Review

Drugging the Plasmodium kinome: thebenefits of academia–industry synergyDidier Leroy1 and Christian Doerig2

1 Merck-Serono International S.A., Geneva Research Center, 9, Chemin des Mines, Case postale 54, CH-1211 Geneve 20,

Switzerland2 INSERM U609, Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, 120 University Place,

Glasgow, G12 8TA, UK

Malaria remains a major killer in many parts of the world.Recently, the development of nonprofit organisationsaimed at fighting this deadly scourge incited academicand industrial scientists to merge their expertise in drug-target validation and lead discovery. Expectations areclear: identification and characterisation of new mol-ecules showing high efficacy, low toxicity and littlepropensity to induce resistance in the parasite. In thiscontext, protein kinase inhibitors represent an attractivepossibility. Here, we compare traditional target-baseddrug-discovery approaches with innovative exploratorypaths (parallel screening, cell-based assays, integratedsystems biology and allosteric inhibition) and discussthe benefits of acadaemia–industry cooperation. Earlycharacterisation of distribution, metabolism, pharmaco-kinetic (DMPK) and toxicology parameters are con-sidered as well.

IntroductionFour to eight thousand people, most of them children insub-Saharan Africa, die every day from malaria. Themortality and morbidity burden inflicted by this diseaserepresents a serious hindrance to the socioeconomic de-velopment of afflicted countries [1]. The spread of drugresistance in Plasmodium falciparum, the parasitic proto-zoan responsible for the vast majority of lethal cases ofmalaria, is a cause for grave concernwith respect to diseasecontrol and renders the development of novel chemother-apeutic agents an urgent task [2].

P. falciparum infection of the human host is initiated byinjection of sporozoites into the bloodstream by an infectedAnophelesmosquito. The sporozoites rapidly gain access tothe liver and invade hepatocytes, where asymptomaticasexualmultiplication (exoerythrocytic schizogony) occurs,leading to the production of several thousand merozoites.These are released into the bloodstream and invade eryth-rocytes that become the site of another round of asexualmultiplication, producing 8 to 24 new merozoites. Thisphase of the infection (erythrocytic schizogony) is respon-sible for malaria pathogenesis and therefore is the targetfor most compounds in the antimalarial chemotherapeuticarsenal. Some of the merozoites, upon red blood cell inva-sion, arrest their cell cycle and differentiate into male orfemale gametocytes instead of initiating a new round ofschizogony. Ingestion of male and female gametocytes by a

Corresponding authors: Leroy, D. (didier.leroy@merckserono.net); Doerig, C.(cdoer001@udcf.gla.ac.uk).

0165-6147/$ – see front matter � 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2008.

mosquito is required for transmission into the insectvector, in the midgut of which these cells mature into maleand female gametes. These gametes then fuse into azygote, the only diploid stage in the parasite’s life cycle.Meiotic reduction occurs in the ookinete, the motile forminto which the zygote rapidly develops. After crossing themidgut epithelium, the ookinete undergoes further devel-opment into an oocyst, the site of intense asexual multi-plication producing several thousands of sporozoites thatthen accumulate in the mosquito’s salivary glands andbecome ready to be injected into a vertebrate host duringa subsequent blood meal [see the Malaria FoundationInternational website (http://www.malaria.org) for generalinformation on malaria].

Modern drug discovery relies on a variety of complemen-tary strategies to optimise identification rates of potentcompounds and subsequent qualification of hit and leadmolecules. The latest breakthroughs in genomic, proteomicand metabolomic studies have been applied fairly system-atically to identify human targets in major disorders suchas cancer and neurodegenerative or inflammation-baseddiseases and now are available for identifying parasite-specific targets (Figure 1). The sequencing of the P. falci-parum genome was a major milestone in this respect [3,4].

