Drug-resistant malaria - an insight

Abstract

Despite intensive research extending back to the 1930s, when the first synthetic antimalarial drugs made their appearance, the repertoire of clinically licensed formulations remains very limited. Moreover, widespread and increasing resistance to these drugs contributes enormously to the difficulties in controlling malaria, posing considerable intellectual, technical and humanitarian challenges. A detailed understanding of the molecular mechanisms underlying resistance to these agents is emerging that should permit new drugs to be rationally developed and older ones to be engineered to regain their efficacy. This review summarizes recent progress in analysing the causes of resistance to the major antimalarial drugs and its spread.

Malaria has a broad distribution throughout tropical and subtropical areas, affecting both indigenous populations and increasing numbers of travellers. The disease is caused by protozoan parasites of the genus Plasmodium and is transmitted via the bite of Anopheles mosquitoes. These parasites have a complex life cycle in both the mosquito and human hosts; in the latter, sporozoite forms injected by the mosquito during its blood meal migrate to liver cells, where, after extensive replication, merozoites are released into the bloodstream to start the cycles of erythrocyte invasion, intracellular growth and division, followed by host-cell lysis and reinvasion, which give rise to the clinical symptoms of the disease. Plasmodium falciparum is the most dangerous of the four species of Plasmodium that cause human malaria, leading to a death rate conservatively estimated as 1-2 million people per year. Such lethality stems in part from the way this particular species modifies its host erythrocyte, such that the more mature stages in this part of the life cycle adhere to endothelial surfaces and progressively block microcapillaries in major organs, such as the brain. Although malaria is still found in over 100 countries, the major burden of disease occurs in sub-Saharan Africa, where over 90% of all deaths are recorded, largely among those aged under-five, and where intense morbidity and transmission have profound consequences for public health and economic infrastructures [1].

In the continuing absence of clinically proven vaccines, preventing or treating malaria parasite infections in the human host has always depended heavily upon chemoprophylaxis and chemotherapy. Since the advent of the first synthetic antimalarials in the 1930s, only a small number of compounds has proved suitable for licensing as drugs for human use, and several of these are now greatly compromised by the inexorable spread of drug-resistant parasite strains, including the cheapest formulations that are so important to Africa, namely chloroquine and pyrimethamine–sulfadoxine (a coformulated combination marketed as Fansidar®). Resistance of malaria parasites arises from several factors, including overuse of antimalarial drugs for prophylaxis, inadequate or incomplete therapeutic treatments of active infections, a high level of parasite adaptability at the genetic and metabolic levels, and a massive proliferation rate that permits selected populations to emerge relatively rapidly. With the development of sophisticated methods of molecular analysis and the ability to manipulate genes of Plasmodium in transfection systems, albeit with some difficulty, considerable progress has been made in understanding how P. falciparum has been able to subvert the chemical attacks made upon it, which in turn is suggesting new strategies for continuing the fight. Several reviews of this area with greater molecular detail and historical perspectives have been published in recent years [2-8]. This short article will focus on core mechanisms and some of the most recent developments concerning resistance to the major antimalarial drugs, as summarized in Table 1.

Table 1.

