Belgium

Received 2 August 1999; received in revised form 11 October 1999; accepted 11 October 1999

Abstract

Glycolysis is the only ATP-generating process in bloodstream form trypanosomes and is therefore a promising drugtarget. Inhibitors which decrease significantly the glycolytic flux will kill the parasites. Both computer simulation andexperimental studies of glycolysis in bloodstream form Trypanosoma brucei indicated that the control of the glycolyticflux is shared by several steps in the pathway. The results of these analyses provide quantitative information aboutthe prospects of decreasing the flux by inhibition of any individual enzyme. The plasma membrane glucosetransporter appears the most promising target from this perspective, followed by aldolase, glyceraldehyde-3-phos-phate dehydrogenase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase. Non-competitive or irre-versible inhibitors would be most effective, but it is argued that potent competitive inhibitors can be suitable,provided that the concentration of the competing substrate cannot increase unrestrictedly. Such is the case forinhibitors that compete with coenzymes or with blood glucose. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Trypanosoma brucei ; Glycolysis; Metabolic control analysis; Inhibitors; Drugs; Review

www.elsevier.com/locate/parasitology

Abbre6iations: IC50, concentration of inhibitor which gives 50% inhibition; SHAM, salicylhydroxamic acid.* Corresponding author. Tel.: +32-2-7647473; fax: +32-2-7626853.E-mail address: michels@trop.ucl.ac.be (P.A.M. Michels)

0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S 0166 -6851 (99 )00197 -8

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–102

1. Introduction

In humans, African trypanosomiasis or sleepingsickness is caused by two subspecies of Try-panosoma brucei. A usually chronic form, pre-dominant in West Africa is due to infection by T.brucei gambiense and a more acute, East Africanform is caused by T. brucei rhodesiense. Bothforms of the disease are fatal if left untreated.Treatment is still very unsatisfactory [1,2]. Pen-tamidine, introduced in 1937, and suramin, ad-ministered since 1922, are used for the treatmentof early-stage infections since they do not readilyenter the cerebrospinal fluid. Late-stage diseasewith central nervous involvement is treated withdrugs such as melarsoprol, a trivalent organo-ar-senical introduced in 1949, and eflornithine (D,L-a-difluoromethylornithine), the only drug thatbecame available in the recent past, in 1990. How-ever, these drugs cannot be used for all cases. Asprimary resistance of T. brucei rhodesiense againstpentamidine has been observed, this drug is usedonly against T. brucei gambiense. Eflornithine isineffective against T. brucei rhodesiense. In recentyears serious problems in treatment of the diseasehave been encountered due to the emergence ofresistance of trypanosomes against melarsoprol.Furthermore, pentamidine, suramin and particu-larly melarsoprol have serious side effects on thehost. Eflornithine requires a hospital setting and isoften unaffordable because of its high costs.Therefore, new drugs against sleeping sickness arein great demand.

The currently available drugs have all beenfound empirically, and their mode of action re-mains largely unknown. In the last two decades,molecular approaches have been introduced todefine targets for drugs in trypanosomes and todesign new drugs [3,4]. Glycolysis has been per-ceived as a potentially excellent target for thedesign of new anti-trypanosome drugs [1,5]. Theglycolytic breakdown of glucose to pyruvate is theonly ATP supply for African trypanosomes livingin the mammalian bloodstream. Inhibition of gly-colysis therefore leads to rapid death of theseparasites. Moreover, the long evolutionary dis-tance between trypanosomes and humans [6], andthe unusual organization of the pathway in the

parasites [7,8] have endowed the trypanosomeenzymes with distinct properties that might beexploited in a structure or catalytic-mechanismbased design of selective inhibitors [3,9].