Among many drug-target classes, protein kinases (PKs)are considered as particularly attractive [5] for severalreasons. First, the catalysis mechanism and overall struc-ture of PKs are conserved, and it is well established thatsmall molecules can bind to their catalytic cleft [6]. Second,reversible protein phosphorylation is one of the mostimportant and pleiotropic modes of regulation of cell physi-ology and is a major mediator in the control of cell pro-liferation, differentiation, migration and homeostasis. Inthe context of drug discovery for diseases such as cancer,diabetes, inflammation and neurodegenerative disorders,most pharmaceutical and biotechnology companies runactive programs aimed at identifying novel PKs as poten-tial drug targets. As a result, many drug candidates tar-geting PKs are in development, and some inhibitors of PKfunction are already on the market (reviewed in Ref. [7]).Third, the development of the analogue-sensitive kinaseallele (ASKA) strategy, also known as the ‘chemicalgenetics’ approach [8–10], allows in vivo validation ofspecific PKs as potential drug targets. ASKA technologyis based on an engineered ATP binding site in the kinase ofinterest that enables it to accommodate bulky inhibitorsthat do not affect wild-type PKs (selected Plasmodium PKs

02.005 241

Figure 1. Classical and new concepts in antimalarial drug discovery. Plasmodium contains various PKs whose expression and function in the life cycle can be assessed

experimentally (i.e. via genomics, transcriptomics and reverse genetics). Similarly at the protein level, the presence, integrity and activity of PKs along this cycle (assessed

via proteomics and phosphoproteomics), as well as the proteasome-mediated degradation of ubiquitinated phosphoproteins (metabolomics), also can be used to discover

or better understand the roles that PKs play in live parasites. Structural studies (i.e. X-Ray, RMN) and databases or phylogenetic approaches are complementary

investigations that support the kinome-directed drug-discovery strategy. Finally, direct pharmacological validation is achievable thanks to well-characterised PK inhibitors

whose low specificity across species might allow to bridge human and plasmodial targets. Recently, erythrocytic PKs and some of their upstream activating proteins

(GPCRs) were shown to participate actively in a parasite’s invasion process. This opened a new avenue in the antiparasitic research field in which new small molecules like

GPCR antagonists or human PK inhibitors are expected to become promising drug candidates. The effect of reference molecules on the infection of red blood cells and the

subsequent change of their shape could be measured directly by label-free technologies (e.g. bioimpedance) and would provide high-content information related to the

intracellular signalling pathways that are involved.

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carrying such mutations and expressed as active enzymesin E. coli actually do display the expected hypersensitivityto bulky inhibitors; allelic replacement in live parasites isin progress (C. Doerig et al., unpublished).

In this article we first briefly review P. falciparumkinomics as well as the opportunities that divergencesbetween parasite and host PKs offer in the search ofselective inhibitors. Then we consider various drug-discov-ery strategies aimed at identifying kinase inhibitors asantimalarial leads, emphasising the benefits that can bederived from academia–industry cooperation.

Targeting the Plasmodium kinomeThe human kinome comprises approximately 500sequences belonging to the eukaryotic protein kinase(ePK) family (reviewed in Ref. [11]). These sequences areclassified into seven large groups: CK1 (casein kinase 1),CMGC (CDK-, MAPK-, GSK3- and CDK-like), TKL (tyro-sine kinase-like), AGC (PKA, PKG, PKC), CamK (calcium/calmodulin-dependent kinases), STE (PKs acting as reg-ulators of MAPKs, first identified in a genetic screen ofsterile yeast mutants) and TyrK (tyrosine kinases), withthe PKs not fitting in any of these groups being describedas ‘other protein kinases’ (OPKs) [11]. Examination of theP. falciparum kinome, which comprises 85–99 enzymesdepending on the study [12,13], has revealed or confirmedconsiderable divergences between P. falciparum andmam-malian cells at the following levels (reviewed in Ref. [14]).

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(i) P

K families. P. falciparum possesses members ofmost of the established PK groups (e.g. CK1, CMGC,TKL, AGC, CamK and OPKs such as NIMA).However, it lacks TyrKs (like many unicellularorganisms) as well as any clear member of the STEgroup, which includes MAPKKs (mitogen-activatedprotein kinase kinases) and other enzymes acting inthe MAPK cascades. This is surprising because theparasite does express two MAPK homologues, theregulation of which, in view of the absence ofMAPKKs, is not understood at present [15]. Further-more, malaria parasites possess a complement ofcalcium-dependent protein kinases (CDPKs), a familyfound in plants and ciliates but not in mammaliancells [16]. In addition to PKs that clearly belong toestablished groups, the plasmodial kinome includesseveral so-called ‘orphan’ enzymes, which do notcluster with any of the groups/families characterisedin other eukaryotes. A striking example is given by anovel group of 21 members that appears to be strictlyspecific to Apicomplexan parasites. All members ofthis new family possess a novel type of conserved C-terminal PK domain called ‘FIKK kinase’ and anonconserved N-terminal region of unknown func-tion, although it contains a peptide directing theprotein to the host erythrocyte [17], a location thathas been experimentally confirmed for somemembersof the family [18].