P. falciparum proteins with a proven role in resistance to clinical antimalarial drugs

Protein	Function	Location	Principal drugs affecteda	Comments	Refb
CRT	Transporter	Membrane of food vacuole	Chloroquine Mefloquine, halofantrine, lumefantrine, artemisinins, quinine	Major determinant Minor determinant	[3,6,8,18]
Pgh1 (P-glycoprotein homologue 1) or MDR1 (multidrug resistance 1)	Transporter	Membrane of food vacuole	Mefloquine, halofantrine, lumefantrine, quinine (possibly)	Major determinant	[8,18,23]
			Minor determinant	Chloroquine, artemisinins	[8,18,23]
DHPS	Folate pathway enzyme	Cytoplasm (principally)	Sulfadoxine, dapsone	DHPS and DHFR targeted simultaneously in synergistic combinations of antifolates	[2,3,5]
DHFR	Folate pathway enzyme	Cytoplasm (principally)	Pyrimethamine, proguanil, chlorproguanil	DHPS and DHFR targeted simultaneously in synergistic combinations of antifolates	[2,3,5]
Cytochrome b	Subunit of complex III (cytochrome bc1 complex) electron transport chain	Mitochondrion	Atovaquone		[40,46]
ATP6 (sarco/endoplasmic reticulum calcium-dependent ATPase [SERCA] orthologue)	Membrane-bound Ca2+ -transporting ATPase	Membranous structures within cytoplasm	Artemisinins	Likely major determinant	[53,55]

^aOnly drugs discussed in the review are listed.

^bReferences (mainly reviews) that describe the polymorphisms affecting each protein in detail.

Chloroquine and related compounds

The 4-aminoquinoline, chloroquine, a synthetic relative of the plant-derived and quite toxic drug quinine, was for several decades the antimalarial agent of choice, as it was safe, highly effective and cheap. More recent related drugs include mefloquine (a quinoline methanol derivative), halofantrine (a phenanthrene methanol) and lumefantrine (a more complex aryl-substituted methanol), introduced in the 1980s to counter parasites that had become resistant to chloroquine and the antifolates (see below). Chloroquine resistance was first observed in Thailand in 1957 and on the Colombian-Venezuelan border in 1959. By 1988 resistance had spread to essentially all of sub-Saharan Africa and today chloroquine has lost its efficacy in all but a few areas of the world. Chloroquine was known to exert its effect by compromising the sequestration of harmful haem moieties produced as a by-product of haemoglobin digestion by the parasite, the major source of its amino acids. This process occurs in a lysosome-like acidic food (or digestive) vacuole (pH ~ 4.5-5.0), in which chloroquine accumulates to high levels in sensitive parasites, but to a much reduced degree in resistant strains. The genetic basis of this latter phenomenon was revealed by careful and extensive recombination mapping of the progeny of a genetic cross of two cloned parasite populations, one chloroquine-sensitive and the other chloroquine-resistant. The key gene, named pfcrt for ‘chloroquine resistance transporter’, resides on chromosome 7, encodes a 49 kDa protein with 10 predicted transmembrane domains, and exhibits mutations that showed complete linkage to the chloroquine-resistance phenotype in the 40 laboratory-adapted strains of P. falciparum originally examined [9]. The polymorphism in this gene documented to date is considerable, indicative of several independent origins of drug-resistant alleles. It encompasses at least 16 single, nonsynonymous nucleotide substitutions in parasites from field samples, but only one amino acid change, K76T, located in the first transmembrane domain, is found consistently in chloroquine-resistant parasites, although never on its own [6,10]. At least three other changes (and usually several more) are always seen in combination with K76T, relative to the canonical wild-type sequence, possibly compensating for unfavourable changes in the normal function of the chloroquine resistance transporter (CRT) protein induced by K76T and/or as a response to pressure from antimalarial drugs other than chloroquine that also pass through this transporter. These mutations lie mainly, but not exclusively, in the transmembrane domains, of which domains 1, 4 and 9 are thought to play particularly important roles in determining the transport characteristics of CRT with respect to different drugs [11]. cDNA versions of pfcrt from resistant parasites transfected into sensitive parasites bestow the resistance phenotype, and confirm the central role of codon 76 [10]. This is supported by numerous field studies conducted across the world, for example in West Africa [12] and Cambodia [13], where all chloroquine-resistant strains examined carried the K76T, and to date, no chloroquine-resistant isolate has been found that carries the wild-type lysine at position 76.

