The parasite Plasmodium vivax is a major public health

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1 Pyrimethamine and WR99210 exert opposing selection on dihydrofolate reductase from Plasmodium vivax Michele D. Hastings and Carol Hopkins Sibley* Department of Genome Sciences, University of Washington, Box , Seattle, WA Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved July 18, 2002 (received for review May 16, 2002) Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent. Until very recently, the parasite has been effectively treated with chloroquine, but resistance to this drug has now been reported in several areas. Affordable alternative treatments for vivax malaria are urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate reductase (DHFR) that has been widely used to treat chloroquine-resistant Plasmodium falciparum malaria. DHFR inhibitors have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax. P. vivax cannot be grown in culture; the reason for its resistance to DHFR inhibitors is unknown. We show that, like P. falciparum, point mutations in the dhfr gene can cause resistance to pyrimethamine in P. vivax. WR99210 is a novel inhibitor of DHFR, effective even against the most pyrimethamine-resistant P. falciparum strains. We have found that it is also an extremely effective inhibitor of the P. vivax DHFR, and mutations that confer high-level resistance to pyrimethamine render the P. vivax enzyme exquisitely sensitive to WR These data suggest that pyrimethamine and WR99210 would exert opposing selective forces on the P. vivax population. If used in combination, these two drugs could greatly slow the selection of parasites resistant to both drugs. If that is the case, this novel class of DHFR inhibitors could provide effective and affordable treatment for chloroquine- and pyrimethamine-resistant vivax and falciparum malaria for many years to come. The parasite Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent (1). Until very recently, the parasite has been effectively treated with chloroquine, but resistance to this drug has now been reported in several areas (1 13). Affordable alternative treatments for vivax malaria are urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate reductase (DHFR) that has been widely used to treat chloroquineresistant Plasmodium falciparum malaria (14). DHFR inhibitors have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax (15, 16). P. vivax cannot be grown in culture; the reason for its resistance to DHFR inhibitors is unknown. DHFR (E.C ) is a key enzyme in the metabolism of all cells. Inhibition of DHFR activity depletes the cellular pool of tetrahydrofolate, a cofactor that is essential for both DNA and protein synthesis, and specific inhibitors have been designed for both prokaryotic and eukaryotic pathogens (17). Pyrimethamine is a specific competitive inhibitor of DHFR from P. falciparum, (18) and with sulfadoxine, is one component of the antimalaria drug, Fansidar. In P. falciparum, extensive field and laboratory studies have shown that resistance to pyrimethamine is caused by point mutations in the dhfr gene (reviewed in refs. 15 and 19 21). However, in contrast to P. falciparum, P. vivax cannot be maintained in culture and so the mechanism of resistance to pyrimethamine has not been established for this species. The dhfr gene from P. vivax was recently cloned, and a number of alternative of the gene were identified (22, 23). Fig. 1 compares the alignment of the dhfr coding regions from P. falciparum and P. vivax. The alignment demonstrates that the polymorphisms observed at positions 58, 117, and 173 of the P. vivax sequence correspond to the changes in codons 