2. Glycolysis in T. brucei

In bloodstream form T. brucei, the seven gly-colytic enzymes converting glucose to 3-phospho-glycerate are present in peroxisome-like organellescalled glycosomes (Fig. 1) [7,8]. The glycosomalmembrane is hardly permeable to metabolites[10,11]. The NADH produced by glyceraldehyde-3-phosphate dehydrogenase in glycolysis is reoxi-dized by molecular oxygen via a mitochondrialglycerol-3-phosphate oxidase to which the elec-trons are transferred through a glycosomal glyc-erol-3-phosphate dehydrogenase and a putativeglycerol-3-phosphate: dihydroxyacetone phos-phate transporter across the glycosomal mem-brane. As a result of this organization, theintraglycosomal milieu is in redox and ATP bal-ance. The net ATP synthesis that is coupled toglycolysis occurs in the cytosol, in the reactioncatalysed by pyruvate kinase. Under anaerobicconditions, glycerol-3-phosphate is converted intoglycerol with concomitant synthesis of ATP viathe reverse action of a glycosomal glycerol kinase.The glycosomal ATP and NAD balance are thusmaintained also under anaerobiosis. The differ-ences with aerobiosis are that only one moleculeeach of glycerol and pyruvate are produced permolecule of glucose, instead of two molecules ofpyruvate, and that net ATP production is lowerby one, i.e. only one per molecule of glucose.

3. Inhibition of trypanosome glycolytic enzymes

Bloodstream form trypanosomes starved at37°C without glucose, or incubated with an in-hibitor of the plasma membrane glucose trans-porter such as phloretin, die within severalminutes [12]. The parasites were also killed whenincubated with pentalenolactone, a specific in-hibitor of glyceraldehyde-3-phosphate dehydroge-nase [13]. Also bromopyruvate, which was shown

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–10 3

to inhibit glyceraldehyde-3-phosphate dehydroge-nase [14], and specific inhibitors of pyruvate effluxsuch as a-cyanocinnamic acid derivatives, causelysis of these parasites [15]. However, none ofthese inhibitors is selective for parasite enzymes.A well-known manner to inhibit selectively try-panosome glycolysis in vitro, resulting in death ofthe parasites, is the inhibition of glycerol-3-phos-phate oxidase by salicylhydroxamic acid (SHAM)

whilst preventing the reversal of the glycerol ki-nase reaction by adding glycerol as well [16]. Thiscombination also has a trypanocidal effect ininfected rats [17]. However, the required concen-trations of SHAM (1 mM) are unpractically high,and those of glycerol (5 mM) are toxic for thehost. Recently, an alternative, highly specific,non-competitive glycerol-3-phosphate oxidase in-hibitor has been identified, i.e. the antibiotic asco-

Fig. 1. The stoichiometric scheme of glycolysis in bloodstream form T. brucei. (1) Hexokinase; (2) glucose-6-phosphate isomerase;(3) phosphofructokinase; (4) aldolase; (5) triosephosphate isomerase; (6) glyceraldehyde-3-phosphate dehydrogenase; (7) phospho-glycerate kinase; (8) glycerol-3-phosphate dehydrogenase; (9) glycerol kinase; (10) phosphoglycerate mutase; (11) enolase; (12)pyruvate kinase; (13) glycerol-3-phosphate oxidase. Substrate and metabolite transporters in membranes are indicated by circles; theinvolvement of specific carrier molecules in metabolite transport across the glycosomal membrane has not yet been establishedunambiguously. Abbreviations: 1,3-BPGA, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; G-3-P, glyceraldehyde-3-phosphate; Gly-3-P, glycerol-3-phosphate; PEP, phosphoenolpyruvate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate.