More recent research has concentrated on classifying the CRT protein encoded by pfcrt and understanding its normal function, as well as the mechanism(s) by which the observed mutations might modulate chloroquine susceptibility, together with the details of drug transport across the food vacuole membrane. Bioinformatics analyses place CRT within a new subgroup of proteins belonging to a superfamily of drug and metabolite transporters (the DMT superfamily) [14,15]. Immunolocalization has confirmed that the molecule is bound into the membrane of the food vacuole and the critical K76T mutation is predicted to face the cytoplasmic side of the first transmembrane domain, where it could alter the selectivity of CRT such that chloroquine more efficiently exits the food vacuole [16,17]. Several models have been proposed to account for the observed distributions of drug between the food vacuole and cytoplasm in sensitive and resistant parasites [18]. One plausible scenario is that the positive charge of K76 normally prevents, or severely limits, the efflux of chloroquine in its diprotonated form (which predominates in the acidic vacuole), an effect that is lost after mutation to T or another nonbasic residue. However, mutations that restore a positive charge to the pore can compensate for the K76T mutation and thus reverse the chloroquine-resistance phenotype, as has been found in at least one apparently anomalous sensitive strain from South East Asia, where an S163R alteration (in the fourth transmembrane domain) is combined with K76T [16]. This ‘proton-trapping’ model, although providing an explanation for the more rapid loss of chloroquine from the vacuole of resistant parasites, does not account for the anomalously high level of drug accumulation within it that is seen in wild-type parasites. It is thus thought likely that binding of chloroquine to ferriprotoporphyrin IX (haem), once it is inside the food vacuole, will contribute significantly to the observed accumulation [19]. The level of chloroquine import into the parasite has recently been shown to depend upon the integrity of the proton gradient maintained across the vacuolar membrane [19], but the pH of the vacuole appears not to play a significant role in resistance, as both chloroquine-resistant and chloroquine-sensitive parasites have closely comparable values, when measured with a variety of molecular probes [20]. However, efflux from chloroquine-resistant lines, but not sensitive lines, appears to be energy independent, implying that the mutant form of PfCRT takes on the function of a gated channel or pore (rather than an active transporter), through which the charged chloroquine species can escape from the vacuole [19]. Attempts to exploit the above findings include studies on a range of compounds that are able to reverse chloroquine resistance, by virtue of molecular structures that are often amphipathic, with both lipophilic and positively charged regions that allow the compound to compete with chloroquine for binding to PfCRT, and hence for exit from the food vacuole [10,21].

The membrane of the food vacuole is expected to carry a number of different transporters, and another identified type is encoded by pfmdr1 on chromosome 5, which shows similarity to the P-glycoprotein product of the human multidrug resistance gene mdr. The malarial protein, Pgh1 or MDR1, is a 162 kDa ABC-type transporter with 12 predicted transmembrane domains and two ATP-binding folds, thought to be involved in importing solutes into the food vacuole, including the drugs mefloquine, halofantrine and artemisinin [22]. It is now known that the mutation status of pfmdr1 can contribute to the precise levels of chloroquine resistance, but on its own, in a pfcrt wild-type background, is unable to confer resistance, a conclusion supported both by transfection studies and recent field studies [18]. However, numerous reports find a reciprocal relationship between chloroquine resistance and resistance to other important quinoline- or methanol-based drugs, such as mefloquine and halofantrine, which also act on the parasite food vacuole [23]. Importantly, amplification of pfmdr1 to copy-numbers of up to five appears to be a primary mechanism whereby parasites become resistant to these drugs, particularly in Asia [24-27], although amino acid polymorphisms in Pgh1 can also have a significant effect [28], as can such changes in PfCRT alone [16]. It has also been proposed that putative parasite transporters other than PfCRT and Pgh1 influence the precise level of sensitivity to these drugs [29,30], but more recent work analysing the genes of nine such transporters from large numbers of parasite samples collected at a single site in South East Asia has thus far failed to support these associations [31].