59, 108, and 164 that are known to cause pyrimethamine resistance in the P. falciparum enzyme (24, 25). Several of these mutant P. vivax enzymes were purified and showed less sensitivity to inhibition by pyrimethamine in vitro (26, 27). In addition, a recent study showed that polymorphisms in amino acids 57, 58, and 117 are very common in Thailand where pyrimethamine-sulfadoxine has been used extensively, but less prevalent in India and Madagascar where antifolate use has been lower (28). These studies suggested that amino acid substitutions might be responsible for pyrimethamine resistance in P. vivax. Because P. vivax cannot be maintained in culture, there is no way to test reference strains in vitro to set criteria for relative drug sensitivity; drug testing can be accomplished only by short-term treatment of parasites newly isolated from patients. This requirement has severely hampered studies of drug efficacy and has made it difficult to determine the relative importance of point mutations in the P. vivax dhfr gene for antifolate resistance. We have developed a system for expression of DHFR enzymes from pathogens in strains of the budding yeast, Saccharomyces cerevisiae, that lack endogenous DHFR activity (29, 30). This approach allows rapid assessment of the relative sensitivity of heterologous DHFR enzymes to inhibitors, simply by measuring the growth of the yeast in the presence of potential inhibitors of the enzyme. We and others have shown that for a range of of P. falciparum the growth of the yeast strain in the presence of the inhibitor reflects the in vivo drug sensitivity of the DHFR enzyme that is expressed (14, 30 32). This approach has been particularly useful in situations where a novel allele of dhfr is of interest, but only the DNA of the parasite is available (31 34). We have adapted the system to assess the relative sensitivity of various P. vivax DHFR to both approved and investigational DHFR inhibitors. Materials and Methods Strains and Plasmids. The S. cerevisiae yeast strain used, TH5 (Mata ura3 52 leu2 3,112 trp1 tup1 dfr1::ura3), is null for the yeast dfr1 gene and requires supplemental dtmp, adenine, histidine, and methionine for growth (35). The expression vector is a shuttle plasmid that can be propagated in both Escherichia coli and S. cerevisiae. It has a yeast centromere, and so is maintained at approximately one copy per yeast cell, and trp1 and bla genes for selection of transformants in either E. coli or S. cerevisiae (36). We modified the plasmid for expression of the P. vivax dhfr coding sequence: it carries a 600-bp fragment from the region 5 of the yeast dfr1 coding sequence, which acts as the Pvdhfr promoter, and the terminator is a 400-bp fragment from 3 of the yeast dfr1 coding sequence (30). E. coli strain DH5 is This paper was submitted directly (Track II) to the PNAS office. Abbreviation: DHFR, dihydrofolate reductase. *To whom reprint requests should be addressed. sibley@gs.washington.edu. cgi doi pnas PNAS October 1, 2002 vol. 99 no

2 Fig. 1. The alignment of amino acid sequences of the P. vivax DHFR and P. falciparum DHFR domains. Amino acids correlated with resistance to pyrimethamine in PfDHFR and the corresponding amino acid in PvDHFR are shown in bold. used for propagation and preparation of the shuttle plasmid. Strains are maintained according to standard lab protocols. Recombinant DHFR Expression. We obtained genomic P. vivax DNA from a line that has been maintained in vivo in Aotus monkeys, a gift from William Collins, Centers for Disease Control and Prevention, Atlanta. We used the published P. vivax dhfr-ts sequence (GenBank accession no. X98123) to design PCR primers for amplification