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–104

Fig. 2. Three possible correlations between the steady stateflux through a metabolic pathway and the activity of one of itsenzymes. Flux and activity are expressed as a percentage ofuninhibited flux and activity at the condition under which theflux control coefficient is evaluated.

lated entities, but interact through the concentra-tions of metabolites. Consequently, the primaryinhibition of an enzyme in the glycolytic pathwaywill be counteracted by an increase of the concen-tration of its substrates and/or a decrease of theconcentrations of its products. After relaxation toa new steady state the glycolytic flux is thus oftenmuch less inhibited than the enzyme was initially.To understand such physiological effects in aquantitative manner, metabolic control analysishas been developed [23,24]. In this section theprinciples of metabolic control analysis will beoutlined (For more details, see the comprehensivereviews [25–27], or the textbook [28]). Further-more, it will be discussed how this method canhelp in choosing target enzymes for trypanocidaldrugs and in improving the selectivity of suchdrugs.

Fig. 2 shows three possible correlations betweena steady state metabolic flux J, e.g. the glycolyticflux, and the activity of one of its enzymes. Theflux control coefficient Ci

J of an enzyme i isdefined as the relative change of the flux J dividedby the small, relative change of the enzyme activ-ity 6i that is responsible for this change in flux, atconstant activities of all other enzymes. In mathe-matical terms [29]:

CiJ=

(d ln J/dp)ss

( ln 6i/(p=6

J·(dJ/dp)ss

((6i/(p)(1)

Here p is a parameter, for instance an inhibitorconcentration, which specifically affects the activ-ity of enzyme i. The symbol d represents a totalderivative, i.e. the effect of a parameter modula-tion on the steady state (ss) flux in the intactsystem is monitored. The symbol ( represents apartial derivative, where the effect of the parame-ter modulation on the enzyme rate ((6i/(p) isanalysed at constant concentrations of all othersubstrates/effectors of the enzyme without allow-ing relaxation to a new steady state. This is simi-lar to in vitro enzyme kinetics. Graphically, theflux control coefficients are the slopes to thecurves in Fig. 2, at 100% activity. When an en-zyme has a flux control coefficient of 1, it is trulyrate limiting (curve 1 in Fig. 2). If it has a fluxcontrol coefficient of 0, it is not limiting at all

furanone (Ki�2 nM) [18], but no effective andselective glycerol kinase inhibitors are available asyet.

Guided by the crystal structures of human andtrypanosomatid glyceraldehyde-3-phosphate de-hydrogenase, adenosine analogues were designedas tight-binding inhibitors to occupy the pocket ofthe parasite enzyme that accomodates the adeno-syl moiety of the NAD+ cofactor. Such com-pounds displayed competitive inhibition, selectivefor trypanosomatid glyceraldehyde-3-phosphatedehydrogenase, with IC50 values as low as 0.3 mM.They inhibit growth of cultured bloodstream formT. brucei, Leishmania promastigotes and T. cruziamastigotes in the low micromolar range, withoutaffecting human fibroblast growth [19,20].

4. Metabolic control analysis

Molecular genetics and structural biology haverevolutionized trypanosome research as they havemany other fields. Either by directly determinedX-ray structures of trypanosome enzymes [21,22],or by making use of homologies with enzymes ofother organisms for which the structure has beendetermined, structure-based drug design or dis-covery at the single enzyme level is now in fullswing [19,20,22]. Enzymes are, however, not iso-

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–10 5

(curve 3 in Fig. 2). Experimentally all valuesbetween 0 and 1 can be found (e.g. curve 2 in Fig.2). An interesting feature of flux control coeffi-cients is expressed by the summation theorem: inan ideal pathway the sum of the flux controlcoefficients of all enzymes is 1 [23,24].Mathematically:

%i

CiJ=1 (2)

A consequence of the summation theorem is thatif one enzyme has a high flux control coefficient,then the others must have low control coefficientsand vice versa. Also, the theorem replaces thenotion that total control must reside in a single(rate limiting) step. Although it can be confined toa single step, control is more often distributedamong several, or even all enzymes in a pathway.