Antifolates

Analysis of resistance to the antifolate drugs has been considerably more straightforward than was the case with chloroquine, as the primary molecular targets of these compounds were originally established from studies of bacteria in the 1930s and 1940s. The principal antimalarial antifolate drugs are pyrimethamine, proguanil (metabolized in vivo to the active form cycloguanil), the sulfonamides, such as sulfadoxine and sulfalene and the sulfone, dapsone (collectively known as the sulfa drugs). Pyrimethamine and cycloguanil target the dihydrofolate reductase (DHFR) activity of the bifunctional DHFR–thymidylate synthetase (TS) protein, while the sulfa drugs inhibit the dihydropteroate synthetase (DHPS) activity of the bifunctional hydroxymethylpterin pyrophosphokinase (HPPK)–DHPS protein. DHPS is an enzyme unique to the parasite that is used in the biosynthesis of key folate coenzymes, starting from GTP, whereas DHFR is present in both host and parasite, being essential to maintain a constant supply of fully reduced (tetra-hydro) forms of such cofactors for key one-carbon transfer reactions, including the provision of nucleotides for DNA synthesis and the metabolism of certain amino acids. The drugs that target this enzyme bind several hundred times more strongly to the parasite protein than to the human orthologue. Although pyrimethamine and proguanil were initially used alone after their introduction in the 1940/1950s, resistance arose rapidly and it was only in strongly synergistic combinations with sulfa drugs that formulations with longer term utility were produced. The main effect of such combinations is to block the parasite as it undertakes DNA replication. Despite a rapid spread of pyrimethamine–sulfadoxine resistance in South East Asia from the mid-1960s onwards, this affordable combination has been extensively used, albeit with diminishing efficacy, to combat chloroquine-resistant parasites in Africa since the early 1990s.

The genetic basis of resistance to the antifolate drugs is similar to that underlying chloroquine resistance, in that a small number of point mutations in these two target genes appear to be responsible for the major part of resistance. As well as classical drug-binding and kinetic studies on recombinant enzymes, more recent parasite transfection studies, where the resistant-type dhfr and dhps sequences are inserted into the genomes of sensitive parasites, prove directly that the mutations observed in field samples do indeed result in drug resistance in live parasites [32]. High-level pyrimethamine resistance results from the accumulation of mutations in the dhfr domain of pfdhfr-ts, principally at codons 108, 59 and 51, where the most common allelic variants encode S108N, N51I and C59R. Similarly, common patterns of mutation in dhps that compromise the efficacy of sulfadoxine include the codon changes A437G/K540E in Africa and A437G/A581G or A437G/K540E/A581G in South East Asia, with the last of these also often observed in areas of South America [2,5]. The most common genotypes at present found in parasites highly resistant to pyrimethamine-sulfadoxine in Africa, where such resistance is most relevant, combine triply mutated dhfr with doubly mutated dhps, a pattern that acts as a reliable marker for this phenotype [33] and a strong indicator of likely clinical outcome. In South East Asia and South America, a fourth alteration, I164L, is often found in DHFR; in combination with the above changes, this renders the mutant parasites completely resistant to achievable physiological concentrations of pyrimethamine-sulfadoxine [34]. A widespread distribution of such quadruple dhfr mutant parasites into Africa would be a major disaster, given that numerous countries are now using pyrimethamine-sulfadoxine as their first-line defence, and that the most recent antifolate combination to be introduced into the field only in 2003 (chlorproguanil/dapsone, LapDap®) would also be compromised. Ominously, such parasites are now beginning to be reported from areas of East Africa [35].

An important recent milestone in malarial antifolate research was the determination of the crystal structures of DHFR-TS from both pyrimethamine-sensitive and pyrimethamine-resistant parasites, in association with anti-DHFR drugs [36]. This has boosted the search for new compounds inhibitory to this activity and increased the reliability of computer-modelled inhibitor specificities [37]. The crystal structures also help to rationalize how the resistance mutations described above alter the enzyme conformation such that drug binding is greatly reduced, while permitting sufficient processing of normal substrate [37]. As yet, no crystal structure for the HPPK–DHPS enzyme of P. falciparum has been reported, although homology modelling has provided insight into the role of the key resistance mutations in the DHPS domain [38].