of the Pvdhfr domain, plus 19 downstream nt. In addition, the 5 end of each primer (italicized) was complementary to the sequence of the shuttle plasmid at the desired insertion position to facilitate homologous recombination in the yeast (forward: 5 -CGACGAGCGGATCCATTTAT- ATTTTCTCCTTTTTATGGAGGACCTTTCAGATGTA- TT-3 ; reverse: 5 -CATTTACGATTGTATAGAGTGAATTCG- AAGCCCGTCAACTCCCCCCCCCACCTTGCTGTAAA-3 ). Yeast were transformed by using a high-efficiency lithium acetate protocol (37), then were plated onto medium lacking tryptophan to select for the plasmid and lacking dtmp to select for functional DHFR activity. Construction of Mutants. Site-directed mutagenesis (QuikChange, Stratagene) was used to generate point mutations at codons 50, 57, 58, 117, and 173 of the Pvdhfr coding sequence. The desired point mutation was designed into complementary primers, and PCR was performed to incorporate the mutation into the target DNA sequence. The wild-type Pvdhfr coding sequence on the shuttle plasmid was mutagenized and then transformed into E. coli. The plasmid from 2 4 transformants was isolated and sequenced to ensure that only the desired mutation was present. Sequencing was conducted via fluorescent dye chemistry (Mega- BACE, Amersham Pharmacia Biotech) and analyzed with SE- QUENCHER software (Gene Codes, Ann Arbor, MI). The plasmid with the desired Pvdhfr mutation was then transformed back into TH5 yeast for drug sensitivity assays. Determination of Drug Sensitivity. The pyrimethamine, chlorcycloguanil, and WR99210 were gifts from Jacobus Pharmaceuticals, Princeton, NJ. Drugs were dissolved in DMSO and spread on plates to attain the desired final concentration or dissolved in DMSO for assay of yeast growth in liquid culture as described (29). An IC 50 assay was performed for each allele to obtain a quantitative measure of drug sensitivity. The IC 50 value is defined as the concentration of drug at which the growth of the yeast is inhibited by 50% relative to the untreated control. Transformed yeast were grown overnight in a 96-well culture dish in complete medium (yeast extract peptone dextrose broth) lacking dtmp. The first row of wells served as the control and contained yeast in the presence of the drug solvent (DMSO), but no drug. The next seven rows contained yeast in the presence of increasing concentrations of drug dissolved in DMSO. Four were measured in each 96-well dish, with three columns per allele. The growth of the yeast in each well was measured by reading the optical density at 650 nm after approximately 24 h incubation time. The average reading for each allele at each drug concentration was then used to plot the percent growth relative to the yeast extract peptone dextrose DMSO (no drug) control. The IC 50 value was calculated from the slope and intercept of the line defined by the two data points that bracket 50% relative growth. Comparisons of the IC 50 values of the mutant to the IC 50 value for the wild-type allele for a drug were used to assess the relative resistance level of each allele to the drug. Results and Discussion In P. vivax, the DHFR enzyme comprises the N-terminal domain of a bifunctional protein that also carries the thymidylate syn cgi doi pnas Hastings and Sibley

