Protein kinases of malaria parasites: an update

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Protein kinases of malaria parasites: an update Christian Doerig1, Oliver Billker2, Timothy Haystead3, Pushkar Sharma4, Andrew B Tobin5 and Norman C Waters6 1

INSERM U609, Wellcome Centre for Molecular Parasitology, University of Glasgow Biomedical Research Centre, Glasgow G12 8TA, Scotland, UK 2 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK 3 Department of Pharmacology, Duke University Medical Center, C119 LSRC Research Drive, Durham, NC 27710, USA 4 Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi-110067, India 5 Department of Cell Physiology and Pharmacology, University of Leicester, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK 6 Australian Army Malaria Institute, Gallipoli Barracks, Enoggera, QLD APO, AP 96553, Australia

Protein kinases (PKs) play crucial roles in the control of proliferation and differentiation in eukaryotic cells. Research on protein phosphorylation has expanded tremendously in the past few years, in part as a consequence of the realization that PKs represent attractive drug targets in a variety of diseases. Activity in Plasmodium PK research has followed this trend, and several reports on various aspects of this subject were delivered at the Molecular Approaches to Malaria 2008 meeting (MAM2008), a sharp increase from the previous meeting. Here, the authors of most of these communications join to propose an integrated update of the development of the rapidly expanding field of Plasmodium kinomics. Eukaryotic protein kinases of malaria parasites The importance of protein phosphorylation in cellular processes in eukaryotes is reflected by the size of the protein kinase (PK) family: more than 500 PKs are encoded in the human genome [1,2], and this number represents 2% of the total number of genes. Most of these (the eukaryotic PKs, or ePKs) share conserved amino acid sequence elements and a common structural fold (Figure 1), whereas only a few (the atypical PKs, or aPKs) do not conform to this group. In phylogenetic trees, the ePKs distribute in seven main clusters [3]:  CK1 (casein kinase 1)  CMGC (CDK [cyclin-dependent kinases], MAPK [mitogen-activated protein kinases], GSK3 [glycogen synthase kinase 3] and CLKs [CDK-like kinases])  TKL (tyrosine-kinase-like)  AGC (PKA [cyclic-adenosine-monophosphate-dependent protein kinase], PKG [cyclic-guanosine-monophosphate-dependent protein kinase], PKC [protein kinase C] and related proteins)  CamK (calcium/calmodulin-dependent kinases)  STE (PKs acting as regulators of MAPKs and first identified in a genetic screen of sterile yeast mutants)  TyrK (tyrosine kinases).

Corresponding author: Doerig, C. ([email protected]).

The ePKs that do not fit into any of these groups are described as other protein kinases (OPKs). The Plasmodium falciparum kinome is much smaller than that of metazoans, with 86 or 99 ePK-related enzymes, depending on the stringency applied to include borderline sequences [4,5]. There is no space here to discuss the plasmodial kinome in any detail (for a recent review, see Ref. [6]), but it is important to emphasize some important divergences from the human kinome:  Absence of members of the TyrK and the STE family.  Presence of ‘orphan’ PKs with no orthologues in mammalian cells. Some of these atypical enzymes possess features of more than one PK family in a single enzyme (‘composite’ kinases).  Presence of a family of calcium-dependent kinases (CDPKs) carrying a kinase domain fused to a calmodulin-like domain, a configuration found in plants and alveolates but not in metazoans [7].  Paucity of clear orthologues of mammalian ePKs. Even for those plasmodial enzymes that clearly cluster within an established PK group, it is generally very difficult to ‘pair’ these with individual human or yeast ePKs.

