Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs

Microbes and Infection 4 (2002) 165–174 www.elsevier.com/locate/micinf

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Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs John E. Hyde * Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), P.O. Box 88, Manchester M60 1QD, UK

Abstract Chemotherapy and chemoprophylaxis are the principal means of combating malaria parasite infections in the human host. In the last 75 years, since the introduction of synthetic antimalarials, only a small number of compounds have been found suitable for clinical usage, and this limited armoury is now greatly compromised by the spread of drug-resistant parasite strains. Our current knowledge of the molecular mechanisms underlying resistance in the lethal species Plasmodium falciparum is reviewed here. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Antifolates; Chloroquine; Mefloquine; Quinine; Halofantrine; Artemisinin; Atovaquone

1. Introduction Malaria affects the populations of tropical and subtropical areas world-wide, as well as an increasing number of travellers to these areas. Of the four species of Plasmodium that cause malaria in humans, Plasmodium falciparum is the most dangerous, as the pathology it induces often leads to death. Although malaria is found in over 100 countries, the major burden of disease is carried by the nations of Africa, where over 90% of all deaths from falciparum malaria are recorded, and where the high levels of morbidity and transmission place considerable strains on public health services and economic infrastructure. In the absence of effective vaccines, management of the disease has depended largely upon chemotherapy and chemoprophylaxis. Of the various antimalarial drugs available, the aminoquinoline chloroquine was for several decades the agent of choice, as it was safe, effective and cheap. Parasite resistance to this drug was first observed in Thailand in 1957 and then on the border of Colombia and Venezuela in 1959. By the late 1970s it had spread to East Africa and by the mid-1980s had become a major problem in several areas of the continent [1]. An increasing number of countries have been forced to adopt a different class of drug, the antifolates,

* Corresponding author. Tel.: +44-161-200-4185; fax: +44-161-2360409. E-mail address: [email protected] (J.E. Hyde). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 1 2 8 6 - 4 5 7 9 ( 0 1 ) 0 1 5 2 4 - 6

as the first-line alternative to chloroquine. The most widely used combination of this type consists of pyrimethamine (PYR) and sulfadoxine (SDX), known as Fansidar or SP, which is also cheap and, until recently, was effective against the chloroquine-resistant parasites found in Africa. However, resistance to this formulation, long established in parts of South-East Asia and South America [2], now threatens to leave Africa with no treatment affordable on a mass scale. Further combination of antifolates with newer drugs such as the artemisinin derivatives, or the development of alternative combinations, may be the only way to limit the pace of parasite resistance to chemotherapy. For example, the antifolate prodrug proguanil has now been formulated together with a new type of inhibitor, atovaquone, to yield Malarone, recently licensed for clinical use. What is known about the mechanisms of parasite resistance to these and other important antimalarial drugs is reviewed below.

2. The antifolates In contrast to other antimalarial drugs, the primary molecular targets of the antifolates have long been established, and much progress in understanding how resistance arises has been made since 1987, when the first gene encoding such a target was cloned [3]. The principal antifolate drugs are pyrimethamine (PYR), proguanil (PG; metabolised in vivo to the active form cycloguanil, CG), the sulfonamides, such as sulfadoxine (SDX) and sulfalene, and