3 Table 1. List of mutant constructed Table 2. Calculated IC 50 values for each mutant allele Single mutant Double mutant Triple mutant Allele IC 50 M (SD) N Relative resistance N50I N50I S117N S58R S117N I173L F57L S58R S117N S58R S58R I173L S117N S117N I173L I173L Abbreviations are the standard amino acid code. thase enzyme activity (EC ). We amplified the dhfr domain from genomic DNA and cloned the fragment by homologous recombination into a yeast shuttle vector. The vector has a centromere sequence so that the plasmid is stably maintained at about one copy per cell (31, 36). Under these conditions, the expression of the parasite DHFR is just sufficient to maintain cell growth; inhibition of the enzyme is reflected in a proportional decrease in cell growth. Using the wild-type sequence as a baseline, we engineered a set of P. vivax dhfr that reflect polymorphisms that had been observed in the field (22, 23, 28) or changes that correspond to mutations that have been associated with pyrimethamine resistance in P. falciparum (15, 19 21). The that were constructed are listed in Table 1. The response of each yeast strain to pyrimethamine was measured by its relative growth in 0 to M pyrimethamine. We used the IC 50 value (the concentration of drug required to inhibit growth by 50%) to measure the relative sensitivity of the DHFR enzymes, and these data are collected in Table 2 and Fig. 2A. The color-coding of the in Fig. 2 represents the relative sensitivity as a rainbow from least pyrimethamine-resistant (S58R; blue) to most pyrimethamineresistant ( ; red). This analysis showed that the wild-type allele encodes an enzyme that is the most sensitive to pyrimethamine with an IC 50 value of M. However, all of the mutations tested show increased resistance to pyrimethamine. Fig. 2A and Table 2 reveal that there are three discernable groups: with a modest effect, increasing the IC 50 value by less than 6-fold, that increase the IC 50 by approximately 50-fold, and those that are essentially insensitive to the drug. Most striking, a single base pair mutation, resulting in the substitution of serine to asparagine at codon 117, increased the IC 50 value more than 80-fold. Addition of a second mutation, resulting in the substitution of serine to arginine at codon 58, produced an enzyme that was more than 400-fold more resistant to pyrimethamine, and further addition of the isoleucine to leucine mutation at codon 173 made the triple mutant approximately 500-fold more resistant than the wild-type allele. All of these mutations have been found in field samples, and the 58R 117N double mutant allele is present in particularly high frequencies in Southeast Asian isolates (28). These data strongly support the idea that point mutations in the dhfr domain can cause resistance to pyrimethamine in P. vivax and can explain the very rapid selection of resistance seen in this species when pyrimethamine-sulfadoxine was tested in the 1950s (16). Mutations at codons 57, 58, 117, and 173 were found in the 30 isolates whose complete dhfr sequences have been determined. The isolates were from a variety of sites in Asia and South America, but carried the double 58R 117N mutation and one carried the triple mutant 58R 117N 173L genotype (23). In a more extensive study, Imwong and her colleagues (28) used allele-specific methods to identify that carried mutations at codons 57, 58 or 117. They found that samples from Thailand had mutations at one or more of these codons, and the Pyrimethamine Wild type 0.53 (0.11) S58R 0.98 (0.17) I173L 2.1 (1.0) N50I 2.5 (0.5) S58R I173L 3.0 (1.1) F57L 28 (2) 4 52 N50I S117N 30 (13) 5 57 S117N 46 (10) 7 87 S58R S117N 250 (30) S58R S117N I173L 260 (80) S117N I173L 370 (100) Chlorcycloguanil Wild type 0.62 (0.09) S58R 3.2 (0.8) I173L 2.0 (0.1) N50I 2.2 (0.1) S58R I173L 6.2 (1.2) F57L 160 (90) N50I S117N 30 (6) 5 49 S117N 8.5 (1.4) 3 14 S58R S117N 220 (80) S58R S117N I173L 370 (70) S117N I173L 290 (120) WR99210 Wild type 0.38 (0.07) S58R 2.6 (0.1) I173L 0.35 (0.03) N50I 0.36 (0.02) S58R I173L 2.2 (0.1) F57L 0.92 (0.65) N50I S117N 0.07 ( 0.01) S117N 0.03 ( 0.01) S58R S117N 0.47 (0.05) S58R S117N