Pyrimethamine and WR99210 Exert Opposing Selection on Dihydrofolate Reductase from Plasmodium Vivax

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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 357730, Seattle, WA 98195-7730

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 falciparum and P. vivax. The alignment demonstrates that the South and Central America where it is most prevalent. Until very polymorphisms observed at positions 58, 117, and 173 of the P. recently, the parasite has been effectively treated with chloro- vivax sequence correspond to the changes in codons 59, 108, and quine, but resistance to this drug has now been reported in several 164 that are known to cause pyrimethamine resistance in the P. areas. Affordable alternative treatments for vivax malaria are falciparum enzyme (24, 25). Several of these mutant P. vivax urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of enzymes were purified and showed less sensitivity to inhibition dihydrofolate reductase (DHFR) that has been widely used to treat by pyrimethamine in vitro (26, 27). In addition, a recent study chloroquine-resistant Plasmodium falciparum malaria. DHFR inhib- showed that polymorphisms in amino acids 57, 58, and 117 are itors have not been considered for treatment of vivax malaria, very common in Thailand where pyrimethamine-sulfadoxine has because initial trials showed poor efﬁcacy against P. vivax. P. vivax been used extensively, but less prevalent in India and Madagas- cannot be grown in culture; the reason for its resistance to DHFR car where antifolate use has been lower (28). These studies inhibitors is unknown. We show that, like P. falciparum, point suggested that amino acid substitutions might be responsible for mutations in the dhfr gene can cause resistance to pyrimethamine pyrimethamine resistance in P. vivax. in P. vivax. WR99210 is a novel inhibitor of DHFR, effective even Because P. vivax cannot be maintained in culture, there is no against the most pyrimethamine-resistant P. falciparum strains. We way to test reference strains in vitro to set criteria for relative have found that it is also an extremely effective inhibitor of the P. drug sensitivity; drug testing can be accomplished only by vivax DHFR, and mutations that confer high-level resistance to short-term treatment of parasites newly isolated from patients. pyrimethamine render the P. vivax enzyme exquisitely sensitive to This requirement has severely hampered studies of drug efficacy WR99210. These data suggest that pyrimethamine and WR99210 and has made it difficult to determine the relative importance of would exert opposing selective forces on the P. vivax population. point mutations in the P. vivax dhfr gene for antifolate resistance. If used in combination, these two drugs could greatly slow the We have developed a system for expression of DHFR enzymes selection of parasites resistant to both drugs. If that is the case, this from pathogens in strains of the budding yeast, Saccharomyces