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the sulfone, dapsone. Although PYR and PG were used alone for a period after their introduction, resistance arose rapidly and it was only in combination with sulfa drugs that effective formulations were achieved that are still widely used today. This arises from the profound synergy observed between the components of these combinations [4]. PYR and SDX inhibit the folate biosynthetic pathway, targeting, respectively, dihydrofolate reductase (part of a bifunctional protein with thymidylate synthetase, DHFR-TS [3]), and dihydropteroate synthetase (part of a bifunctional protein with hydroxymethylpterin pyrophosphokinase, PPPK-DHPS [5,6]). DHPS links p-aminobenzoic acid (pABA) to a 7,8-dihydropterin, forming dihydropteroate in the step preceding dihydrofolate synthesis. DHFR activity is essential in maintaining a constant supply of folate cofactors for key one-carbon transfer reactions, in which only the fully reduced (tetrahydro) forms are functional. These reactions are essential for provision of nucleotides for DNA synthesis and in the metabolism of certain amino acids. 2.1. Mutations in the dhfr gene The detailed molecular basis of clinical PYR/SDX failure is not yet completely clear, although thanks to a combination of genetic analyses, biochemical assays, mutagenesis and transformation studies, we now have a good grasp of molecular events involved in resistance to the individual components. Variant sequences of P. falciparum DHFR were first described in the late 1980s [7–9], and it is now well established that high-level PYR resistance results from the accumulation of mutations in the dhfr domain, principally at codons 108, 59 and 51, where allelic variation gives rise to S108N, N51I and C59R. Positions 16 and 164 (A16V and I164L), as well as an alternative alteration in position 108 (S108T), are also involved in resistance to anti-DHFR inhibitors, particularly in the case of CG, the active metabolite of PG (reviewed in [10]). This relationship between resistance and a structurally modified enzyme was first suggested by correlations between the mutant sequences found in different parasites and levels of PYR resistance in in vitro cultures thereof. Subsequently, active DHFR-TS was isolated from several parasite strains whose level of resistance could be inversely correlated with the degree of drug binding to DHFR [11]. This type of study was considerably extended by constructing recombinant genes carrying the known mutations, either singly or in combination, placing them in an Escherichia coli expression system, and measuring drug binding and other kinetic parameters on the purified expressed protein [12]. Similarly, relative IC50 values for a range of antifolates have been determined in a yeast expression system carrying mutant forms of the pfdhfr domain [13,14]. Both of these types of study demonstrate marked decreases in drug susceptibility as mutations accumulate. Conclusive proof of their importance was provided by the recently developed technique of in vitro transformation of drug-sensitive parasites with vector constructs bear-

ing mutant forms of dhfr, and showing that they acquired the predicted level of PYR resistance [15]. However, it would be unwise to conclude that we now know everything about PYR resistance in the field. Studies in vitro where cultures were subjected to step-wise increases in drug pressure over long periods (up to a year) demonstrate that chromosomal rearrangement and gene amplification can occur which affect the level of expression of the DHFR target [16], and there is heterogeneity in the promoter region of the dhfr-ts gene from different parasite lines (P. Wang and J.E. Hyde, unpublished). Although expression levels of mRNA and protein in field isolates have not been systematically measured, these phenomena cannot be ruled out as potential contributory factors to clinical drug resistance. 2.2. Mutations in the dhps gene In 1994, the gene encoding PPPK-DHPS was characterised [5,6], allowing significant progress to be made in exploring the basis of resistance to SDX and related sulfa compounds. In these two initial studies, variations were reported in codons 436, 437, 581 and 613 in the dhps domain of cultured parasite lines, which could be broadly correlated with estimated levels of SDX resistance. Later studies revealed that variability in codon 540 was also commonly observed in field samples [17–19]. To date, only changes in these five codons have been observed in a large number of field samples of diverse geographical origins, e.g. [17–20]. Position 436 appears to be tetramorphic, with mutation in the 436 codon giving rise to Ala, Phe or Cys, from the wild-type Ser. For the 613 codon, trimorphism is seen, giving rise to Ser or Thr from Ala. Positions 437, 540 and 581 appear to be dimorphic, with codons 437 and 581 both showing a change from Ala to Gly, while codon 540 changes from Lys to Glu. The most common alteration is A437G which, in one large field study where the dhps genes of all samples were fully sequenced, was seen in 84/141 samples [19]. Of the 13 fully characterised mutant alleles described to date, 10 involve this alteration, although samples carrying just this mutation appear to be rare. Common in East Africa are the A437G, K540E and A437G, A581G combinations [17,19,21]. The latter is also common in South-East Asia [19], while in areas of South America, samples with all three of these mutations have been frequently observed [17,22]. While the mutations affecting positions 436, 437 and 540 can each occur singly, the data so far suggest that the A581G alteration is always found with A437G, and that the A613S/T alterations must also be coupled to changes in either residue 436 or 437, presumably reflecting steric constraints of the enzyme. As for dhfr, further studies have explored the relationship of the dhps mutations with drug inhibition. After overcoming the major technical difficulties of reliably quantitating SDX resistance [23], the parents and 16 progeny of a genetic cross between two cloned lines [24,25], one highly sensitive to SDX (HB3) and one highly resistant (Dd2),