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(ii) I

ndividual enzymes. Most parasite PKs that clearlycluster within known families neverthelessdisplay atypical characteristics, e.g. nonconservedregulatory sites or presence in the same enzyme ofsubdomains with maximal homology to distinct PKfamilies (‘composite kinases’). Although classifiableinto established families, such ‘semiorphan’ enzymesdo not have identifiable orthologues in mammaliancells.

(iii) O

rganisation of signalling pathways. Together withthe microsporidium Encephalitozoon cuniculi, whichpossess the most reduced genome and kinomedescribed to date in the Eukaryotic kingdom [19],malaria parasites are the only eukaryotes describedso far as lacking the 3-component MAPK pathways[15]. Another striking example of the peculiarity ofsome signalling pathways in P. falciparum is given bythe PKB-related enzyme PfPKB. Whereas mamma-lian PKB is regulated through a pleckstrin-homology(PH) domain and PDK1-dependent phosphorylation,PfPKB was found unexpectedly to be regulated bycalmodulin binding [20] and to play a calcium-dependent role in erythrocyte invasion [21]. Moregenerally, drawing on knowledge gained from othersystems to reconstruct phosphorylation cascadesfrom the sequences of plasmodial PKs is very difficultbecause a majority of these enzymes are orphans orsemiorphans.

The differences between phosphosignalling pathways of

parasites and those of their hosts suggest that specificinhibition of the former can be achieved, a notion thatrecently has been compounded in the case of Plasmodiumby structural data demonstrating exploitable divergencesbetween host and parasite PKs [22,23]. Many plasmodialkinases display activity in vitro as recombinant enzymesand can be used in medium/high throughput screeningoperations. Optimising the success rate of drug discoveryat early stages implies a good biochemical understandingof the target candidate, which facilitates the choice of theadequate molecular assay for high-throughput screening.Guided by 3D-based modeling (or experimentally solvedstructures, when available) of PKs, medicinal chemistrythen allows fast structure–activity relationship (SAR)-based progression of active molecules. In an alternativestrategy phenotypic screening of kinase-directed librarieson parasite cultures directly identifiesmolecules inhibitingparasite growth, whose molecular targets can sub-sequently be elucidated (see below).

Other strategies for antiparasitic chemotherapeuticintervention using interference with phosphosignallingalso can be considered. Plasmodium is an obligate intra-cellular parasite, and essential roles for host-cell signallingin parasite survival have been documented; hence, target-ing host-cell signalling is a possibility (see below). Target-ing transmission by the mosquito vector also can beenvisioned: complex developmental transitions of the para-site’s life cycle occur within the vector [24]; inhibiting PKsinvolved in these processes would block transmission andmight prove useful in the context of disease control(reviewed in Ref. [14]).

Identification of new drugsConventional target-based screening

After publication of the P. falciparum genome [4],databases such as PlasmoDB [3] and the WHO/TDR data-base of targets in neglected diseases (http://TDRtargets.org) have provided research teams with efficient in silicotools for the identification, comparison, evaluation andselection of potential drug targets (Figure 1). A crucialdeterminant for selection of a PK as a drug target is thedemonstration of its essential role in the erythrocyticasexual cycle (curative drug targets) or in the sexual de-velopment (transmission-blocking drug targets). Onestrategy for target validation consists of inactivating thegenomic locus in the Plasmodium genome by homologousrecombination (reviewed in Ref. [25]). Reports on PK geneinactivation in P. berghei [14,24,26–31] and, more recently,P. falciparum [32,33], have been published.