novel class of DHFR inhibitors could provide effective and afford- cerevisiae, that lack endogenous DHFR activity (29, 30). This MICROBIOLOGY able treatment for chloroquine- and pyrimethamine-resistant approach allows rapid assessment of the relative sensitivity of vivax and falciparum malaria for many years to come. heterologous DHFR enzymes to inhibitors, simply by measuring the growth of the yeast in the presence of potential inhibitors of he parasite Plasmodium vivax is a major public health the enzyme. We and others have shown that for a range of alleles Tproblem in Asia and South and Central America where it is of P. falciparum the growth of the yeast strain in the presence of most prevalent (1). Until very recently, the parasite has been the inhibitor reflects the in vivo drug sensitivity of the DHFR effectively treated with chloroquine, but resistance to this drug enzyme that is expressed (14, 30–32). This approach has been has now been reported in several areas (1–13). Affordable particularly useful in situations where a novel allele of dhfr is of alternative treatments for vivax malaria are urgently needed. interest, but only the DNA of the parasite is available (31–34). Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate re- We have adapted the system to assess the relative sensitivity of ductase (DHFR) that has been widely used to treat chloroquine- various P. vivax DHFR alleles to both approved and investiga- resistant Plasmodium falciparum malaria (14). DHFR inhibitors tional DHFR inhibitors. have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax (15, 16). P. Materials and Methods vivax cannot be grown in culture; the reason for its resistance to Strains and Plasmids. The S. cerevisiae yeast strain used, TH5 DHFR inhibitors is unknown. (Mata ura3–52 leu2–3,112 trp1 tup1 dfr1::URA3), is null for the DHFR (E.C. 1.5.1.3) is a key enzyme in the metabolism of all yeast dfr1 gene and requires supplemental dTMP, adenine, cells. Inhibition of DHFR activity depletes the cellular pool of histidine, and methionine for growth (35). The expression vector tetrahydrofolate, a cofactor that is essential for both DNA and is a shuttle plasmid that can be propagated in both Escherichia protein synthesis, and specific inhibitors have been designed for coli and S. cerevisiae. It has a yeast centromere, and so is both prokaryotic and eukaryotic pathogens (17). Pyrimethamine maintained at approximately one copy per yeast cell, and trp1 is a specific competitive inhibitor of DHFR from P. falciparum, and bla genes for selection of transformants in either E. coli or (18) and with sulfadoxine, is one component of the antimalaria S. cerevisiae (36). We modified the plasmid for expression of the drug, Fansidar. In P. falciparum, extensive field and laboratory P. vivax dhfr coding sequence: it carries a 600-bp fragment from studies have shown that resistance to pyrimethamine is caused by the region 5Ј of the yeast dfr1 coding sequence, which acts as the point mutations in the dhfr gene (reviewed in refs. 15 and 19–21). Pvdhfr promoter, and the terminator is a 400-bp fragment from However, in contrast to P. falciparum, P. vivax cannot be 3Ј of the yeast dfr1 coding sequence (30). E. coli strain DH5␣ is 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 This paper was submitted directly (Track II) to the PNAS ofﬁce. of alternative alleles of the gene were identified (22, 23). Fig. 1 Abbreviation: DHFR, dihydrofolate reductase. compares the alignment of the dhfr coding regions from P. *To whom reprint requests should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.182295999 PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 13137–13141 Downloaded by guest on September 23, 2021 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. mid with the desired Pvdhfr mutation was then transformed back Strains are maintained according to standard lab protocols. into TH5 yeast for drug sensitivity assays.