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were used to demonstrate that the inheritance of mutations in the dhps gene conferred upon the progeny a similar level of SDX resistance to that shown by the mutant parent. Moreover, in these and a series of unrelated lines, the degree of SDX resistance correlated well with the number of altered residues in DHPS [26]. The most SDX-resistant parasites assayed in vitro carried the mutations S436F, A437G and A613S, and displayed IC50 values ca. three orders of magnitude higher than parasites with wild-type DHPS. Parallel to this work, engineered forms of the parasite enzyme carrying the various altered amino acids have been purified from E. coli and their drug-binding characteristics examined [18]. These studies provided powerful evidence that altered amino-acid residues in DHPS convey high levels of SDX resistance. As with dhfr, this conclusion was eventually put beyond doubt by measurements of SDX resistance levels in sensitive parasites stably transformed in vitro with different mutant forms of the dhps gene, which showed that expected levels of resistance were acquired by the recipient parasites [27]. 2.3. Physiological folate levels and antifolate synergy The effectiveness of PYR/SDX and similar combinations was originally thought to derive from the dependency of the parasite on folate biosynthesis and the sequential blocking of two steps in the pathway to fully reduced folate. Early indications that the situation was more complex arose from observations that the action of sulfa drugs was strongly antagonised by the presence of preformed folate in the medium [28,29]. This antagonism and the difficulty in controlling folate concentrations in culture were the major reasons why a sensitive and reproducible quantitative assay for SDX resistance was so difficult to develop, and IC50 values for the effects of the various dhps mutations on SDX resistance have all necessarily been derived in near-zero folate conditions [26,27]. However, in the human host, which is dependent upon preformed folate in the diet, parasites are exposed to significant levels in the plasma. It was found that different parasite lines can vary greatly in their susceptibility to this so-called ‘folate effect’ in vitro [23,26] and that concentrations of folate comparable to those found in human plasma were capable, for some parasite lines, of conferring levels of SDX resistance even higher than those arising from triply mutated dhps. This apparent resistance arises from the ability of some, if not most, strains of the parasite to salvage pre-formed folate, thus by-passing the SDX blockage of de novo folate synthesis. A more detailed study of this phenomenon, where parasites were assayed in the presence of varying concentration of both PYR and SDX simultaneously, indicated that the presence of PYR significantly reduces the folate effect compared to its level when parasites are challenged with SDX alone [30]. As well as providing a putative explanation for the observed synergy between these drugs, it follows that the status of the dhps gene becomes important once

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mutations have occurred in dhfr to a degree where inhibition of DHFR alone by normal therapeutic levels of PYR is insufficient in itself to kill the parasites [31]. In practice, it appears that parasites that are at least triply mutant in dhfr (encoding 108N, 59R, 51I), combined with at least doubly mutant dhps (encoding 437G, with either 540E or 581G) will result in PYR/SDX failure, whereas predicting the outcome for parasites with lower levels of mutation in these genes is much less certain, being more highly dependent upon additional factors such as the immune status of the host and the rate at which the latter metabolises the drugs. 2.4. Structural studies of DHFR and DHPS Although neither the parasite DHFR-TS protein nor the isolated DHFR domain has yet been crystallised, DHFR from other organisms represents one of the best studied enzymes at the level of tertiary structure, as revealed by X-ray diffraction and NMR data. It has thus been possible to employ homology modelling to predict a structure for P. falciparum DHFR [32–34]. Although the degree of amino-acid sequence similarity between parasite DHFR and its homologues is not particularly high overall, active site residues are well conserved and sites of mutation in the parasite DHFR correspond to residues close to the active site in bacterial homologues [10,35]. The recent determination of the first X-ray diffraction patterns of DHPS molecules, from E. coli [36], Staphylococcus aureus [37] and Mycobacterium tuberculosis [38], should also help to rationalise how the mutations described above structurally alter this enzyme so that drug binding is greatly reduced, while permitting sufficient processing of normal substrate. It was apparent from simple alignments that the changes in residues 436 and 437 occur in regions of the P. falciparum molecule that are highly likely to be intimately involved with substrate binding [27], but more recent computer modelling now suggests that all five of the polymorphic amino acid residues observed are likely to form part of the only solvent-accessible channel connecting to the catalytic centre [38,39]. While extrapolation from DHFR and DHPS molecules of other organisms can provide important insights, crystallisation and/or NMR studies of the actual molecules of interest are still ultimately required. Detailed structural knowledge of this type can permit the design of improved inhibitors that are able to overcome resistance to the original compound, as has already been achieved using homology modelling for P. falciparum DHFR [32,33].