The above described theory can help to predictwhich enzymes are the most effective targets foranti-parasitic drugs. Because the control coeffi-cients depend on the kinetics of all the enzymes,the structure of the metabolic network and theconditions under which they are studied, they maydiffer between organisms. Therefore, it is possiblethat the distribution of flux control in try-panosomes differs markedly from that in theirhost, i.e. in the parasite most of control mayreside in other enzymes of the pathway than inthe host. We have proposed that the selectivity ofglycolytic inhibitors is enhanced, if they affect anenzyme with a high flux control coefficient in theparasite, but a low flux control coefficient in thehost [30]. Determining the flux control distribu-tion in the parasite in comparison to that in thehost is called differential control analysis.

5. Enhancing drug selectivity by differentialcontrol analysis

A little over half the above described strategyhas been applied to trypanosomiasis by a com-bined theoretical and experimental approach. Theflux control coefficients of the glycolytic enzymesin bloodstream form T. brucei have been mea-sured and/or calculated. In order to identifypromising drug targets, the results were compared

to what is known about control of mammalianglycolysis. A detailed control analysis of the latteris still lacking, however.

5.1. Model construction and 6alidation

To calculate the flux control coefficients of theglycolytic enzymes in bloodstream form T. brucei,a detailed kinetic model of this pathway wasconstructed [31]. It contained measured kineticsfor most of the T. brucei enzymes. Also the com-partmentation of glycolysis was included in themodel. The glycosomal membrane is assumed tobe impermeable for metabolites, except to thosethat need to be transported to allow glycolysis toproceed (Fig. 1). This is supported by the resultsof pulse-labelling studies [10]. For lack of kineticinformation, it was assumed that the metabolitesthat are transported across the glycosomal mem-brane, such as glucose and 3-phosphoglycerate(Fig. 1), fully equilibrate across this membrane.Because this is an assumption, any control exertedby the glycosomal metabolite transporters willhave been missed in the analysis.

The model predicted the steady state glycolyticflux and the steady state metabolite concentra-tions as a function of the enzyme kinetic parame-ters and of the concentrations of metabolites inthe host blood [31]. To validate the model, itspredictions were compared to fluxes and metabo-lite concentrations as measured in bloodstreamform T. brucei [31]. The model mimicked a shiftfrom aerobic conditions to anaerobic conditionsreasonably well. Also the measured biphasic in-crease of intracellular glucose as a function of anincreasing extracellular glucose concentration wasaccurately predicted. More problematic was thesensitivity of the anaerobic glycolytic flux to glyc-erol. Since the standard free energy of the glycerolkinase reaction favours production of glycerol-3-phosphate, glycerol inhibits glycolysis underanaerobic conditions [16]. Qualitatively this wasreproduced by model calculations, but the glyc-erol concentration required to inhibit glycolysiswas some 10-fold lower in the model than underexperimental conditions [32]. Therefore, the modelcan only be used to predict physiological be-haviour under conditions when the blood glycerol

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–106

concentration is zero or negligibly low. In generalit should be mentioned that it has not been possi-ble to subject the model to stringent tests, espe-cially because uncertainties concerning theglycosomal volume preclude accurate experimen-tal determination of the concentrations of glyco-somal metabolites. As always, models and theiractions should be treated with caution.

5.2. Distribution of flux control in bloodstreamform T. brucei

By modulating the activity (6i) of each enzymein the model independently, the flux control co-efficients of the glycolytic enzymes and the in-cluded transporters were calculated [32]. At 5 mMof extracellular glucose, the normal concentrationin blood, most of the flux control was exerted bythe transport of glucose into the cells (Table 1).This result however, was rather sensitive to the

values of the kinetic parameters of the glucosetransporter. If the glucose transporter was madeslightly more active than had been measured, itlost control in favour of a group of four enzymes:aldolase, glyceraldehyde-3-phosphate dehydroge-nase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase (Table 1). Smallchanges in other parameters, e.g. in the glycoso-mal volume, led to the same shift of control fromglucose transport to the above group of fourenzymes. The other kinases, hexokinase, phospho-fructokinase and pyruvate kinase are oftenthought to be rate limiting steps of glycolysis. Inthe trypanosome model they did not assume anycontrol unless they were inhibited strongly. Weconcluded that the trypanosome model yieldsthree alternative possibilities: the glycolytic flux iscontrolled either by the glucose transporter or bya group of four enzymes, or by all five proteins[32]. To decide which of these predictions is true,modelling alone was not sufficient, but additionalmeasurements were required.