After enzymatic characterisation, assay development,primary screening and data analysis [34–36] (Figure 2),the potency of identified hit molecules is tested directly onlive parasites in the academic laboratory or in specialisednonprofit institutes. The capacity of the molecules to inhi-bit the growth of other parasites such as Trypanosomabrucei and T. cruzi can be evaluated in parallel.

New approaches in malaria drug discovery

Computer-assisted drug design and virtual screening

Several academic laboratories and consortia [e.g. theStructural Genomics Consortium (http://www.thesgc.com)] are now engaged in crystallography of plasmodialtargets, including PKs. Currently, the PfPK5 cyclin-dependent kinase and the ‘orphan’ kinase PfPK7 are theonly Plasmodium PKs whose 3D coordinates are depositedin the Protein Data Bank (PDB entries 1OB3 and 2PML,and 2PMN and 2PMO, respectively). Small molecules werecocrystallised with both kinases. Subtle differences in themode of binding of indirubin-5-sulfonate and purvalanol Bto PfPK5 as compared with human CDK2 explained theobserved differential susceptibility of the two enzymes tothese inhibitors [22]. Although purine-based purvalanol Bis more potent on CDK2 than on PfPK5, both enzymes areinhibited in the nanomolar range by this compound (IC50

values are 6 nM and 130 nM, respectively), whereas thelarger inhibitors, staurosporine and indirubin derivatives,are much less active (by approximately three orders ofmagnitude) against PfPK5 than against CDK2 [22].Together with docking data, these results provide proofof concept that despite overall high levels of sequencesimilarity, the structure of the Plasmodium kinasediffers sufficiently from that of its human homologueCDK2 to allow differential effects of small-moleculeinhibitors. Likewise, structure determination of selectedcompounds bound to PfPK7 has revealed the molecularinteractions that mediate specific inhibitor binding [23].There is little doubt that many similar datasets willbecome available in the next few years.

Based on the 3D structure of the kinase and kinase-inhibitor cocrystals, a computer-assisted drug-design(CADD) strategy can identify the spatial constraints andstabilising interactions requested to support a medicinal-chemistry-based SAR refinement [37]. For example, in

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Figure 2. Discovery of PK inhibitors: a shared effort between academia and industry. Screening of molecules on purified targets or on live parasites are described as in vitro

activities. Pharmacokinetics, ADME, toxicity studies and measurement of potency in disease models (i.e. rodent, monkey), are in vivo activities. Drug discovery is initiated

by either a molecular-based screening in industry (up to 106 small molecules and eventually natural products) or by a phenotypic approach (103–104 small molecules

applied on cultured parasites) performed more frequently in academic institutions. In both cases, assay quality and analysis tools [35,36] are key for success. Modern

technologies enable parallel screening of multiple PKs of the same family at low and high ATP concentrations, allowing the search for ATP-competitive and ATP-

noncompetitive molecules, respectively, at the same time. Qualification of lead molecules requires selectivity studies on other parasites (in academia), determination of

physicochemistry parameters (i.e. solubility, stability, Ka, Kd, analysed in industry) in vitro toxicity [i.e. inhibition of cytochromes P450 (CYP450) and gentoxicity] and

molecular profiling (kinases and receptors panel). For highly potent molecules whose chemical tractability and novelty are clearly both established, a more detailed

molecular profiling is either performed within the industry’s targets portfolio or might be outsourced to contract research organisations. Once lead status is reached, in vivo

characterisation is initiated in industry with DMPK and toxicology studies. Promising drug candidates are then subjected to rodent disease model (academia) and

toxicology determination. Parasitemia clearance in a non-human primate model is the ultimate criterion to qualify a molecule for clinical development.

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cancer research virtual screening allowed the identifi-cation of inhibitors of the erythropoietin-producing hepa-tocellular B2 receptor tyrosine kinase domain (reviewed inRef. [38]). In addition, a ‘target-hopping’ process mightcomplement the selected set of compounds: conserved resi-dues in the catalytic pocket of human PKs that are suscept-ible to a given inhibitor series might help to identifypotential targets in the Plasmodium homologue.