I173L 0.26 (0.04) S117N I173L 0.05 (0.02) Values were determined as shown in Fig. 2. The relative resistance was calculated in comparison to the response of the wild-type allele. majority were double or triple mutants. Pyrimethaminesulfadoxine would almost certainly have poor efficacy in any P. vivax population in which these were prevalent, and even these small samples suggest that mutant may be common in many regions, especially in Southeast Asia. Chlorproguanil is a biguanide prodrug that is metabolized to chlorcycloguanil, another specific inhibitor of the P. falciparum DHFR (38, 39). In P. falciparum, chlorcycloguanil is a more effective inhibitor of DHFR than pyrimethamine, and phase III trials of chlorcycloguanil-dapsone have recently been completed (40). To further define the effect of this set of mutations on the P. vivax DHFR enzyme, we also assayed the sensitivity of each yeast strain to 0 to M chlorcycloguanil. Table 2 and Fig. 2B compile these data. In contrast to the results in P. falciparum (40 42), chlorcycloguanil is not a more effective inhibitor of the P. vivax enzyme than is pyrimethamine; the range of IC 50 values against the set of mutant is the same for both drugs. In P. falciparum, each point mutation that increases resistance to pyrimethamine also increases resistance to chlorcycloguanil by roughly the same degree (42). We observed the same trend when the P. vivax enzymes were assayed. This is apparent from the spectrum of colors in Fig. 2B. There was one exception, however. A single mutation (F57L) produces an enzyme that is about Hastings and Sibley PNAS October 1, 2002 vol. 99 no

4 Fig. 2. Sensitivity of P. vivax DHFR to (A) pyrimethamine, (B) chlorcycloguanil, and (C) WR Drugs were dissolved in DMSO for assay of yeast growth in liquid culture as described in Materials and Methods. 250-fold more resistant to chlorcycloguanil than the wild-type allele, but only about 50-fold more resistant to pyrimethamine. This difference may provide some useful leads for development of drugs specifically targeted to the P. vivax enzyme. WR99210 is a triazine inhibitor of the P. falciparum DHFR. Like chlorcycloguanil, it is cyclized in vivo from its prodrug, PS-15 (43). A series of compounds based on PS-15 are in preclinical development because they are extremely active against the most pyrimethamine-resistant of P. falciparum dhfr, both in vivo and in vitro (42 44). Fig. 2C shows the effectiveness of 0 to M WR99210 against the set of mutant P. vivax, and these IC 50 values are also listed in Table 2. There are two important observations. First, WR99210 is a more potent inhibitor than pyrimethamine or chlorcycloguanil of all of the P. vivax dhfr except S58R, which is equally sensitive to all three drugs. This S58R allele showed the highest level of resistance to WR99210, but it is still only 7-fold more resistant to WR99210 than the wild-type allele. Second, the that are most resistant to pyrimethamine are among the most sensitive to WR The color spectrum in Fig. 2C emphasizes the striking reversal of sensitivity to the two drugs when the IC 50 data are compared. Three (117N 173L, 117N, and 50I 117N) are 5- to 10-fold more sensitive to WR99210 than the wild-type allele. The S117N substitution, which seems to be a critical mutation for the development of high-level pyrimethamine resistance, also appears to render the enzyme particularly sensitive to the action of WR Because P. vivax cannot be grown in culture, there were no in vitro data on the sensitivity of the parasite to pyrimethamine on which to base our study. The yeast system offers one way to compare the relative sensitivity of particular DHFR enzymes to standard drugs and