3. Resistance to chloroquine, quinine, mefloquine and halofantrine Although chloroquine (CQ) resistance, like antifolate resistance, has been apparent for over 40 years, progress in understanding the underlying mechanism(s) has been less

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rapid. It was clear from the much slower rate of appearance and spread of CQ resistance that it was likely to be more complex genetically than antifolate resistance. As with any drug, knowledge of its mode of action is an essential component of understanding how an organism becomes resistant, although its ultimate target and the molecules mediating resistance to it need not be directly connected, as they clearly are in the case of the antifolates. Chloroquine, a weak base, was known for some time to accumulate to a high level in the acidic food vacuole of the parasite. In its non-protonated form, it can diffuse across the vacuolar membrane where it becomes protonated. This inhibits exit of the molecule, which accumulates at mM levels in the vacuole from nM levels in the plasma [40], although such a high degree of accumulation may depend on factors other than just the pH gradient. This concentration of the drug prevents the proper sequestration of toxic haem moieties that arise from parasite digestion of haemoglobin in the erythrocyte, so the parasite effectively dies from exposure to its own waste. In untreated parasites, the haem is polymerised into an inert form called haemozoin or malaria pigment, although some may also be removed in a degradative pathway [41]. In resistant parasites, CQ is observed to accumulate to much lower levels [42]. This could in principle arise by a lower rate of influx, a higher rate of efflux, or a combination of both, and would also be influenced by any reduction in binding affinity of the drug for its ultimate target. While a higher rate of efflux was originally proposed to be the major factor [43], later studies indicated that changes in uptake better explained the observed kinetics [44–46]. Earlier studies had reported that cross-resistance was apparent among some parasites resistant to CQ with antimalarials related to CQ, such as the arylaminoalcohols quinine and mefloquine, and the phenanthrene alcohol halofantrine [47–49]. Morphological studies indicated that they too were acting in the food vacuole, and protease inhibitors have recently been used to investigate the effects of haemoglobin degradation on the action of these drugs [50,51]. The inhibitors were found to antagonise the antiparasitic activity of the drugs and severely reduce their incorporation into malarial pigment, illustrating the close connection between haemoglobin degradation and drug action, whereby the haem-quinoline complexes that are formed interact with the growing haem polymer, thus ‘capping’ it and preventing further incorporation of haem. In a complementary study [52], the binding of such complexes with the haem polymer was shown to be specific and of high affinity. Moreover, the relative binding affinities correlated well with the ability of the individual drugs to inhibit further haem polymerisation. Haemozoin released from both CQsensitive and CQ-resistant parasites showed similar affinities for the CQ-haem complex [52], consistent with the idea that CQ resistance results rather from a reduced level of access to the haem target [46].