Atovaquone and the artemisinins

Two other drug types are of particular importance as recently licensed antimalarials, although both also have a long history. The 2-hydroxynaphthoquinone derivative atovaquone stems from research into this class of mitochondrial inhibitors from the 1940s, but was only certified for use as a clinical antimalarial in combination with proguanil (as Malarone®) in 1997. Atovaquone is a structural analogue of coenzyme Q in the electron transport chain and acts to collapse the organellar membrane potential, thus arresting parasite respiration and essential pyrimidine biosynthesis. This results from inhibition of cytochrome b in complex III of the chain, without affecting human mitochondria, which employ a CoQ₁₀ complex (i.e. one carrying 10 isoprene units on the aromatic ring), that differs from the CoQ₈ type found in the parasite. Atovaquone action is proposed to arise from its blockage of a large-scale domain movement of the iron–sulfur protein subunit that is required for electron transfer to cytochrome c₁ from ubihydroquinone bound to cytochrome b within complex III [39]. Where atovaquone has been used in monotherapy, resistance rapidly emerges, with point mutations in the cytochrome b gene, principally affecting Y268 (converted to S, N or C), associated with drastic reductions in parasite susceptibility to the drug both in vitro and in vivo [40,41]. However, in combination with proguanil, the onset of resistance is markedly retarded and Malarone has now been successfully used for some time, especially for travellers to South East Asia where multidrug-resistant parasites are common. Although the mechanism of synergy between the two components of Malarone is not yet fully understood, it appears not to be associated with the role of proguanil as an antifolate precursor, described above, but rather with its ability to sensitize the mitochondrion, possibly by blocking a secondary mechanism for maintaining its membrane potential [42]. Although apparently still completely effective as a prophylactic, some cases of resistance to Malarone treatment have been reported [43,44], not all of which were associated with mutation in the cytochrome b gene [45], and thus suggestive of possible alternative resistance mechanisms. Moreover, a recent study discovered the cyt b Y268N mutation in several P. falciparum isolates from Nigeria, where the population has not been exposed to atovaquone. One such isolate had an additional mutation (P266T) located at the ubiquinone reduction site of the enzyme [46]. Clearly, such observations are of concern and ongoing surveillance again will be essential to help prolong the effective life of this drug.

The artemisinins are sesquiterpene lactones that derive ultimately from the Chinese herb qinghao (Artemisia annua), used for centuries to treat malaria and other parasitic diseases. Their considerable potency stems in part from a highly reactive epoxide bridge across the seven-membered component of the triplering system, which is essential for antiparasitic activity. The most effective of these compounds thus far is sodium artesunate, which can rapidly reduce parasite numbers ~ 10⁴-fold in a single 48-h erythrocytic cycle. However, the (predictable) danger of using this type of drug in monotherapy, first observed in Chinese field trials in the 1970s, was emphasized by a recent study where decreased sensitivity was observed in recrudescent parasites after 7 days of treatment with artesunate alone [47]. Artesunate was originally deployed in combination with mefloquine in areas of multidrug resistance from 1994 onwards in South East Asia, where it is still used successfully, and artemisinin derivatives are now being evaluated in many other endemic areas in combinations with other antimalarials, such as lumefantrine, piperaquine, amodiaquine and sulfadoxine-pyrimethamine (so-called artemisinin combination therapy; ACT), all of which operate on longer time scales [48]. These formulations are able to eradicate the small fraction of parasites that escape the rapidly metabolized artemisinin (which has a plasma half-life of only about 4 h) and help reduce the likelihood of drug resistant forms emerging. However, there is doubt as to the effectiveness of certain of these combinations where the partner drug is already compromised [49] and there is recent evidence that resistance to the major coformulated combination of artemether-lumefantrine (Coartem®) may already be developing in parts of East Africa [50,51].