To measure the flux control coefficient of theglucose transporter, its activity was modulated bythe transport inhibitor phloretin. The inhibitionof the steady state flux (d ln J/dp in Eq. (1)) andthe primary inhibition of the transporter itself(( ln 6i/(p in Eq. (1)) were monitored indepen-dently [33]. This led to an estimation of the fluxcontrol coefficient of the transporter between 0.3and 0.5 at a normal blood glucose concentration(5 mM). At a low extracellular glucose concentra-tion (0.4 mM), the transporter assumed all control(flux control coefficient 1.290.2) [33], in accor-dance with the model prediction [32].

The above result shows that under physiologi-cal conditions glucose transport is one of the stepscontrolling the glycolytic flux in T. brucei, but itshares control with other steps. From the mod-elling work, we concluded that these other stepsare aldolase, glyceraldehyde-3-phosphate dehy-drogenase, phosphoglycerate kinase and glycerol-3-phosphate dehydrogenase. If they share thecontrol more or less equally, their individual fluxcontrol coefficients are 0.15 approximately (Eq.(2)), which is substantial, but lower than that ofthe glucose transporter.

Table 1Flux control coefficients of glycolysis in bloodstream form T.bruceia

Vmaxtransport=106 Vmax

transport=143.4nmol min−1 (mgnmol min−1 (mg

protein)−1 protein)−1

0.90Glucose transport 0.080.02Hexokinase 0.05

0.01Phosphofructoki- 0.00nase

0.280.02Aldolase0.23Glyceraldehyde-3- 0.02

phosphate de-hydrogenase

0.02Phosphoglycerate 0.15kinase

Pyruvate kinase 0.00 0.01Pyruvate trans- 0.000.00

port0.02Glycerol-3-phos- 0.17

phate dehydro-genase

Glycerol-3-phos- 0.00 0.02phate oxidase

0.000.00ATP utilization

a Extracellular glucose concentration was 5 mM and theVmax of glucose transport was either the measured value [106.2nmol min−1 (mg protein−1)] or increased by 35%. Data from[32].

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–10 7

5.3. Identification of drug targets from theperspecti6e of metabolic control analysis

By their nature of being derivatives, flux con-trol coefficients can only give precise, quantitativeinformation about small modulations of flux. Tokill trypanosomes, however, a substantial inhibi-tion of glycolytic flux may be required. Howstrong this inhibition should be is not knownexactly, but it may be expected that it is notnecessary to inhibit the glycolytic flux completely,because the mammalian immune system also com-bats the parasites. The extent to which each indi-vidual enzyme should be inhibited by anon-competitive inhibitor to obtain a 50% inhibi-tion of the glycolytic flux was calculated. It turnedout that the hierarchy of effectiveness of thetargets parallelled the hierarchy of flux controlcoefficients [32]. Glucose transport, the step withthe highest flux control coefficient, needed to beinhibited by only 51% to obtain a 50% flux inhibi-tion. Aldolase, glyceraldehyde-3-phosphate dehy-drogenase, phosphoglycerate kinase andglycerol-3-phosphate dehydrogenase with lower,but substantial flux control coefficients, were in-termediate drug targets, requiring to be inhibitedby 76–85% in order to inhibit the flux by 50%.The other kinases (hexokinase, phosphofructoki-nase and pyruvate kinase), which did not partici-pate in the control, had to be inhibited by93–97% to obtain a 50% flux reduction.