3.1. A CQ-resistance determinant on chromosome 7 To elucidate the genetic basis of CQ resistance, two main approaches were adopted in the late 1980s, which have continued to yield increasingly detailed insights, not only into CQ resistance, but also into resistance to the other drugs mentioned above. A highly systematic and technically demanding approach involved crossing two cloned parasite populations, respectively CQ-sensitive (HB3) and CQresistant (Dd2), during the sexual stage in the mosquito and isolating recombinant progeny at the asexual stage in a primate host [24]. Phenotypic typing of progeny and mapping of their recombination loci using both RFLP [25,53] and microsatellite markers [54] localised a key gene to a region of about 36 kb on chromosome 7. Sequencing along this region led to the identification of several potential open reading frames (ORFs), named cgX for ‘candidate gene’. These were analysed for polymorphisms that might correlate with the CQ-resistance phenotype in a large array of P. falciparum strains from around the world. An initially promising candidate, cg2, showed a complex pattern of polymorphisms that were tightly, but not perfectly, linked with CQ resistance. This ORF encoded a large (∼330 kDa) protein located at the parasite periphery and associated with haemozoin, and was thus potentially involved in CQ action [55]. However, transfection studies where the alleles of both the cg2 and cg1 genes in CQ-resistant parasites were replaced by their variant counterparts from CQ-sensitive parasites showed that they had no effect on the level of resistance of the transformed parasites [56]. The observed strong linkage thus indicated that another gene must lie in very close proximity whose product was more relevant to resistance. Further transcriptional analysis of the surrounding DNA sequence eventually revealed a highly fragmented ORF (13 exons), polymorphism in which showed complete linkage to the CQ-resistance phenotype in 40 strains examined [57]. This gene, named pfcrt (for ‘chloroquine resistance transporter’), is polymorphic in 10 codon positions, but only one amino-acid change, K76T, was found consistently only in CQ-resistant parasites, although a second alteration, A220S, shows almost complete linkage. cDNA versions of this gene from resistant parasites have been inserted by transformation into sensitive parasites and shown to bestow the resistance phenotype with the status of position 76 being especially critical. This is reflected in a recent field study in West Africa where all CQ-resistant strains carried both the K76T and A220S changes compared to about 40% prevalence in random pre-treatment samples [58]. Further field studies in different regions should determine the reliability of these polymorphisms as indicators of clinical outcome. Transfection of mutant pfcrt alleles also led to increased acidification of the food vacuole in recipient parasites as measured by acridine orange fluorescence [57]. While this would appear at first sight to lead to an increased accumulation of the dibasic CQ, it is suggested that this may be outweighed by the fact that the pH drop drives soluble

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haem into an insoluble form, steeply reducing the number of CQ-binding sites and thus the level of toxic haem/CQ complex formation, consistent with earlier hypotheses [42]. It is also possible that the mutations, which occur largely within predicted transmembrane segments, somehow reduce the inward flux of CQ across the actual membrane. 3.2. The role of the P-glycoprotein homologue, Pgh1, in resistance The second approach was to assume that an ABC-type membrane transporter would be involved in altering flux of CQ into the food vacuole, by analogy with mammalian tumour cells exhibiting a multidrug-resistance (mdr) phenotype, where a gene product called P-glycoprotein is upregulated and mediates an ATP-dependent expulsion of a wide range of inhibitors. This expulsion can itself be reversed by the use of Ca2+ channel blockers like verapamil, and the discovery that CQ resistance levels in P. falciparum were also diminished by verapamil [59] lent strong credence to this approach. Two genes, pfmdr1 and pfmdr2, encoding homologues of a human mdr-type gene were initially identified [60,61]. The pfmdr2 gene was eventually eliminated as a candidate as expression levels were unchanged between CQ-resistant and CQ-sensitive parasites [62,63], no polymorphism among resistant and sensitive parasites has been observed, and the predicted architecture of its gene product differed significantly from that of mammalian mdr-encoded proteins [64]. However, polymorphisms apparently strongly linked to CQ-resistance in the pfmdr1 gene were identified in a number of samples soon after its isolation [65], although correlations of these alterations with CQ resistance were not always evident in further surveys of geographically diverse samples [66–68]. Moreover, the pfmdr1 gene mapped to chromosome 5 [60] and so was not easily reconciled with the importance of the locus on chromosome 7 identified from the genetic cross study described above. Nevertheless, the location of its gene product Pgh1 (for ‘P-glycoprotein homologue’) on the surface of the food vacuole of mature parasites, and the much closer similarity of this protein to mammalian P-glycoproteins, indicated that it might modulate intracellular chloroquine concentrations in some way [69]. The changes that affect Pgh1 all appear to be dimorphic: N86Y, Y184F, S1034C, N1042D and D1246Y. Although it has been difficult to discern a simple pattern of differences involving these residues between CQ-resistant and CQsensitive strains, strong but imperfect statistical associations between 86Y and CQ resistance in West and Central Africa have been reported [58,70]. However, this mutation has not been observed in a large number of South American strains resistant to CQ [65,71]. Conversely, 1246Y is particularly common in such strains, but has also been seen in CQsensitive parasites. Recently, the situation has at least been partially clarified, again by using the power of transfection studies. Variant pfmdr1 genes carrying three of the above