In the absence of actual crystallography data, in silicoapproaches can be implemented by using tridimensionalmodeling of the target based on crystal data from relatedPKs. Waters and colleagues have thus characterised sev-eral classes of compounds including quinolines and oxi-ndoles as selective inhibitors of Pfmrk, a plasmodial cyclin-dependent kinase [39]. As observed with PfPK5 (seeabove), detailed structural analyses suggest that evensubtle differences in amino acid composition within theactive sites are responsible for conferring selectivity ofisoquinoline derivatives for Pfmrk over mammaliankinases [40].

Although highly challenging, the structure-baseddesign of molecules that would disrupt functional inter-actions between subunits of multimeric PKs is applicabletoPlasmodiumPKs. Such structure-based designwas usedsuccessfully, for example, to disrupt human CK2 catalyticand regulatory subunits [41] or the SH3-mediated Grb–Sam68 interation [42]. The Plasmodium kinome comprisesseveral multimeric PKs that are regulated by bindingpartners such as PfCK2 catalytic and regulatory subunits(Z. Holland and C. Doerig, unpublished), PfPKB/CaM[20,21], cyclin/CDKs [23] and PfPKA [43,44].

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Parallel screening of multiple targets of the same class

Contrary to genetic loss-of-function studies,which generallyfocus on a single PK, inhibition of several enzymes withinthe same family could prevent complementation ofindividual PKs by other members of the same PK family.As mentioned above, the plasmodial kinome comprisesenzymes that cluster together on phylogenetic trees suchas CDKs, Neks, TKLs or the CDPK family, which is ofparticular interest because it lacks mammalianhomologues. Even if specific inhibition of one familymember does not translate into a clear inhibition ofparasite growth (possibly because of functionalcomplementation by other members of the same family),targeting several family members simultaneously mightwell have parasitocidal effects. In support of this notion,it was shown recently that one of the genes encoding one ofthe two MAPKs, Pfmap-1, can be inactivated by reversegenetics; however, the pfmap-1� parasite cloneoverexpresses Pfmap-2, the second MAPK, indicating thatfunctional compensation is occurring [32]. Pharmaceuticalcompanies can easily evaluate several PKs in parallel; oncekinetic parametershavebeendetermined, themost suitabletechnology is chosen for all kinases of the same class. Anadded benefit of this approach is that it optimises theeffort:output ratio of the assay development phase.

ATP competition versus allosteric inhibition The vastmajority of PK inhibitors in development consist of ATP-competitive molecules for the following reasons: first, thedesign of chemical libraries is inspired by moleculestargeting the ATP-binding pocket, a well-characterised

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3D space for which molecular docking information isavailable, and second, classical molecular screens rely onlow concentrations of ATP (Km values of 5–20 mM),precluding allosterical regulations of the kinase catalysisefficiency (kcat) or of substrate affinity [3]. Seekingallosteric inhibitors of PKs is now of high interest, andmolecules inhibiting PKs via non/uncompetitive modes ofaction have been developed successfully [45–48]. Small-molecule binding sites have been shown recently to belocated close to the ATP-binding pocket [49]. Molecularmodelling demonstrates that both the ATP molecule andthe inhibitor could coexist in this large pocket in a non-mutually-exclusive manner. One can hypothesise thatother binding sites might exist elsewhere at the surfaceof the kinase and could impact, for instance, on theflexibility of the kinase activation loop, locking it in aninactive conformation.