Recombinant DHFR Expression. We obtained genomic P. vivax DNA Determination of Drug Sensitivity. The pyrimethamine, chlorcy- from a line that has been maintained in vivo in Aotus monkeys, cloguanil, and WR99210 were gifts from Jacobus Pharmaceuticals, a gift from William Collins, Centers for Disease Control and Princeton, NJ. Drugs were dissolved in DMSO and spread on plates Prevention, Atlanta. We used the published P. vivax dhfr-ts to attain the desired final concentration or dissolved in DMSO for sequence (GenBank accession no. X98123) to design PCR assay of yeast growth in liquid culture as described (29). primers for amplification of the Pvdhfr domain, plus 19 down- An IC50 assay was performed for each allele to obtain a stream nt. In addition, the 5Ј end of each primer (italicized) was quantitative measure of drug sensitivity. The IC50 value is complementary to the sequence of the shuttle plasmid at the defined as the concentration of drug at which the growth of the desired insertion position to facilitate homologous recombina- yeast is inhibited by 50% relative to the untreated control. Ј Transformed yeast were grown overnight in a 96-well culture tion in the yeast (forward: 5 -CGACGAGCGGATCCATTTAT- ͞ ͞ ATTTTCTCCTTTTTATGGAGGACCTTTCAGATGTA- dish in complete medium (yeast extract peptone dextrose TT-3Ј; reverse: 5Ј-CATTTACGATTGTATAGAGTGAATTCG- broth) lacking dTMP. The first row of wells served as the control and contained yeast in the presence of the drug solvent (DMSO), AAGCCCGTCAACTCCCCCCCCCACCTTGCTGTAAA-3Ј). but no drug. The next seven rows contained yeast in the presence Yeast were transformed by using a high-efficiency lithium of increasing concentrations of drug dissolved in DMSO. Four acetate protocol (37), then were plated onto medium lacking alleles were measured in each 96-well dish, with three columns tryptophan to select for the plasmid and lacking dTMP to select per allele. The growth of the yeast in each well was measured by for functional DHFR activity. reading the optical density at 650 nM after approximately 24 h incubation time. The average reading for each allele at each drug Construction of Mutants. Site-directed mutagenesis (QuikChange, concentration was then used to plot the percent growth relative Stratagene) was used to generate point mutations at codons 50, to the yeast extract͞peptone͞dextrose ϩ DMSO (no drug) 57, 58, 117, and 173 of the Pvdhfr coding sequence. The desired control. The IC50 value was calculated from the slope and point mutation was designed into complementary primers, and intercept of the line defined by the two data points that bracket PCR was performed to incorporate the mutation into the target 50% relative growth. Comparisons of the IC50 values of the DNA sequence. The wild-type Pvdhfr coding sequence on the mutant alleles to the IC50 value for the wild-type allele for a drug shuttle plasmid was mutagenized and then transformed into E. were used to assess the relative resistance level of each allele to coli. The plasmid from 2–4 transformants was isolated and the drug. sequenced to ensure that only the desired mutation was present. Sequencing was conducted via fluorescent dye chemistry (Mega- Results and Discussion BACE, Amersham Pharmacia Biotech) and analyzed with SE- In P. vivax, the DHFR enzyme comprises the N-terminal domain QUENCHER software (Gene Codes, Ann Arbor, MI). The plas- of a bifunctional protein that also carries the thymidylate syn-