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mutations (1034C, 1042D and 1246Y) have been transfected into CQ-sensitive parasites (D10), which carry the allelic variant encoding 1034S, 1042N and 1246D [72]. Interestingly, acquisition of these altered residues had no effect on the level of CQ resistance, but removal of the same mutations from a CQ-resistant strain (7G8) resulted in a two-fold drop in the resistance level. Thus, alteration of residues 1034, 1042 and 1246 appears insufficient to confer CQ resistance alone, but must presumably act in concert with another gene product to give rise to the level seen in 7G8. Accumulation of the drug was measured in the various transfected lines and found to be lower in the mutant 7G8 line than in 7G8 modified to restore the ‘wild-type’ residues in the three positions. This provides direct evidence for the involvement of Pgh1 in CQ accumulation. Given the similarities of quinine, halofantrine and mefloquine to CQ in their structures and site of action, some effect of these mutations on their efficacy might also be expected, and this was confirmed in the same study [72], though with subtly varying results. Alteration of the 1034, 1042 and 1246 residues in the sensitive D10 strain doubled the IC50 level for quinine, which reverted to the ‘wild-type’ level when these residues were engineered back in 7G8. For mefloquine and halofantrine, however, the opposite effect was observed. In general, mutation of these residues in D10 led to an increase in sensitivity to these drugs, most marked if only the D1246Y mutation was implemented (4–5-fold effect), implicating this residue as the most critical in influencing mefloquine and halofantrine accumulation. This sensitivity was ablated to a comparable degree when 7G8 was mutated back to ‘wild-type’. In a separate study, complete linkage was seen between the mefloquine resistance status and the pfmdr1 allelic type in a genetic cross between the HB3 and 3D7 clones [73], both of which are CQ-sensitive, emphasising that the pattern of mutations in any given field sample may reflect a complex interplay of selection pressures arising from differing degrees of exposure to the various quinoline-type drugs and differences in their precise mode of interaction with the parasites. As well as polymorphisms arising from point mutations, variation in copy number of the pfmdr1 gene has been observed in a number of studies, either in samples isolated from the field [60,66,74], or arising through deliberate selection of highly resistant progeny of laboratory strains [61,75–77]. Although amplification of pfmdr1 was observed in a number of CQ-resistant isolates, this correlation could not be substantiated across a wide variety of strains [24,67]. Moreover, adapting laboratory strains carrying multiple copies of pfmdr1 to high concentrations of CQ resulted in de-amplification of this gene, indicating that high levels of the P-glycoprotein were in fact incompatible with pronounced CQ resistance [75]. Conversely, resistance to mefloquine, halofantrine and quinine does seem to be causally linked to pfmdr1 amplification [66,76], although, as described above, intragenic point mutations also influence levels of resistance to these inhibitors. It has also been

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observed, however, that one parasite line selected for high-level halofantrine resistance and showing a concomitant enhancement of mefloquine resistance showed no alteration of pfmdr1, either in sequence or in copy number [78]. Thus, it seems clear that there are several possible contributory factors and mechanisms involved in the acquisition of resistance to these drugs. It is interesting to note in this regard that in CQ-resistant forms of Plasmodium vivax, the other major, but normally non-lethal human malaria parasite, variation in the homologue of pfcrt does not appear to play a role in conferring resistance [79].