Phenotypic screening and target(s) identification Thisstrategy consists of screening kinase-inhibitor libraries onlive parasites. Compounds can be selected based on theirsimilarity with known PK inhibitors or on diversity ofpharmacophores, and a phenotypic screening approachallows the identification of effective molecules and theirfurther optimisation. The molecular basis for growthinhibition (i.e. the target) is subsequently identified inorder for chemists to develop new inhibitors based onthe same mode and site of action. For targetidentification, a SAR is first performed to pinpoint allthe sites that can be chemically modified within theactive scaffold without impacting negatively on potencyagainst parasite growth. On the basis of SAR-derivedinformation, an aliphatic chain is introduced as achemical linker to couple the molecule covalently to anactivated matrix and thereby constitute an affinitychromatography phase. Binding proteins are purifiedfrom parasite soluble lysates and identified by massspectrometry [50]. Using this approach, it was foundthat the major parasite-protein binding to purvalanol B,a purine derivative with potent activity againstmammalian cyclin-dependent kinases, and activeagainst P. falciparum growth in vitro [51] was(unexpectedly) the parasite’s Casein kinase 1 [52].Another example of the successful use of this approachis provided by Compound 1, a molecule first identified in acellular screen against Eimeria tenella (a Plasmodium-related apicomplexan parasite that infects chickens andbears considerable impact on the poultry industry). Usinga variety of biochemical techniques, the target wasidentified as the cGMP-dependent protein kinase (PKG)of the parasite. Interestingly, the potency of Compound 1was three orders of magnitude higher on the parasite PKGthan on the host PKG [53]. This was subsequentlyexplained by the observation that a bulky residue(methionine) in the chicken PKG catalytic cleftinterferes with binding of the inhibitor (this residue is athreonine in the parasite enzyme). Transgenic parasitesexpressing a Thr!Met-mutated PKG, a mutation thatrenders the enzyme insensitive to Compound 1, wereresistant to the inhibitor in cellular tests, confirmingthat parasite PKG is indeed the target [54].

Integrated systems The target-based approach offers theadvantage of detecting a direct interaction between smallmolecules and their protein target but does not considerthe physiological environment. However, among the newlyidentified positive molecules, some will be unable to accessthe target. Conversely, cell-based assays and phenotypicscreening lead to the identification of effective moleculeswithout unraveling the molecular mechanism of action. Insome cases, a cell-free system might represent a goodcompromise between the ‘target-based’ and ‘phenotypic-screening’ approaches because it preserves, to some extent,the biochemical context of the target but is not dependenton cell permeability of the compounds. Efficacy of cellpenetration subsequently can be optimised further viaSAR studies and combinatorial chemical synthesis.

The complexity of protein–protein interaction networksand subcellular structures must be taken into accountwhen measuring the activity of inhibitors throughoutthe drug-screening process. One possibility is to rely onwhole-parasite lysates as a source of protein target(s) thatpotentially conserves, at least in part, the physiologicalenvironment of the PK in question. Likewise, intact orga-nelles, vesicles and signalling machineries associated withmembrane fragments better reflect biologically relevanttarget conformation and can be useful in mode-of-actionstudies [55].

Targeting the Plasmodium–erythrocyte interface

Erythrocyte signalling molecules essential for parasitesurvival have been identified (Figure 1). For example,pharmacological and biochemical studies revealed animportant role for the host erythrocyte PKC in P.falciparum infection [56]. More recently, erythrocyte b2-adrenoceptor G-protein-coupled receptors (GPCRs) wereshown to be implicated in Plasmodium infection [57].Eukaryotic GPCRs sit upstream of many phosphorylationcascades (reviewed in Ref. [58]), and molecules targetingthese GPCRs or downstream pathways, identified in thecontext of drug discovery for diseases affecting the Westernworld, additionally could exhibit antimalarial properties(see Concluding remarks).

Plasmodium ‘nonprotein kinases’: additional classes of

potential targets Recently, hexadecyltrimethylammoniumbromide (HDTAB) was shown to be a potent inhibitor of Pfcholine kinase. HDTAB also offered very potentantimalarial activity in vitro against P. falciparumwithout affecting viability of red blood cells. Thissuggests that selectivity of small molecules towards Pfkinases is achievable [59]. Similarly, a series ofpantothenic acid analogs were shown to inhibitpantothenate kinase in a substrate-competitive mode andthe proliferation of intraerythrocytic P. falciparumparasites in vitro. The compounds generally wereselective, inhibiting the proliferation of Jurkat cells onlyat concentrations several fold higher than those requiredfor inhibition of parasite growth [60].

As a third example, PK1 and PK2 are pyruvate kinasesexpressed during the intraerythrocytic stages of P. falci-parum. Phylogenetic analysis revealed that the ‘PK2’ typeof enzyme appears to be confined to Apicomplexans,

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similar to the orphan PKs discussed above [61]. Amino acidresidues of the effector binding sites of PK1 and PK2 differfrom those of the human pyruvate kinases, suggestingagain that significant differences could serve as a rationalefor the design of selective inhibitors against these kinases[61]. The parasite also encodes several nucleotide kinasesthat might represent potential targets [62].