13138 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.182295999 Hastings and Sibley Downloaded by guest on September 23, 2021 Table 1. List of mutant alleles constructed Table 2. Calculated IC50 values for each mutant allele Single mutant Double mutant Triple mutant Relative

alleles alleles alleles Allele IC50 ␮M (SD) N resistance

N50I N50I ϩ S117N S58RϩS117NϩI173L Pyrimethamine F57L S58R ϩ S117N Wild type 0.53 (0.11) 4 1.0 S58R S58R ϩ I173L S58R 0.98 (0.17) 6 1.9 S117N S117N ϩ I173L I173L 2.1 (1.0) 6 4.0 I173L N50I 2.5 (0.5) 4 4.6 S58R͞I173L 3.0 (1.1) 5 5.6 Abbreviations are the standard amino acid code. F57L 28 (2) 4 52 N50I͞S117N 30 (13) 5 57 thase enzyme activity (EC 2.1.1.45). We amplified the dhfr S117N 46 (10) 7 87 ͞ domain from genomic DNA and cloned the fragment by ho- S58R S117N 250 (30) 2 460 S58R͞S117N͞I173L 260 (80) 4 500 mologous recombination into a yeast shuttle vector. The vector S117N͞I173L 370 (100) 2 700 has a centromere sequence so that the plasmid is stably maintained at about one copy per cell (31, 36). Under these condi- Chlorcycloguanil tions, the expression of the parasite DHFR is just sufficient to Wild type 0.62 (0.09) 5 1.0 maintain cell growth; inhibition of the enzyme is reflected in a S58R 3.2 (0.8) 5 5.2 proportional decrease in cell growth. Using the wild-type se- I173L 2.0 (0.1) 3 3.1 quence as a baseline, we engineered a set of P. vivax dhfr alleles N50I 2.2 (0.1) 4 3.5 that reflect polymorphisms that had been observed in the field S58R͞I173L 6.2 (1.2) 3 9.9 (22, 23, 28) or changes that correspond to mutations that have F57L 160 (90) 6 250 ͞ been associated with pyrimethamine resistance in P. falciparum N50I S117N 30 (6) 5 49 (15, 19–21). The alleles that were constructed are listed in Table 1. S117N 8.5 (1.4) 3 14 ͞ The response of each yeast strain to pyrimethamine was S58R S117N 220 (80) 6 350 Ϫ S58R͞S117N͞I173L 370 (70) 5 600 measured by its relative growth in 0 to 2.5 ϫ 10 4 M py- S117N͞I173L 290 (120) 3 460 rimethamine. We used the IC50 value (the concentration of drug required to inhibit growth by 50%) to measure the relative WR99210 sensitivity of the DHFR enzymes, and these data are collected Wild type 0.38 (0.07) 5 1.0 in Table 2 and Fig. 2A. The color-coding of the alleles in Fig. 2 S58R 2.6 (0.1) 6 7.0 represents the relative sensitivity as a rainbow from least py- I173L 0.35 (0.03) 3 0.9 rimethamine-resistant (S58R; blue) to most pyrimethamine- N50I 0.36 (0.02) 3 1.0 resistant (117͞173; red). S58R͞I173L 2.2 (0.1) 3 5.8 This analysis showed that the wild-type allele encodes an F57L 0.92 (0.65) 6 2.4 MICROBIOLOGY ͞ Ͻ enzyme that is the most sensitive to pyrimethamine with an IC N50I S117N 0.07 ( 0.01) 3 0.2 50 Ͻ value of 5.3 ϫ 10Ϫ7 M. However, all of the mutations tested show S117N 0.03 ( 0.01) 2 0.1 ͞ increased resistance to pyrimethamine. Fig. 2A and Table 2 S58R S117N 0.47 (0.05) 3 1.3 S58R͞S117N͞I173L 0.26 (0.04) 3 0.7 reveal that there are three discernable groups: alleles with a S117N͞I173L 0.05 (0.02) 3 0.1 modest effect, increasing the IC50 value by less than 6-fold, alleles that increase the IC50 by approximately 50-fold, and those that Values were determined as shown in Fig. 2. The relative resistance was are essentially insensitive to the drug. Most striking, a single base calculated in comparison to the response of the wild-type allele. pair mutation, resulting in the substitution of serine to asparagine at codon 117, increased the IC50 value more than 80-fold. Addition of a second mutation, resulting in the substitution of majority were double or triple mutants. Pyrimethamine- serine to arginine at codon 58, produced an enzyme that was sulfadoxine would almost certainly have poor efficacy in any P. more than 400-fold more resistant to pyrimethamine, and further vivax population in which these alleles were prevalent, and even these small samples suggest that mutant alleles may be common addition of the isoleucine to leucine mutation at codon 173 made in many regions, especially in Southeast Asia. the triple mutant approximately 500-fold more resistant than the Chlorproguanil is a biguanide prodrug that is metabolized to wild-type allele. All of these mutations have been found in field chlorcycloguanil, another specific inhibitor of the P. falciparum samples, and the 58R͞117N double mutant allele is present in DHFR (38, 39). In P. falciparum, chlorcycloguanil is a more particularly high frequencies in Southeast Asian isolates (28). effective inhibitor of DHFR than pyrimethamine, and phase III These data strongly support the idea that point mutations in the trials of chlorcycloguanil-dapsone have recently been completed dhfr domain can cause resistance to pyrimethamine in P. vivax (40). To further define the effect of this set of mutations on the and can explain the very rapid selection of resistance seen in P. vivax DHFR enzyme, we also assayed the sensitivity of each this species when pyrimethamine-sulfadoxine was tested in the yeast strain to 0 to 5 ϫ 10Ϫ4 M chlorcycloguanil. Table 2 and Fig. 1950s (16). 2B compile these data. In contrast to the results in P. falciparum Mutations at codons 57, 58, 117, and 173 were found in the 30 (40–42), chlorcycloguanil is not a more effective inhibitor of the isolates whose complete dhfr sequences have been determined. P. vivax enzyme than is pyrimethamine; the range of IC50 values The isolates were from a variety of sites in Asia and South against the set of mutant alleles is the same for both drugs. In P. America, but 13͞30 carried the double 58R͞117N mutation and falciparum, each point mutation that increases resistance to one carried the triple mutant 58R͞117N͞173L genotype (23). In pyrimethamine also increases resistance to chlorcycloguanil by a more extensive study, Imwong and her colleagues (28) used roughly the same degree (42). We observed the same trend when allele-specific methods to identify alleles that carried mutations the P. vivax enzymes were assayed. This is apparent from the at codons 57, 58 or 117. They found that 99͞100 samples from spectrum of colors in Fig. 2B. There was one exception, however. Thailand had mutations at one or more of these codons, and the A single mutation (F57L) produces an enzyme that is about