4. Artemisinin derivatives An entirely different class of compound originates from the Chinese herb qinghao (Artemisia annua), used for centuries to treat malaria and other parasitic diseases. The parent compound is artemisinin (qinghaosu), first isolated in the 1970s, from which several analogues, such as sodium artesunate, dihydroartemisinin, arteether and artemether, have been derived with varying pharmacokinetic properties. These sesquiterpene lactones carry an epoxide bridge across the seven-membered ring, and act rapidly on the asexual blood stages. They are effective against parasites multiply resistant to other antimalarials, and are thus already in widespread use in South-East Asia. In vitro studies indicate that resistance can develop, but as yet seems not to be of clinical significance, a situation unlikely to continue indefinitely. These compounds also have the considerable advantage over other antimalarials of possessing gametocytocidal action [80], which acts to limit transmission to new hosts and slow down the spread of resistant forms. This differs from the situation with CQ, where drug-resistant parasites have been reported to have higher gametocyte densities [81], favouring the transmission of these forms over sensitive strains. Moreover, they can inhibit parasite forms at an earlier stage in the asexual cycle than the quinoline-based drugs, reducing the number of parasites that will mature sufficiently to sequester in the microcapillaries of vital organs. A number of studies have shown that the endoperoxide bridge in these compounds can be cleaved by a reductive interaction with iron, as Fe(II), provided either as ferrous haem or in other (exogenous) forms, yielding one or more types of free radical that could alkylate or otherwise covalently modify malarial proteins [82,83]. Additional experiments indicated that the mode of action showed similarities to that of the quinolines, in that the drug also accumulates in the food vacuole and interacts with haemozoin [84]. The drug can form covalent adducts to haem itself [84,85] and to proteins of the parasite [86,87] or host cell [88], while in a Plasmodium yoelii model, it has been proposed to interfere with the haemoglobin catabolic pathway, apparently via inhibition of the cysteine protease activity of the food vacuole, leading to accumulation of

undegraded haemoglobin in treated parasites [89]. Moreover, artemisinin induced a concentration-dependent breakdown of purified malarial haemozoin in vitro, which, if reproduced in vivo, could further reduce the efficacy of the detoxification system, increase the level of free haem and thus promote further free radical reactions and damage to the parasite [89]. There are conflicting reports as to whether artemisinin additionally inhibits the actual polymerisation of haem [89,90] and the precise details of the activation of the artemisinin drugs and their most important targets are still to be settled [83]. However, although the mode of action does show some similarities to that of the quinolines, the quite different molecular structures of these classes of compounds means that mechanisms of altered susceptibility might well involve a different subset of malarial proteins. One common protein, though, is Pgh1, in which the S1034C, N1042D and D1246Y mutations can increase the sensitivity to artemisinin about 2-fold, as revealed by the transfection study with engineered pfmdr1 genes described earlier [72]. An increase of similar magnitude has been linked to the N86Y alteration in a study of the parents and progeny of the HB3-3D7 genetic cross, together with a range of unrelated lines [73]. Although no significant resistance to these compounds in the field seems yet to have appeared, monotherapy does lead to recrudescence, possibly due to suboptimal drug levels. It has also been suggested that some parasites respond to the drug pressure by entering a dormant state that is resistant to further inhibition [91]. It is therefore preferred to administer a combination of the artemisinin derivative with a longer acting drug of a different family, to increase the likely demise of any residual parasites [92].

5. Naphthoquinones Atovaquone is a highly substituted naphthoquinone derivative and a structural analogue of coenzyme Q (ubiquinone) in the mitochondrial electron transport chain. It is thought to interfere with the transfer of electrons generated by pyrimidine synthesis when dihydroorotate dehydrogenase oxidises dihydroorotate to orotate. These electrons are involved in maintaining the membrane potential of the mitochondrion, essential for the transport of proteins and small molecules in and out of the organelle [93]. Atovaquone has been shown to collapse the membrane potential of P. yoelii, which remains unaffected when exposed to other malarial inhibitors such as CQ and tetracycline [94]. The rapid effect on respiration appears to arise from inhibition by the drug of the cytochrome bc1 complex of the electron transport chain of the parasite [95]. Specifically, independent resistant lines of P. yoelii [95] or of P. falciparum [96] showed either single or double point mutations in the cytochrome b gene, which have been correlated with atovaquone resistance in vitro. One of these mutations was also found in parasites isolated from a