to chlorcycloguanil and WR99210 that are still under development. At the outset, we chose to construct mutants based on the initial DNA sequences published by de Pecoulas and his colleagues (23) and on the additional assumption that the P. falciparum and P. vivax enzymes would be similar in their sensitivity to this set of drugs. In general, that assumption is supported by the data, even for mutations that have not been observed yet in P. vivax isolates. For example, a mutation from asparagine to isoleucine at amino acid 51 is common in P. falciparum isolates from widely divergent geographic locations (45), but has not been reported in the few published studies of P. vivax. However, we constructed the equivalent mutation, N50I in P. vivax and observed that it does confer modest levels of resistance to pyrimethamine and chlorcycloguanil. This finding suggests that studies of field isolates might include methods for detecting this change. Although the sensitivity of the P. falciparum and P. vivax enzymes to pyrimethamine shows considerable similarity, the results of studies with chlorcycloguanil and WR99210 yielded some interesting surprises. First, in laboratory studies of P. falciparum, chlorcycloguanil is more effective against the wild type and canonical set of pyrimethamine-resistant than pyrimethamine (31, 42). Furthermore, in combination with dapsone, chlorproguanil has been shown to be clinically more effective than sulfadoxine-pyrimethamine (38, 40, 41, 46). Thus, it was a surprise that the effectiveness of chlorcycloguanil against the wild type and mutant enzymes of P. vivax was indistinguishable from pyrimethamine. Second, the prodrug of WR99210, PS-15, is highly effective against the most pyrimethamine-resistant of P. falciparum DHFR (42). A crystal structure of the P. falciparum DHFR has not been published, but homology models have been used to further understand the structural differences that underlie the effectiveness of WR99210 (47 49). The 117 position is equivalent to amino acid 108 in P. falciparum, and this position is clearly critical for drug resistance and for the catalytic function of DHFR from P. falciparum and a wide variety of species (21, 50). In P. falciparum, substitution of any amino acid but serine, asparagine, or threonine at amino acid 108 produces an enzyme with little or no activity (51). The fact that WR99210 is about 10-fold more effective against P. vivax DHFR enzymes that carry the 117N mutation is in striking contrast to P. falciparum. When the structures of the P. vivax and P. falciparum DHFR enzymes are solved to sufficient resolution, the data on drug efficacy in mutant DHFR enzymes of both species will be a rich source of information for further development of highly effective inhibitors. This remarkable efficacy of WR99210 raises two exciting possibilities. The fact that WR99210 is most effective against the that show the highest levels of resistance to pyrimethamine strongly suggests that WR99210 would be effective even in areas where these are common. For example, the S117N mutation was found in samples from Thailand (28), and it seems likely that WR99210 would be particularly effective against the parasites from that region. In fact, this observation suggests that pyrimethamine and WR99210 could be used together, forcing the selection of resistance by some mechanism other than the simple point mutations in the dhfr gene. Combining them with yet another drug with a different mechanism of action altogether could greatly retard the selection of resistance. Second, mixed infections of P. vivax and P. falciparum are frequently observed, especially in Southeast Asia (52 54). When chloroquine was still effective against both species, patients with mixed infections were treated effectively with chloroquine alone, because the drug could be expected to clear both infections. The rise of chloroquine-resistant P. falciparum rendered this strategy cgi doi pnas Hastings and Sibley