Brief overview of recent reports on P. falciparum PK biology Here, we recapitulate essential available data concerning plasmodial PKs, according to the established group classification. CK1 group Although this family is vastly expanded in various organisms (e.g. 80 members in nematodes), the parasite possesses a single member of this group. The recombinant enzyme is active in vitro and can phosphorylate several proteins in parasite extracts [8]. The role of this enzyme in the life cycle of the parasite has to our knowledge not been investigated. CMGC group This group includes (i) the CDKs, which are major regulators of cell-cycle progression, (ii) MAPKs, which are

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Figure 1. Structure of ePKs. (a) The catalytic domain of ePKs is characterized by highly conserved amino acids distributed in 11 subdomains. The indicated residues are: the three glycine residues (GxGxxG) forming a hairpin enclosing part of the ATP molecule in subdomain I; a lysine (K) in subdomain II, which orientates ATP through contacts with the a- and b-phosphates; a glutamate (E) in subdomain III that forms a salt bridge with the former residue; aspartate (D) and asparagine (N) within the HRDXXXXN signature motif of ePKs in subdomain VIB, in which the D is thought to be the catalytic residue acting as a base acceptor; D, the aspartate in the DFG motif of subdomain VII, which binds to the cation (Mg2+ or Mn2+) associated with ATP; the glutamate (E) in subdomain VIII, which forms a salt bond with the arginine (R) in subdomain XI and provides structural stability of the C-terminal lobe; and the aspartate in subdomain IX, which is involved in structural stability of the catalytic loop of subdomain VI through hydrogen bonding with the backbone. Adapted, with permission, from Ref. [5]. (b) The three-dimensional structure of PfPK7 as an example of the ePK fold, presented in ribbon representation and colour ramped from blue at the N-terminus (N) through green to red at the C-terminus (C). The bi-lobal ePK fold is characterized by a b-sheet-rich N-terminal lobe and a largely a-helical C-terminal lobe, between which the catalytic cleft is located (shown with AMPPNP, an ATP analogue, in magenta; the so-called C helix, which forms the back of the ATP-binding cleft, is indicated). PfPK7 provides an example of a plasmodial ePK with atypical insertions (I1–I4); these insertions are usually located between conserved structural elements such as b-sheets and a-helices and do not disrupt the overall fold. For example, the stretches labelled I1 and I2 are much shorter in typical ePKs. Adapted, with permission, from Ref. [49], which should be consulted for details.

crucial transducers of extra- or intracellular signals to effectors such as cell cycle control element or transcription factors, (iii) enzymes of the GSK3 family, which are also major regulators of cell proliferation and, (iv) the CLKs, which play important roles in RNA metabolism. CDKs. Several CDKs have been identified in P. falciparum [5,9], and at least two of these, PfPK5 and Pfmrk, are regulated by the binding of cyclins – to date four plasmodial cyclins, Pfcyc1–Pfcyc4 and one CDK effector molecule (PfMAT1) have been characterized [10–12]. The activity of mammalian and yeast CDKs is also regulated by phosphorylation, either positively via the action of CAK (CDK-activating kinase, of which Pfmrk has been proposed as a functional homologue) or negatively via a Wee1-like kinase. Such regulation has not been demonstrated in malaria parasites, although the target residues are conserved in most plasmodial CDKs [9]. Several plasmodial CDKs undergo autophosphorylation in vitro, a characteristic that requires investigation in vivo before it is assigned as a bona fide novel regulatory mechanism [9]. CDK inhibitors (CDIs), conserved small proteins that inhibit CDKs in response to environmental stress and DNA damage, have not been identified in Plasmodium species. Nevertheless, PfCDKs can be inhibited by mammalian CDIs in vitro, which demonstrates that structural features required for inhibition are conserved in the PfCDKs and that cryptic CDI functional homologues might be encoded 2