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recrudescent patient following atovaquone treatment [96]. In both studies, these mutations were associated with up to a 103–104-fold reduction in parasite susceptibility to the drug. Molecular modelling suggests that the affected residues are clustered within a highly conserved region of the molecule which is involved in the ubiquinol oxidation step and is thus a putative atovaquone-binding site whose affinity for the drug is diminished after mutation. Using cloned lines and PCR analysis in the P. yoelii study [95], it was concluded that such mutations were arising spontaneously during treatment of the mice. Similarly, monotherapy with atovaquone against P. falciparum was shown to result in clinical resistance very rapidly [97]; however, a combination of atovaquone with PG (Malarone) provides a powerful and effective treatment [98]. The role of the PG in this combination appears to be unrelated to its known properties as an antifolate precursor. Thus, although CG, the metabolite of PG, is a DHFR inhibitor, PG itself has an intrinsic activity against parasites. This was suggested from observations of patients with poor metabolism of PG, compared to controls, where synergism with atovaquone appeared not to be affected [99]. Moreover, in transfected parasites where the DHFR activity was provided from a highly drug-resistant human gene, PG, but not CG or PYR, was still able to inhibit parasite growth [100]. Similarly, in vitro IC50 studies show that PG inhibit parasites with a wide range of genotypes in the 1–20 µM range (P. Wang and J.E. Hyde, unpublished). An analysis of proguanil action found that the drug alone had no effect on either electron transport or the mitochondrial membrane potential, although it increased the ability of atovaquone to collapse the potential when used in combination [101]. The basis of this synergistic effect is thus still unclear and Malarone-resistant parasites have yet to be collected and analysed.

6. The accelerated resistance to multiple drugs (ARMD) phenotype The experience of many field workers has been that parasites prevalent in areas of South-East Asia, particularly regions of Thailand and Cambodia, appear to become resistant to the various antimalarial drugs faster than is the case in other parts of the world. This phenomenon has been systematically investigated by challenging parasite lines of diverse origins with inhibitors to which they had never been previously exposed [102]. The drugs chosen were 5-fluoroorotate, a compound that has never been used as a clinical antimalarial drug, and atovaquone, which at the time of the study had also not been used in the field. These drugs were initially equally effective on parasite lines of diverse origins at low nM concentrations. However, the Indochina strain W2, which is highly resistant to a range of clinical antimalarials, developed resistance independently to both of these drugs at a rate ca. 1000-fold that of an African strain (D6) sensitive to all antimalarials. This suggests that

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some parasites may have acquired something akin to the well known ‘mutator’ phenotype observed in E. coli and other cell types [103], whereby DNA editing or repair mechanisms are relaxed, permitting a greater range of mutant genotypes to be tested for fitness, in a given time period, against particular environmental challenges.

7. Conclusion Drug-resistant falciparum malaria is a major killer and one of the most important obstacles to world health, especially in the developing countries of Africa. Although the strenuous efforts of many workers over the last 10–15 years have shed considerable light upon mechanisms of drug resistance in P. falciparum and other malaria species, we still have a fairly narrow and selective view of events taking place in parasites that have adapted to drug challenge. The imminent completion of the P. falciparum genome sequence, together with the rapidly enlarging sequence databases for other human, simian and rodent species of Plasmodium, should allow for the first time more global assays of differences between drug-resistant and drug-sensitive parasites. This should soon give us a better understanding of the molecular subtleties involved in resistance to current antimalarials, as well as suggesting other areas of parasite metabolism that could potentially be targeted by novel inhibitors.

Acknowledgements Work from the author’s laboratory referred to in this review was supported by the MRC, UK, and grants 037986, 046643 and 056845 from the Wellcome Trust, UK.

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