5 ineffective and forced the use of more expensive alternatives like mefloquine. The PS-15 series is already in preclinical development for P. falciparum treatment (43, 44), and a PS-15 analogue is likely to be combined with dapsone to make an affordable antimalarial drug. Our results suggest that when it is deployed, mixed P. vivax P. falciparum infections would again be sensitive to a single, affordable drug treatment. The life of this formulation could be further prolonged by combination with yet a third drug. One might choose one with an independent mode of action, like amodiaquine or artesunate, or even pyrimethamine to exploit the opposing selection that is exerted by pyrimethamine and WR We thank Dr. Jane Carlton and Dr. William Collins for their gifts of the P. vivax DNA and Jacobus Pharmaceutical for its gifts of pyrimethamine, chlorcycloguanil, and WR This work was supported by Public Health Service Grant AI (to C.H.S.). M.D.H. is supported by a National Science Foundation training fellowship. 1. Mendis, K., Sina, B. J., Marchesini, P. & Carter, R. (2001) Am. J. Trop. Med. Hyg. 64, Barat, L. M. & Bloland, P. B. (1997) Infect. Dis. Clin. North Am. 11, Singh, R. K. (2000) Trans. R. Soc. Trop. Med. Hyg. 94, Taylor, W. R., Widjaja, H., Richie, T. L., Basri, H., Ohrt, C., Tjitra, E., Taufik, E., Jones, T. R., Kain, K. C. & Hoffman, S. L. (2001) Am. J. Trop. Med. Hyg. 64, Nomura, T., Carlton, J. M., Baird, J. K., del Portillo, H. A., Fryauff, D. J., Rathore, D., Fidock, D. A., Su, X., Collins, W. E., McCutchan, T. F., et al. (2001) J. Infect. Dis. 183, Soto, J., Toledo, J., Gutierrez, P., Luzz, M., Llinas, N., Cedeno, N., Dunne, M. & Berman, J. (2001) Am. J. Trop. Med. Hyg. 65, Collins, W. E., Sullivan, J. S., Fryauff, D. J., Kendall, J., Jennings, V., Galland, G. G. & Morris, C. L. (2000) Am. J. Trop. Med. Hyg. 62, Kain, K. C., Shanks, G. D. & Keystone, J. S. (2001) Clin. Infect. Dis. 33, Baird, J. K., Basri, H., Purnomo, Bangs, M. J., Subianto, B., Patchen, L. C. & Hoffman, S. L. (1991) Am. J. Trop. Med. Hyg. 44, Baird, J. K., Sustriayu Nalim, M. F., Basri, H., Masbar, S., Leksana, B., Tjitra, E., Dewi, R. M., Khairani, M. & Wignall, F. S. (1996) Trans. R. Soc. Trop. Med. Hyg. 90, Garg, M., Gopinathan, N., Bodhe, P. & Kshirsagar, N. A. (1995) Trans. R. Soc. Trop. Med. Hyg. 89, Marlar, T., Myat Phone, K., Aye Yu, S., Khaing Khaing, G., Ma, S. & Myint, O. (1995) Trans. R. Soc. Trop. Med. Hyg. 89, Murphy, G. S., Basri, H., Purnomo, Andersen, E. M., Bangs, M. J., Mount, D. L., Gorden, J., Lal, A. A., Purwokusumo, A. R., Harjosuwarno, S., et al. (1993) Lancet 341, Sibley, C. H., Hyde, J. E., Sims, P. F. G., Plowe, C. V., Kublin, J. G., Mberu, E. K., Cowman, A. F., Winstanley, P. A., Watkins, W. M. & Nzila, A. M. (2001) Trends Parasitol. 17, Peters, W. (1998) Adv. Parasitol. 41, Young, M. D. & Burgess, R. W. (1959) Bull. W.H.O. 20, Hitchings, G. S. & Burchall, J. J. (1965) Adv. Enzymol. 27, Peters, W. (1987) Chemotherapy and Drug Resistance in Malaria (Academic, London). 19. Wang, P., Lee, C. S., Bayoumi, R., Djimde, A., Doumbo, O., Swedberg, G., Dao, L. D., Mshinda, H., Tanner, M., Watkins, W. M., et al. (1997) Mol. Biochem. Parasitol. 89, Plowe, C. V., Kublin, J. G. & Doumbo, O. K. (1998) Drug Resistance Updates 1, Hyde, J. E. (1990) Pharmacol. Ther. 48, de Pecoulas, P. E., Basco, L. K., Tahar, R., Ouatas, T. & Mazabraud, A. (1998) Gene 211, de Pecoulas, P. E., Tahar, R., Ouatas, T., Mazabraud, A. & Basco, L. K. (1998) Mol. Biochem. Parasitol. 