in the Plasmodium genome [13]. PfPK5 is the only PfCDK (and one of only two PfPKs, the other one being the orphan enzyme PfPK7, see below) for which a crystal structure has been reported; information from this structure as well as future structural studies might reveal unique regulatory mechanisms of malarial CDKs [14]. In view of the peculiarities of Plasmodium schizogony, which involves endoreduplication (i.e. multiple genome replications in the absence of cytokinesis), it has to be expected that PfCDKs might have adopted unique regulatory mechanisms. The distinct roles that these kinases play in the control of the Plasmodium cell cycle (the organization of which is still largely not understood) remain to be elucidated; some insight has been gained using chemical CDK inhibitors [15] but (as with all studies based on kinase inhibitors) must be considered with caution until it is corroborated by reverse genetics [16]. MAPKs. The MAPKs regulate cell proliferation and differentiation in response to a variety of stimuli [17]. P. falciparum possesses two atypical MAPK homologues, Pfmap-1 and Pfmap-2. Inactivation of the pfmap-1 gene does not cause any detectable developmental phenotype between erythrocytic schizogony and sporogony; however, overexpression of Pfmap-2 in the pfmap-1 parasites suggests that Pfmap-1 fulfils an important function in wild-type parasites and that this function must be taken over by the other MAPK when Pfmap-1 is absent. Pfmap-2,

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Figure 2. Functions of Plasmodium PKs during the life cycle of malaria parasites. Infection of the human host is initiated by the delivery of sporozoites into the bloodstream by an infected Anopheles mosquito. The sporozoites invade hepatocytes where they develop into a schizont producing several thousand merozoites, which are able to infect erythrocytes. The red blood cell is the site of further asexual multiplication (erythrocytic schizogony) and of sexual differentiation into male or female gametocytes. After a blood meal by the mosquito vector, the gametocytes undergo gametogenesis in the midgut of the insect. Fertilization and zygote formation ensue, followed by development into a motile ookinete, which crosses the midgut epithelium and establishes an oocyst at the basal lamina. The oocyst generates sporozoites, which accumulate in the salivary glands and are primed for infection of a new human host. Reverse genetics has revealed roles for PbCDPK6 in sporozoite infectivity [27]; for PfPK7 [50], Pfmap-2 [18], PfCDPK1 [62] and PfPKG [40] in erythrocytic schizogony; for PbCDPK4 [30], Pbmap-2 [19–21] and PfPKG [40] in male gametogenesis; for Pbnek-4 (P. berghei NIMA-related kinase-4) in ookinete maturation [48]; for PbCDPK3 in ookinete motility [28,29]; and for PfPK7 in oocyst development [50]. Parasites lacking Pfmap-1 display no detectable developmental phenotype [18]. Enzymes belonging to the various ePK groups are labelled in the following colours: CMGCs, blue; CamK/CDPK, pink; AGC, orange; OPK, green; orphan kinase, black. This figure is restricted to functional data obtained through reverse genetics; functional information is also available from pharmacological investigations that used specific inhibitors, but this is not included here for the sake of concision and because of selectivity issues regarding kinase inhibitors [63,64]. Adapted, with permission, from Ref. [65].

by contrast, appears to be essential for completion of the erythrocytic asexual cycle of P. falciparum [18]. (See Figure 2 for a summary of the phenotypes observed in parasites lacking individual PKs). Interestingly, the P. berghei orthologue of Pfmap-2 can be deleted without affecting asexual growth or gametocytogenesis, but pbmap-2 microgametocytes are unable to exflagellate [19–21]. The basis for this phenotypic discrepancy between species is unclear. Malaria parasites do not appear to possess classical MAPK kinase (MAPKK) orthologues [22], and the mode of regulation of PfMAPKs remains to be elucidated; a few reports [23,24] suggest that members of the NIMA (never-in-mitosis A; see the section on OPKs later) kinase family could act as atypical regulators of Pfmap-2 (see later). GSK3 and CLKs. The GSK3 cluster contains three P. falciparum sequences, only one of which has been the subject of a published characterization: PfGSK3 appears to colocalize with Maurer’s clefts, and hence to be at least in part exported to host erythrocytes [25]. Apart from a paper