Physical and chemical parameters and early DMPK/

ADME Drug-development projects suffer from highattrition rate, with toxicity issues being a major causefor drug candidate withdrawal. Toxicity, DMPK (drugmetabolism and pharmacokinetics) and ADME(absorption, distribution, metabolism and excretion)studies are essential steps that need to be evaluatedearly in the drug-development process to ensure atimely and efficient detection of possible undesirableside effects or limited efficacy of the lead molecules.

Pharmacokinetic studies investigating stability of thecompounds in physiological environment should be per-formed as soon as submicromolar potencies are reachedwhen compounds are tested on parasites.When PK–inhibi-tor interaction studies are feasible, accurate assessment ofassociation and dissociation parameters (Ka/Kd) are crucialto drive SAR towards scaffold modifications that wouldimprove association rate and decrease dissociation rateconstants.

The drug-resistance issue Preventing drug resistance isone of the biggest challenges in antimalarial drugdiscovery. This issue is worth considering with regard toboth malaria and cancer therapies [63]. First, pointmutations within the kinase gene might affect drug-binding efficiency, as shown in imatinib-resistant [64,65]or dasatinib-insensitive [66] BCR-ABL-kinase mutants.Second, amplification of the targeted kinase genes isanother mechanism for resistance, as described, forexample, in GIST patients with oncogenic KITmutations [67]. Third, upregulation of the glutathione-S-transferase-mediated detoxification mechanism mightimpact on drug sensitivity [68]. Alternative kinasepathways might compensate drug action and restorePlasmodium infectivity. For example, as mentionedabove, transgenic parasites lacking Pfmap-1, one of the

Table 1. A few websites of interest

Public–private partnerships and industrial involvement in malaria researc

European Commission-AntiMal

WHO/TDR

Drugs for Neglected Diseases

Medicines for Malaria Venture

Novartis Institute for Tropical Diseases

Sanofi-Aventis Impact Malaria

GlaxoSmithKline

Palumed

Global Fund to fight AIDS, Tuberculosis and Malaria

Bill and Melinda Gates Foundation

PK-targeted antimalarial drug discovery

Phosphorylation sites

Protein Data Bank

Parasitic targets database

Protein kinases

Plasmodium database

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two MAPKs in the Plasmodium kinome, overexpressPfmap-2, presumably as a compensatory mechanism[32]; such a scenario also might occur in response todrug treatment. Therefore, targeting several enzymes ofthe same family at once would considerably decrease therisk of drug-resistance emergence. Similarly, targetingmultiple pathways by the use of combined therapies isstrongly expected to hamper the emergence and spreadingof resistance phenotypes [69]. Finally, efflux pumps couldwithdraw drugs from the parasite [70,71].

Academia and industry: a joint venture to killthe killerAs previously detailed by Nwaka and Ridley [72], the lastdecade witnessed a significant increase in interactionsbetween academic laboratories working on neglected dis-eases on the one hand and lead discovery departments ofprivate companies on the other hand. Organisations dedi-cated to public–private partnerships (such asMedicines forMalaria Venture), joint projects between the pharmaceu-tical industry and international institutions [e.g. theWorldHealth Organization (WHO)], and academia–industry col-laborations fostered by funding agencies such as theFramework Programs of the European Commission orthe Global Fund to Fight AIDS, Tuberculosis and Malaria,are all expected to generate major synergies [73–76] (seeTable 1). First, small-molecule libraries and infrastruc-tural capacity to store and handle large number of chemi-cal, both of which are available in industry, add a clearvalue to ongoing antiparasitic drug-discovery efforts by theacademic community. Second, industrial middle/high-throughput/high content/screening (M/HT/HC/S) allowsdrug discovery at a much higher scale than feasible inmost academic environments. Antiparasitic drug discoverycan be smoothly integrated within ongoing screeningactivities at industrial organisations. The academic part-ner plays a crucial initial role by identifying the target PK,assessing target essentiality by reverse genetics, generat-ing expression vectors and demonstrating target PKactivity. The industrial partner then provides the expertiseand equipment to express and purify the target in suitableamounts (up to several milligrams) for drug-discoverypurposes. This allows characterisation of enzymatic andkinetic parameters, the development of the screening