Several types of study, including immunolocalization, indicate that, upon activation by Fe²⁺, the artemisinins inhibit the Ca²⁺-dependent SERCA-like ATPase, PfATP6, a transporter found on membranous structures within the parasite cytoplasm [52]. However, such compounds form free radicals in vitro that can modify proteins and other molecules [52,53], which could also play a role in vivo. Studies in a yeast model suggested that disruption of the membrane potential of the mitochondrion might be an important factor [54], although localization of artemisinins to the mitochondria of P. falciparum has not been demonstrated and the relevance of these observations to the parasite system is still unclear [53]. More pertinent to the PfATP6 hypothesis, it has recently been shown in a heterologous (Xenopus oocyte) system that mutations in the key residue L263 close to the inhibitor binding site of this enzyme can abolish sensitivity to artemisinins. Disturbingly, field isolates that show reduced susceptibility to artemether associated with a S769N mutation in PfATP6 (but not with pfcrt or pfmdr1 genotypes) have been found in areas of French Guiana where uncontrolled use of artemisinin derivatives has occurred [55]. This serine residue is thought to be involved in a conformational change around a conserved hinge region of the molecule. These studies support the hypothesis that PfATP6, although possibly not the sole target, is a key player, as well as suggesting how artemisinin derivatives may be further developed in future [56,57]. Stable resistance to artemisinin and artesunate has also been induced in laboratory lines of the rodent parasite Plasmodium chabaudi, but no mutations or copy number changes were apparent in any of the genes that have been potentially implicated in the resistance of P. falciparum and other malarial species, including the homologue of pfatp6 [58]. This may reflect genuine differences between the distantly related human and rodent species of Plasmodium, which may not be relevant to P. falciparum, or, perhaps more likely, a warning that resistance in the latter may also eventually arise in the field by as yet unknown genetic alterations.

Concluding remarks

In general, microorganisms can employ a range of mechanisms to overcome drug challenge, in addition to structural modification of the target protein. These include amplification of the gene encoding the target, or its upregulation during transcription or translation, or by compromising the drug itself by inactivation or sequestration. However, to date, the principal (but not sole) strategy of P. falciparum appears to result in one or a small number of point mutations in genes encoding the key proteins. One lesson that is emerging is that certain of these mutations at the amino acid level directly affect drug binding or transport, whereas others are almost certainly compensatory, to permit the parasite to continue processing native substrates at levels that, although possibly suboptimal, are still compatible with viability. This raises the attractive possibility of administering simultaneously drugs that target the same molecule, but which by subtle structural variation, would select on their own mutually incompatible combinations of mutations, as proposed nearly a decade ago for the antifolates [59]. Proof of principle of such an idea has now been demonstrated for the case of Plasmodium vivax, where the mutations that confer high-level resistance to pyrimethamine cause the DHFR-TS enzyme to become up to 10-fold more sensitive to the powerful (nonclinical) antifolate, WR99210, compared with the wild-type [60]. Studies are currently underway to establish whether this phenomenon can be extended to P. falciparum, as seems likely from earlier work on pyrimethamine and WR99210 selection on randomly mutated pfdhfr libraries [61]. Similarly, the observation that primaquine, used for decades to clear the liver of the long-lived hypnozoite form of the parasite peculiar to P. vivax and Plasmodium ovale, can act as a chloroquine resistance reversal agent in P. falciparum, leads to the suggestion of combining these two extremely cheap drugs [62], and there is also evidence that Coartem and amodiaquine exert opposing selective pressures on the pfmdr1 gene [63]. Such approaches complement the strategy of deploying drug combinations against two (or more) unrelated targets, described above.