reporting identification and preliminary characterization of a gene encoding a LAMMER-like kinase [26], no other report on the four P. falciparum sequences clustering with the CLKs has been published. CamK group Of the 13 Plasmodium ePKs belonging to this group [5], the CDPKs have received most attention. Five PfCDPKs share the typical domain architecture of plant CDPKs, whereas CDPK6 is predicted to have an incomplete C-terminal calmodulin-like domain and a longer N-terminal extension of the kinase domain, with an additional EF hand [27]. Gene knockout experiments in P. berghei have identified distinct functions for CDPKs in ookinete motility [28,29], microgamete formation [30] and hepatocyte invasion by the sporozoite [27]. All these aspects of malaria biology require calcium as a second messenger, and calcium is known to regulate CDPK activity [7]. Subcellular localization of some CDPKs is determined by N-terminal acylation [31], which, together with stage-specific gene 3

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Review expression profiles, is thought to contribute to the specificity of CDPK signalling in response to a ubiquitous second messenger. PfCDPK1 has recently been shown by reverse genetics to be essential for completion of the erythrocytic asexual cycle [32]. Treatment of parasites with a CDPK1 inhibitor blocks merozoite egress, and treatment with the same molecule interferes with host-cell invasion by Toxoplasma gondii [32]. Although at this stage it cannot be excluded that the inhibitor affects other targets in addition to CDPK1, these data suggest a role for CDPK1 in parasite motility, and this contention is consistent with the observation that CDPK1 can phosphorylate MTIP, a glideosome component, in vitro [32]. It must be kept in mind, however, that the physiological relevance of phosphorylation events observed in vitro with recombinant PKs and substrates remains to be established. AGC group Recent work has begun to reveal important cellular functions for cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent signalling pathways at different life-cycle stages. A close crosstalk between cyclic nucleotide and calcium-dependent pathways emerges as a unifying theme from this work. Three P. falciparum PKs that clearly belong to the AGC group have been characterized: PfPKA, PfPKG and PfPKB. PfPKA. This enzyme is the only known cAMP effector kinase in Plasmodium. Inhibitors of PKA block P. falciparum growth in vitro [33], but whether these are selective for parasite PKA or exert their effect through host PKA or other host or parasite kinases, is unclear. Overexpression of the PKA regulatory subunit (PfPKA-R) might offer a more selective way of interfering with PKA. Using this approach, Merckx et al. proposed that PKA modulates an anion channel conductance of the host erythrocyte [34]; how the PfPKA could reach such a target remains unknown. Pharmacological experiments on cultured P. falciparum blood stages suggest that cAMP–PKA-dependent pathways can regulate cytosolic calcium levels, and thereby delay parasite maturation in vitro [35]. Independent evidence for crosstalk between cAMP and calcium signalling comes from P. berghei sporozoites, in which the phenotype of an adenylyl cyclase a knockout parasite combined with pharmacological data demonstrate that cAMP and calcium-dependent signalling pathways both regulate microneme exocytosis and are required for the efficient invasion of hepatocytes [36]. PfPKG. Apicomplexan PKG is selectively inhibited over its mammalian homologue by the trisubstituted pyrrole, Compound 1 [37]. A single amino acid substitution in the ATP-binding site renders T. gondii PKG, which is essential for tachyzoite development, resistant to Compound 1 [38]. Transgenic parasites expressing inhibitor-resistant PKG can thus serve as controls for compound selectivity. By this approach, it was ascertained that T. gondii PKG is important for microneme secretion, gliding motility, host cell attachment and invasion by tachyzoites [39]. At the Molecular Approaches to Malaria 2008 meeting (MAM2008), L. McRobert et al. reported on the allelic replacement in P. falciparum of wild-type PKG with a resistant mutant, and 4