To improve drug selectivity, the target enzymeshould not only have a high flux control coeffi-cient in the parasite, but the corresponding en-zyme in the host cells should have a low fluxcontrol coefficient. Schuster and Holzhutter [34]studied the effects of large-scale enzyme deficien-cies in erythrocyte glycolysis. On the basis of adetailed kinetic model of erythrocyte metabolism,they showed that these cells were most susceptibleto deficiencies of hexokinase, phosphofructoki-nase, enolase, pyruvate kinase, 2,3-bisphospho-glycerate phosphatase and glutathione reductase.In contrast, a decrease of the activities of aldolase,glyceraldehyde-3-phosphate dehydrogenase orphosphoglycerate kinase to 5% of the normal levelwas predicted not to cause any clinical symptoms.These results are almost the opposite of what was

found for T. brucei and therefore point to aldo-lase, glyceraldehyde-3-phosphate dehydrogenaseand phosphoglycerate kinase as promising drugtargets. This is equally so for glycerol-3-phosphatedehydrogenase, which in erythrocytes does notplay a major role in the reoxidation of glycolyti-cally produced NADH because of the presence oflactate dehydrogenase. Hexokinase, phosphofruc-tokinase and pyruvate kinase are less interestingtargets from the perspective of metabolic controlanalysis. Glucose transport was not included inthe erythrocyte model, since glucose is believed toequilibrate readily across the erythrocyte plasmamembrane. Implicitly, this should make the trans-porter an important target, were it not for the factthat in other mammalian cell types glucose trans-port may be an important controlling factor. Acomprehensive study, in which glucose transportwas included, dealt with glucose utilization andglycogen synthesis in perfused rat heart [35]. Con-trol in heart cells was variably distributed amongseveral enzymes, depending upon substrateavailability, hormonal stimulation and otherchanges of conditions. Phosphofructokinase wasnot included in the analysis, however, and glycer-aldehyde-3-phosphate dehydrogenase was as-sumed to be at equilibrium. In the absence ofinsulin most control within the glycolytic branchwas shared by the plasma membrane glucosetransporter and hexokinase (together 0.6). Con-trol by the glucose transport step was abolishedby insulin. The perspective for glucose transportas a drug target is therefore quite subtle. At highblood insulin concentrations it may be a verypromising target from the perspective ofmetabolic control analysis. Furthermore, onecould speculate that any undesired inhibition ofthe mammalian glucose transporters might becompensated by an increase of the blood insulinconcentration, while such a hormonal compensa-tion mechanism should not work in try-panosomes. This then might improve theselectivity of drugs directed against glucose trans-port. A concern is that trypanosomes might com-pensate for an inhibition of THT1, thepredominant glucose transporter in the blood-stream form [36], by expressing the insect-formglucose transporter, THT2 [37]. Therefore, any

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–108

drugs directed against glucose transport, shouldrecognize both THT1 and THT2. Also, from astructural point of view glucose transport hasbeen proposed to be a promising drug target [38].

5.4. On different types of inhibitors

Eisenthal and Cornish-Bowden [39] have ar-gued that there is little prospect of killing try-panosomes by depressing their glycolytic flux.Their analysis focused on the effects of competi-tive inhibitors. Many inhibitors are competitive,since they are often designed as substrate ana-logues. The authors showed that competitive in-hibitors do not always work in vivo, since theycan be easily competed away by an increase of theconcentration of the substrate with which theycompete. Indeed this is a serious concern and ithas implications for drug design. However, wewill demonstrate that it is quite well possible toinhibit glycolysis by competitive inhibition, if oneselects appropriate targets.

In their study Eisenthal and Cornish-Bowden[39] did not allow the inhibitor concentration toexceed the inhibition constant ([I]/Ki=1). Theycalculated that a competitive inhibitor of glucosetransport, the step with the highest flux controlcoefficient, could inhibit the steady state flux byonly 20%. This surprising result was, however,due to the fact that the transporter itself wasinhibited by only 20% at [I]/Ki=1 and at 5 mMglucose (its Km for glucose being 2 mM in thisversion of the model). Especially in the case of theglucose transporter there is no reason why in-hibitor concentrations could only be low, sincethe protein is readily accessible from the outside.Also for intracellular enzymes it is an unnecessaryrestriction, since several inhibitors are known toaccumulate to high concentrations in the cell.Moreover, with inhibitors active in the lownanomolar range it should be quite possible toachieve high [I]/Ki values and thus to inhibit theglycolytic flux.