Although considerable progress has now been made in detailing underlying mechanisms, it would not be surprising if other, as yet unidentified, factors contribute to the precise levels of susceptibility to the various antimalarial drugs. For example, there is a recent suggestion that variation in copy number of the gene encoding GTP cyclohydrolase I, at the entry point of the folate biosynthetic pathway, may modulate levels of antifolate resistance in P. falciparum [64], although varying expression levels of pfcrt appear to have little influence on susceptibility to chloroquine in vitro [13]. As described above, copy number has been established as a major factor in the relationship of the pfmdr1 gene to mefloquine resistance. With regard to the host, their history of exposure to the parasite and hence immune status will influence the clinical outcome of drug treatment, and in the case of the antifolates, nutritional status can play a significant role, as higher levels of blood folate reduce the efficacy of pyrimethamine-sulfadoxine [65]. However, the seeming general predominance of point mutations in underlying resistance in the parasite, now well established for a number of key genes, at least permits relatively straightforward epidemiological surveys based on PCR, which can be multiplexed to provide information on several genes simultaneously [66,67], not only from patient blood samples, but from mosquito populations as well [68]. Importantly, such surveys can also include longitudinal studies of samples stored long ago, before key genes and their allelic variants were identified. For example, it has now been shown that, after chloroquine was withdrawn from use in Malawi in 1993, prevalence of the resistant form of pfcrt decreased from about 85% in 1992 to an undetectable level in 2001 [69]. A similar increase in frequency of the wild-type allele has been observed for the dhfr domain of the pfdhfr-ts gene following reduced usage of pyrimethamine–sulfadoxine in an area of Tanzania, thanks to the deployment of bed-net protection from mosquito bites [70]. In the case of atovaquone resistance, mutations in the cyt b gene also appear to result in loss of fitness [71], as do those in the pfmdr1 gene [72]. However, parasites mutant in dhfr are still prevalent in parts of South East Asia and elsewhere, despite low usage of antifolates in these areas for prolonged periods, suggesting that in some contexts, the mutant parasites retain a small but significant selective advantage [73]. Such studies are highly valuable when considering changes in a given drug regime at a national or more local level [74]. Related to this are the analyses of variable DNA sequences flanking the coding regions of the target genes from large numbers of geographically diverse field samples, which also provide considerable insight into the evolution and spread of drug-resistant strains. Unexpectedly perhaps, the picture that has emerged here, both for the antifolates and chloroquine [75,76], is consistent with the widespread migration of a relatively small number of rare ancestral mutant alleles across countries and continents, rather than the frequent, independent genesis of mutant types in numerous different locations where drug pressure is strong. However, some data on pfdhfr from western Kenya that are more compatible with the latter scenario have also been reported [35], as has a distinct lineage of antifolate-resistant strains originating in the Melanesian islands [77].

The complete genome sequences of several Plasmodium species, including P. falciparum, are now known, and discoveries in this fertile postgenomic era suggest a plethora of new areas to explore, exemplified by the broad spectrum of potential targets that can be envisaged, for example, in just one area of metabolism, that of purine and pyrimidine biochemistry [78]. Other studies, too numerous to detail here, envisage deployment of entirely new classes of drugs, targeted, for example, to recently discovered metabolic pathways in essential organelles of the parasite, the apicoplast [79] and the mitochondrion [80,81]. However, the translation of fundamental research into clinically licensed drugs that operate in new ways or can overcome resistant forms of the parasite is a formidable challenge, and careful assessment of the most promising of these targets is required to validate them as bona fide candidates, before the labour- and cost-intensive commitment to long-term development. The latter of course entails considerable hurdles in the many steps between identification of a seemingly attractive enzyme or transporter target and the eventual formulation of a safe and effective medicine, where problems of solubility, effective delivery, toxicity to the host and other potential pitfalls must first be tackled. Better though to be spoilt for choice, rather than wondering where the next target is coming from, as was the case not so many years ago.

Abbreviations

CRT

chloroquine resistance transporter

DHFR

dihydrofolate reductase

DHPS

dihydropteroate synthetase

HPPK

hydroxymethylpterin pyrophosphokinase

TS

thymidylate synthetase