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this approach allowed them to determine how Compound 1 inhibits the differentiation of gametocytes to gametes*. (This study has now been published [40].) In parasites expressing resistant PKG, Compound 1 failed to block rounding up of gametocytes in vitro, demonstrating a key role for PKG in gametocyte activation. The same transgenic parasites will now allow the functional analysis of Plasmodium PKG in asexual proliferation, which is also affected by Compound 1 [41]. PfPKB. Mammalian PKB is regulated by the action of phosphoinositides (PIs) generated by PtdIns-3 kinase, and PIs interact with the PH domain of PKB [42]. Interestingly, PfPKB lacks a PH domain and is activated by calcium-bound calmodulin through a calmodulin-binding domain (CBD) present in its N-terminal region [43,44]. A novel signalling pathway, which involves the activation of PfPKB via calcium and calmodulin, was recently identified in P. falciparum. Phospholipase C could be an upstream regulator of this pathway, because it is involved in the release of calcium from intracellular stores [45]. Data presented by P. Sharma at MAM2008 suggest that the calcium/calmodulin–PfPKB pathway could be involved in erythrocyte invasion by the parasite*. Pharmacological evidence that PfPKB phosphorylates the glideosome-associated protein 45 (GAP45) and thereby might modulate motor function was also provided. Hence, two different proteins of the inner membrane complex (IMC) of the glideosome appear to be phosphorylated by two different PKs (GAP45 by PfPKB and MTIP by CDPK1), although both observations must at this stage be considered with caution. (The PfPKB inhibitor used in the experiment might affect other targets, and the use of recombinant proteins might not reflect enzyme–substrate relationships in vivo, see above.) It is to be expected that with this type of approach additional phosphorylation events relating to gliding motility will be uncovered in the near future. Dissecting the physiological roles of individual PKs in the phosphorylation of IMC components, and determining how this regulates the gliding mechanisms, represents a serious challenge for the years to come. TKL group Five Plasmodium PK sequences cluster with human TKLs, but no published reports on these enzymes are available. OPKs The NIMA-related kinase (Nek) family is represented by four members in the P. falciparum kinome. Neks play central roles in the eukaryotic cell division process, regulating processes such as centrosome replication [46]. Microarray analysis shows that Pfnek-1 is expressed in asexual and sexual blood stages, whereas the other three Pfneks are expressed predominantly in gametocytes [47]. The presence of a MAPKK-like activation site in Pfnek-1 prompted experiments aimed at determining whether Pfnek-1 might represent a MAPKK functional homologue. (There are no typical MAPKK enzymes in the plasmodial * The abstract of this communication can be found in ‘Abstracts of the Molecular Approaches to Malaria Conference. February 3–7, 2008. Lorne, Victoria, Australia. Int. J. Parasito.l 38, Suppl. 1, S17–98’.

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Review kinome.) Indeed, Pfnek-1 phosphorylates the atypical MAPK Pfmap-2 in vitro, but not Pfmap-1 or mammalian MAPKs, and acts in synergy with Pfmap-2 towards exogenous substrate phosphorylation [24]. The P. berghei orthologue of Pfnek-1 is present in male but not female gametocytes [21], and this finding is consistent with a possible role for Pfnek-1 in male gametogenesis as displayed by its possible target, Pbmap-2. Subsequently, a similar activity on Pfmap-2 was reported for another Pfnek enzyme [23]. The only published study revealing the role of a plasmodial Nek in the life cycle of the parasite is that of Pbnek-4, which is essential for ookinete maturation in P. berghei [48]. Orphan kinases The P. falciparum kinome comprises ePK-related kinases that do not cluster with any of the established ePK groups, or with any family in the OPK group. One of these orphan kinases is PfPK7, a composite enzyme with a C-terminal lobe showing highest homology to MAPKKs, whereas its Nterminal region is more closely related to fungal PKAs [22]. The solution of the PfPK7 crystal structure established that the phylogenetic peculiarities of the enzyme are reflected by atypical structural features [49]. Parasites lacking PfPK7 display a slower parasitaemia growth rate, linked to a smaller number of daughter merozoites produced by schizonts, as well as impairment in oocyst development [50]. PK9 [51] is another orphan kinase that (similar to PfPK7 [22]) is able to autophosphorylate and to phosphorylate exogenous substrates in vitro. Autophosphorylation occurs on three residues, the mutation of which to alanine abolished activity, which points to a potentially complex mode of regulation in vivo. Recombinant PfPK9 was added to P. falciparum extracts in the presence of [32P] g-ATP to investigate physiological function, and labelled proteins were subsequently identified by mass spectrometry. Strikingly, PfPK9 displayed a discrete phosphorylation profile, exclusively labelling a P. falciparum homologue of the E2 ubiquitin-conjugating enzyme 13 (UBC13), thereby suppressing its ubiquitinconjugating activity [51]. This finding indicates that PfPK9 could play a crucial role in proteasome regulation and hence in fundamental cellular processes such as cell cycle progression. The FIKKs (named after a shared Phe-Ile-Lys-Lys motif in the N-terminal region of their kinase domain) constitute a distinct cluster of 21 orphan kinases in P. falciparum [4,5], and these kinases appear to be restricted to apicomplexans, most species of which possess only one member of this novel family [52]. The enzymes possess a Plasmodium export element (PEXEL) motif [52], and localization in the erythrocyte has been verified experimentally for at least some FIKKs [53]. Perspectives in Plasmodium kinomics The phosphoproteome of mammalian cells comprises 30% of all proteins [54,55]. In HeLa cells alone, 2200 phosphoproteins have been identified [54]. Similar analyses in yeast have revealed more than 700 phosphopeptides, a staggering 139 of which are regulated by