h (nonexhaustive list; we apologise for any omissions)

http://www.antimal.eu

http://www.who.int/tdr

http://www.dndi.org

http://www.mmv.org

http://www.nitd.novartis.com

http://www.impact-malaria.com/en/index_prehome.asp

http://www.gsk.com/infocus/malaria.htm

http://www.palumed.com

http://www.theglobalfund.org/en

http://www.gatesfoundation.org/default.htm

http://www.phosphosite.org

http://www.rcsb.org/pdb

http://tdrtargets.org

http://www.kinasenet.org

http://www.plasmodb.org

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assay, the primary screening of large numbers ofcompounds (105–106 molecules) and the confirmation ofhit molecules resulting from IC50 determination.

In addition to scientific activities, two issues must beconsidered in the context of partnerships between industryand academia. The first one is the ownership of intellectualproperty for newly discovered drug candidates (it is gener-ally agreed that the targets themselves should not beprotected, as a way to facilitate screening by multiplecompanies). Usually, companies distinguish two cases:(i) The chemical scaffold of the newly discovered small-molecule series provides an exclusive specificity for thePlasmodium kinase. If this is the case, then this seriesmight not be viewed as relevant to the therapeutic area ofthe core business of the company. In this situation theownership could be shared by both partners. (ii) Thechemical scaffold is of interest to the company and, thus,the intellectual property will remain exclusive to the com-pany that performed the screening, although this is usuallydiscussed in a case-by-case approach. Second, approval ofnew drugs by the FDA is a process that is strictly controlledaccording to safety criteria. Toxicity issues are often thereason for withdrawal of drugs. Recently, the FDA hasrevised toxicity as a strict rejection rule in the context ofmalaria by declaring, ‘‘Because malaria is life-threatening,the risks associated with quinine use are justified forthat condition’’ (http://www.fda.gov/bbs/topics/NEWS/2006/NEW01521.html). We strongly support this stance:considering that saving the life of a patient suffering fromcerebral malaria would necessitate only a very short-termtreatment (as compared with treatment of chronic dis-eases), it would be counterproductive to impose an absolutelack of toxicity.

Concluding remarksOver the recent years, nonprofit organisations, EuropeanFramework Programmes and other funding agencies havestrongly encouraged connections and exchanges betweenacademic and industrial partners. This led to the creationof small exploratory teams in which scientists contribute toopening a new era of antimalarial drug discovery.

Today, fighting against malaria or any other diseaserequires new approaches and criteria, from target selectionto lead identification. However, the quest for innovativestrategies should not exclude traditional target-baseddrug-discovery processes. Demonstrating therapeutic con-cepts and biological rationale is demanding in both timeand highly specialised expertise and is often seen as alimiting factor. Yet it is crucial to secure quality, relevanceand success in the course of drug-discovery phases. Finally,the antimalarial potential of drug candidates that targethuman PKs and are currently in development for other,more profitable, therapeutic areas should be explored. Thiswould allow initiating the process of antimalarial drugdiscovery from a stage in which good pharmacokineticand safety profiles of promising compounds are alreadyavailable, and hence significantly reduce the cost of theearly development phase.

Considering the numerous opportunities that werebriefly alluded to in this review, we believe that PKinhibitors generate true hope and could well bring a

strong contribution in the next decade of antimalarialdrug-discovery research.

AcknowledgementsThe authors are members of the AntiMal project, funded by theFramework Programme 6 of the European Commission and workcollaboratively on Plasmodium kinase inhibition, implementing some ofthe approaches described in this review. We apologise to those colleagueswhose work is not mentioned here because of space constraints. We thankA. Dorr (Merck-Serono, Geneva) for advice on the manuscript and J.Chevalier (Service Scientifique de l’Ambassade de France a Londres) forcontinuing interest and support. Work in C.D.’s laboratory is funded byINSERM, the European Commission (AntiMal Project and BioMalParNetwork of Excellence), and a grant from the Novartis Institute fortropical Diseases (NITD, Singapore). Work in D.L.’s laboratory issupported by Merck-Serono and the European Commission (AntiMalProject).

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