92, Brobey, R. K. B., Sano, G., Itoh, F., Aso, K., Kimura, M., Mitamura, T. & Horii, T. (1996) Mol. Biochem. Parasitol. 81, Sirawaraporn, W., Sathitkul, T., Sirawaraporn, R., Yuthavong, Y. & Santi, D. V. (1997) Proc. Natl. Acad. Sci. USA 94, Tahar, R., de Pecoulas, P. E., Basco, L. K., Chiadmi, M. & Mazabraud, A. (2001) Mol. Biochem. Parasitol. 113, Leartsakulpanich, U., Imwong, M., Pukrittayakamee, S., White, N. J., Snounou, G., Sirawaraporn, W. & Yuthavong, Y. (2002) Mol. Biochem. Parasitol. 119, Imwong, M., Pukrittakayamee, S., Looareesuwan, S., Pasvol, G., Poirreiz, J., White, N. J. & Snounou, G. (2001) Antimicrob. Agents Chemother. 45, Sibley, C. H., Brophy, V. H., Cheesman, S., Hamilton, K. L., Hankins, E. G., Wooden, J. M. & Kilbey, B. (1997) Methods 13, Wooden, J. M., Hartwell, L. H., Vasquez, B. & Sibley, C. H. (1997) Mol. Biochem. Parasitol. 85, Cortese, J. F. & Plowe, C. V. (1998) Mol. Biochem. Parasitol. 94, Iyer, J. K., Milhous, W. K., Cortese, J. F., Kublin, J. G. & Plowe, C. V. (2001) Lancet 358, Brophy, V. H., Vasquez, J., Nelson, R. G., Forney, J. R., Rosowsky, A. & Sibley, C. H. (2000) Antimicrob. Agents Chemother. 44, Lau, H., Ferlan, J. T., Brophy, V. H., Rosowsky, A. & Sibley, C. H. (2001) Antimicrob. Agents Chemother. 45, Huang, T., Barclay, B. J., Kalman, T. I., VonBorstel, R. C. & Hastings, P. J. (1992) Gene 121, Sikorski, R. S. & Hieter, P. (1989) Genetics 122, Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. Bacteriol. 153, Amukoye, E., Winstanley, P. A., Watkins, W. M., Snow, R. W., Hatcher, J., Mosobo, M., Ngumbao, E., Lowe, B., Ton, M., Minyiri, G. & Marsh, K. (1997) Antimicrob. Agents Chemother. 41, Winstanley, P., Watkins, W., Muhia, D., Szwandt, S., Amukoye, E. & Marsh, K. (1997) Trans. R. Soc. Trop. Med. Hyg. 91, Mutabingwa, T. K., Maxwell, C. A., Sia, I. G., Msuya, F. H., Mkongewa, S., Vannithone, S., Curtis, J. & Curtis, C. F. (2001) Trans. R. Soc. Trop. Med. Hyg. 95, Mutabingwa, T., Nzila, A., Mberu, E., Nduati, E., Winstanley, P., Hills, E. & Watkins, W. (2001) Lancet 358, Hankins, E. G., Warhurst, D. C. & Sibley, C. H. (2001) Mol. Biochem. Parasitol. 117, Canfield, C. J., Milhous, W. K., Ager, A. L., Rossan, R. N., Sweeney, T. R., Lewis, N. J. & Jacobus, D. P. (1993) Am. J. Trop. Med. Hyg. 49, Jensen, N. P., Ager, A. L., Bliss, R. A., Canfield, C. J., Kotecka, B. M., Rieckmann, K. H., Terpinski, J. & Jacobus, D. P. (2001) J. Med. Chem. 44, Plowe, C. V., Cortese, J. F., Djimde, A., Nwanyanwu, O. C., Watkins, W. M., Winstanley, P. A., EstradaFranco, J. G., Mollinedo, R. E., Avila, J. C., Cespedes, J. L., et al. (1997) J. Infect. Dis. 176, Nzila-Mounda, A., Mberu, E. K., Sibley, C. H., Plowe, C. V., Winstanley, P. A. & Watkins, W. M. (1998) Antimicrob. Agents Chemother. 42, Rastelli, G., Sirawaraporn, W., Sompornpisut, P., Vilaivan, T., Kamchonwongpaisan, S., Quarrell, R., Lowe, G., Thebtaranonth, Y. & Yuthavong, Y. (2000) Bioorg. Med. Chem. 8, Warhurst, D. C. (1998) Drug Discovery Today 3, Warhurst, D. C. (2002) Sci. Prog. 85, Hyde, J. E. (2002) Microbes Infect. 4, Sirawaraporn, W., Yongkiettrakul, S., Sirawaraporn, R., Yuthavong, Y. & Santi, D. V. (1997) Exp. Parasitol. 87, Singh, B., Cox-Singh, J., Miller, A. O., Abdullah, M. S., Snounou, G. & Rahman, H. A. (1996) Trans. R. Soc. Trop. Med. Hyg. 90, Mehlotra, R. K., Lorry, K., Kastens, W., Miller, S. M., Alpers, M. P., Bockarie, M., Kazura, J. W. & Zimmerman, P. A. (2000) Am. J. Trop. Med. Hyg. 62, Mayxay, M., Pukritrayakamee, S., Chotivanich, K., Imwong, M., Looareesuwan, S. & White, N. J. (2001) Am. J. Trop. Med. Hyg. 65, Hastings and Sibley PNAS October 1, 2002 vol. 99 no