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stimulation of a single cell surface receptor [56]. Furthermore, most phosphoproteins are multiply phosphorylated by a range of protein kinases that act with differential kinetics on their substrate proteins [54]. Despite intense activity devoted to mammalian and yeast cell kinomics, most of the sites of phosphorylation in vivo and their physiological role have yet to be determined [55]. Our understanding of the Plasmodium phosphoproteome is even more rudimentary. The 518 PK genes of the human kinome represent 2% of the genome [2], compared with the 85–100 P. falciparum ePK sequences that amount to 1.1–1.6% of the protein-coding genes of malaria parasites [4,5]. An estimated 60% of coding genes are transcribed in the P. falciparum blood stage parasite [57]. If one assumes that phosphorylation plays a similar role in regulating biological processes in malaria parasites as it does in mammalian cells, then 30% of blood-stage Plasmodium proteins, that is some 1000 polypeptides, will be present as phosphoproteins. Taking multiple phosphorylation into account, the number of phosphorylation states might well run into the many thousands. How will this diversity be unravelled? A good starting point would be to define which proteins are phosphorylated. This can be resolved by a combination of approaches ranging from work focussed on individual protein kinases and their substrates, as illustrated above for PfPK9, to global phosphoproteomic mass spectrometry approaches similar to those that have been applied so successfully to mammalian [55], yeast [56] and bacterial [58] systems. Although Plasmodium proteomics has come of age in recent years [21], no report on the phosphoproteomics of malaria parasites has yet been published; however, preliminary work has identified numerous phosphoproteins in blood stages of P. falciparum (A. Tobin et al. and T. Haystead et al., unpublished). The advent of quantitative proteomic approaches such as stable isotope labelling by amino acids in cell culture (SILAC) [59] and the improvements in the sensitivity of mass spectrometers [60] are inevitably going to make a major impact in this area. This, however, will reveal only the extent of the phosphoproteome. The tough job will be to determine which kinases are responsible for which phosphorylation event, and what are the functional outcomes of each phosphorylation. A full understanding of the role played by phosphorylation will be achieved only with resolution of the mechanisms by which the Plasmodium kinome is regulated. This will require elucidation of the organization of signalling pathways and of the complexes in which the PKs function (scaffolding and regulatory proteins), which in turn is dependent on systematic approaches in reverse genetics (including those allowing inducible phenotypes such as chemical genetics [61]) and phosphoproteomics. The complexity and specificity of the Plasmodium kinome present a challenge to understanding the role played by protein phosphorylation in malaria parasites that is as demanding as anything experienced in mammalian or yeast cell biology. The reward, however, could be a plethora of novel potential drug targets (see Box 1) and the satisfaction of understanding one of biology’s most intriguing unicellular organisms. 5