The argument that the cell can readily over-come competitive inhibition by increasing theconcentration of the competing substrate, doesnot always apply. Clearly, it does not hold for theglucose transporter. The concentration of its sub-

strate, the blood glucose, is regulated by the mam-malian host rather than by the parasites.Furthermore, inhibitors which compete withcoenzymes (or other metabolites engaged in moi-ety conservation [31]), should be effective. Theconcentrations of the competing coenzymes donot increase beyond the total concentration of thecoenzyme couple, at least not as a result of theglycolytic activity itself.

When the expected effects of large concentra-tions of competitive inhibitors were calculated onthe basis of the model [40], it was found againthat glucose transport should be the most effectivedrug target. Glyceraldehyde-3-phosphate dehy-drogenase and phosphoglycerate kinase were in-termediate targets, while unpractically highconcentrations of hexokinase inhibitors were re-quired to inhibit the flux. Qualitatively the samehierarchy of targets was found as for non-compet-itive inhibitors. However, the required concentra-tions of inhibitors of glyceraldehyde-3-phosphatedehydrogenase and phosphoglycerate kinase werean order of magnitude larger for competitive in-hibitors, as compared to non-competitiveinhibitors.

The concentrations of coenzymes in the glyco-some (the sum of ATP, ADP and AMP and thesum of NADH and NAD+) are uncertainparameters of the model. Because the glycosomesoccupy only 4% of the cellular volume [41] andthese coenzymes occur in the cytosol as well, theirglycosomal concentrations are difficult to mea-sure. Initially we made rather high estimates oftheir concentrations (ATP+ADP+AMP=6mM and NADH+NAD+=4 mM), but theymay be lower and in that case lower concentra-tions of competitive inhibitors are needed. Whenthe coenzyme concentrations were decreased to0.5 mM, the effective concentrations of competi-tive and non-competitive inhibitors reached thesame order of magnitude [40].

From the above considerations three conclu-sions can be drawn. First, competitive inhibitorsare capable of inhibiting the glycolytic flux ifsufficiently high (but realistic) concentrations areused, although the concentrations of competitiveinhibitors required to inhibit the flux are some-times higher than might be expected on the basis

B.M. Bakker et al. / Molecular and Biochemical Parasitology 106 (2000) 1–10 9

of their effect on isolated enzymes. Secondly, itmay be expected that inhibitors that compete withcoenzymes are relatively successful in vivo, be-cause the concentrations of the competing coen-zymes cannot increase unrestrictedly. Finally, aninhibitor that competes with a coenzyme is mosteffective if the coenzyme concentration is low.This suggests a new strategy, namely to combinesuch an inhibitor with a compound that inhibitsthe biosynthesis of the corresponding coenzyme.Of course, the selectivity of the inhibitor, and itsspecificity for the target enzyme remain the mostessential aspects to consider in drug design orselection. This is particularly important if theinhibitor competes with a ligand that binds tomany enzymes in both the parasite and mam-malian host cell, as is the case for coenzymes.

We conclude that trypanosome glycolysis oranything else can only be a rational drug target ifthe target enzymes and the type of inhibitors arenot only chosen in view of their activities andeffects in isolation, but also in view of the controlthe inhibited component exerts on an essentialflux.

Acknowledgements

This work was supported by the NetherlandsOrganization for Scientific Research (NWO) andthe Belgian ‘Fonds de la Recherche ScientifiqueMedicale’ (FRSM). The authors would like tothank Dr Michael Gelb (University of Washing-ton, Seattle) for stimulating discussions.

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