Hastings and Sibley PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 13139 Downloaded by guest on September 23, 2021 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 alleles 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 indistinguish- able from pyrimethamine. Second, the prodrug of WR99210, PS-15, is highly effective against the most pyrimethamine-resistant alleles of P. falciparum DHFR (42). A crystal structure of the P. falciparum DHFR has not been published, but homology models have been used to Fig. 2. Sensitivity of P. vivax DHFR to (A) pyrimethamine, (B) chlorcyclogua- further understand the structural differences that underlie the nil, and (C) WR99210. Drugs were dissolved in DMSO for assay of yeast growth in liquid culture as described in Materials and Methods. 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 250-fold more resistant to chlorcycloguanil than the wild-type DHFR from P. falciparum and a wide variety of species (21, 50). allele, but only about 50-fold more resistant to pyrimethamine. In P. falciparum, substitution of any amino acid but serine, This difference may provide some useful leads for development asparagine, or threonine at amino acid 108 produces an enzyme of drugs specifically targeted to the P. vivax enzyme. with little or no activity (51). The fact that WR99210 is about WR99210 is a triazine inhibitor of the P. falciparum DHFR. 10-fold more effective against P. vivax DHFR enzymes that carry Like chlorcycloguanil, it is cyclized in vivo from its prodrug, the 117N mutation is in striking contrast to P. falciparum. When PS-15 (43). A series of compounds based on PS-15 are in the structures of the P. vivax and P. falciparum DHFR enzymes are solved to sufficient resolution, the data on drug efficacy in preclinical development because they are extremely active mutant DHFR enzymes of both species will be a rich source against the most pyrimethamine-resistant alleles of P. falciparum of information for further development of highly effective dhfr, both in vivo and in vitro (42–44). Fig. 2C shows the Ϫ inhibitors. effectiveness of 0 to 1 ϫ 10 4 M WR99210 against the set of This remarkable efficacy of WR99210 raises two exciting possi- mutant P. vivax alleles, and these IC50 values are also listed in bilities. The fact that WR99210 is most effective against the alleles Table 2. There are two important observations. First, WR99210 that show the highest levels of resistance to pyrimethamine strongly is a more potent inhibitor than pyrimethamine or chlorcyclogua- suggests that WR99210 would be effective even in areas where these nil of all of the P. vivax dhfr alleles except S58R, which is equally alleles are common. For example, the S117N mutation was found sensitive to all three drugs. This S58R allele showed the highest in 99͞100 samples from Thailand (28), and it seems likely that level of resistance to WR99210, but it is still only 7-fold more WR99210 would be particularly effective against the parasites from resistant to WR99210 than the wild-type allele. Second, the that region. In fact, this observation suggests that pyrimethamine alleles that are most resistant to pyrimethamine are among the and WR99210 could be used together, forcing the selection of most sensitive to WR99210. The color spectrum in Fig. 2C resistance by some mechanism other than the simple point muta- emphasizes the striking reversal of sensitivity to the two drugs tions in the dhfr gene. Combining them with yet another drug with ͞ when the IC50 data are compared. Three alleles (117N 173L, a different mechanism of action altogether could greatly retard the 117N, and 50I͞117N) are 5- to 10-fold more sensitive to selection of resistance. WR99210 than the wild-type allele. The S117N substitution, Second, mixed infections of P. vivax and P. falciparum are which seems to be a critical mutation for the development of frequently observed, especially in Southeast Asia (52–54). When high-level pyrimethamine resistance, also appears to render the chloroquine was still effective against both species, patients with enzyme particularly sensitive to the action of WR99210. mixed infections were treated effectively with chloroquine alone, Because P. vivax cannot be grown in culture, there were no in because the drug could be expected to clear both infections. The vitro data on the sensitivity of the parasite to pyrimethamine on rise of chloroquine-resistant P. falciparum rendered this strategy

13140 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.182295999 Hastings and Sibley Downloaded by guest on September 23, 2021 ineffective and forced the use of more expensive alternatives like action, like amodiaquine or artesunate, or even pyrimethamine mefloquine. The PS-15 series is already in preclinical develop- to exploit the opposing selection that is exerted by pyri- ment for P. falciparum treatment (43, 44), and a PS-15 analogue methamine and WR99210. is likely to be combined with dapsone to make an affordable antimalarial drug. Our results suggest that when it is deployed, We thank Dr. Jane Carlton and Dr. William Collins for their gifts of the mixed P. vivax͞P. falciparum infections would again be sensitive P. vivax DNA and Jacobus Pharmaceutical for its gifts of pyrimethamine, to a single, affordable drug treatment. The life of this formula- chlorcycloguanil, and WR99210. This work was supported by Public tion could be further prolonged by combination with yet a third Health Service Grant AI 42321 (to C.H.S.). M.D.H. is supported by a drug. One might choose one with an independent mode of National Science Foundation training fellowship.

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