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Box 1. PKs as targets for antimalarial chemotherapy Major points to be considered in antimalarial drug discovery targeting PKs include (i) target validation, (ii) variety of screening platforms, (iii) proper use of counter screens and (iv) insight into the development of drug resistance (Figure I). Target validation must address essentiality of the kinase at the lifecycle stage that is targeted. Inhibition of a single kinase within a kinase cascade pathway could be potentiated by targeting other PKs along the pathway, or across pathways in the event of signalling crosstalk. Such cascades, although divergent from mammalian pathways, appear to exist in Plasmodium parasites (see main text for details). Many P. falciparum PKs are available as active recombinant enzymes and can be used in enzymatic screens. Although many P. falciparum proteins are difficult to express because of high AT content in the coding region and, in several instances, large insertions within the catalytic domain, several recombinant PfPKs have been produced in an active form and used in screening procedures [6,66]. Cellular screens, monitoring the effect of a compound library on a specific life-cycle stage, can also be implemented; obviously, erythrocytic schizogony is a major target. Counterscreens using the human orthologues (if any) of the target should be used and a selectivity index should be determined to identify promising compounds for hit-to-lead transition. However, any panel of human potential targets is likely to be incomplete; therefore, mammalian cell toxicity assays are a crucial counterscreen. Cell-based assays allow the calculation of an in vitro therapeutic index, which assesses activity between parasites and mammalian cells. An understanding of the propensity of kinases to develop resistance to inhibitors and their involvement in general drug resistance mechanisms is important for evaluating the longevity of malaria kinase inhibitors. Recent work presented at MAM2008 by Schiebout et al. suggests that computational tools can be used to predict the acquisition of inhibitor-resistant mutations in malarial kinases*. Malaria kinase drug discovery efforts can be accelerated with the utilization of the vast ‘kinase knowledge’ that has accumulated from

Figure I. P. falciparum PKs as drug targets: overview of the drug discovery process.

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human ePKs. Inhibitory scaffolds and structural data are reported for all major kinase families. This information facilitates grouping of kinases according to their inhibitor sensitivity. Sensitivity clades allow parallel processing of multiple kinases through the drug discovery process and encourage ‘target-hopping’ of a single inhibitor scaffold to exploit common structural and inhibitor sensitivity profiles. The accumulation of Plasmodium PK inhibitor data will facilitate comparisons with ePK clades and might provide initial compound characteristics responsible for differentiating human and plasmodial kinases. Although most PK inhibitors target the ATP-binding site, there are other targetable sites such as protein–protein interaction and regulatory domains, which might be unique to malarial PKs. Another class of PK inhibitors comprises the so-called ‘allosteric inhibitors’, which function by preventing the enzyme from adopting an active conformation [67,68]. It has recently been realized that specific inhibitors targeting a single enzyme are not ideal and that, in most cases, specificity cannot be achieved anyway [16,63,64,69]. A clear advantage of targeting multiple structurally related enzymes would be a decrease in the probability for the parasite to develop resistance [70]. Furthermore, it is possible that members of a given PK family could be inactivated individually without adverse cellular effects because of functional complementation by other members of the family (for example, Pfmap-2 is overexpressed in pfmap-1 parasites, which suggests that Pfmap-1 plays important roles in the parasite even though its gene can be deleted [18]). Targeting several members of the same family might well, therefore, have parasitocidal effects even if the genes can be deleted individually. This presents the advantage of increasing the number of potential targets, because even PKs that appear not to be essential in single-gene inactivation experiments might represent useful targets. The Plasmodium kinome, comprising several families of PKs that are clearly distinct from human PKs, provides a potentially interesting system to test this avenue of research.

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Acknowledgements We thank the organizers of the MAM2008 meeting for providing such an outstanding forum on recent developments in Plasmodium research. We apologize to our colleagues whose work could not be cited here because of space limitations.

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