Conflicting Requirements of Plasmodium falciparum Dihydrofolate Reductase Mutations Conferring Resistance to Pyrimethamine-WR99210 Combination

ABSTRACT Plasmodium falciparum strains bearing quadruple mutations of dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) at codons 51, 59, 108, and 164 are highly resistant to pyrimethamine (PYR), a diaminopyrimidine, but sensitive to WR99210 (WR), a cycloguanil analog, suggesting different enzyme-inhibitor binding interactions. A combination of these inhibitors to delay the onset of antifolate resistance is proposed. Using error-prone PCR, libraries of random mutants of wild-type PfDHFR and PfDHFR-TS were generated and used to transform Escherichia coli, and transformants were then selected for PYR or WR resistance. Mutants highly resistant to either PYR or WR were also generated from libraries obtained from further random mutagenesis of quadruple mutants (QM) with mutations in PfDHFR or PfDHFR-TS. For reversion mutants carrying altered residues I51N, N108S, and L164I, a further mutation of D54N was required to achieve resistance against WR, but these mutants regained sensitivity to PYR. When a combination of PYR and WR was used, fewer resistant mutants were generated from both mutant libraries using the QM gene templates. The effectiveness of the drug combination in reducing the appearance of resistance mutations is likely due to conflicting requirements for mutations conferring resistance to the two drugs. Thus, a combination of inhibitors from these two drug classes should be effective in impeding the emergence of P. falciparum resistance to antifolates.

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Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.164 ◽

1998 ◽

Vol 42 (1) ◽

pp. 164-169 ◽

Cited By ~ 65

Author(s):

A. Nzila-Mounda ◽

E. K. Mberu ◽

C. H. Sibley ◽

C. V. Plowe ◽

P. A. Winstanley ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Dihydrofolate Reductase ◽

Drug Response ◽

Point Mutations ◽

Distinct Group ◽

Wild Type ◽

Field Isolates ◽

Vitro Data ◽

In Vitro Data

ABSTRACT Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P< 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

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Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors

Parasitology ◽

10.1017/s003118200400664x ◽

2005 ◽

Vol 130 (3) ◽

pp. 249-259 ◽

Cited By ~ 65

Author(s):

Y. YUTHAVONG ◽

J. YUVANIYAMA ◽

P. CHITNUMSUB ◽

J. VANICHTANANKUL ◽

S. CHUSACULTANACHAI ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Dihydrofolate Reductase ◽

Thymidylate Synthase ◽

Structural Basis ◽

Antifolate Resistance

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Molecular Correlates of High-Level Antifolate Resistance in Rwandan Children with Plasmodium falciparum Malaria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00498-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 477-483 ◽

Cited By ~ 28

Author(s):

Corine Karema ◽

Mallika Imwong ◽

Caterina I. Fanello ◽

Kasia Stepniewska ◽

Aline Uwimana ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Resistance Mutation ◽

Plasmodium Falciparum Malaria ◽

Drug Susceptibility ◽

Resistance Mutations ◽

Genes Encoding ◽

High Level ◽

Cure Rates ◽

Antifolate Drugs ◽

Antifolate Resistance

ABSTRACT Antifolate drugs have an important role in the treatment of malaria. Polymorphisms in the genes encoding the dihydrofolate reductase and dihydropteroate synthetase enzymes cause resistance to the antifol and sulfa drugs, respectively. Rwanda has the highest levels of antimalarial drug resistance in Africa. We correlated the efficacy of chlorproguanil-dapsone plus artesunate (CPG-DDS+A) and amodiaquine plus sulfadoxine-pyrimethamine (AQ+SP) in children with uncomplicated malaria caused by Plasmodium falciparum parasites with p fdhfr and p fdhps mutations, which are known to confer reduced drug susceptibility, in two areas of Rwanda. In the eastern province, where the cure rates were low, over 75% of isolates had three or more p fdhfr mutations and two or three p fdhps mutations and 11% had the p fdhfr 164-Leu polymorphism. In the western province, where the cure rates were significantly higher (P < 0.001), the prevalence of multiple resistance mutations was lower and the p fdhfr I164L polymorphism was not found. The risk of treatment failure following the administration of AQ+SP more than doubled for each additional p fdhfr resistance mutation (odds ratio [OR] = 2.4; 95% confidence interval [CI] = 1.01 to 5.55; P = 0.048) and each p fdhps mutation (OR = 2.1; 95% CI = 1.21 to 3.54; P = 0.008). The risk of failure following CPG-DDS+A treatment was 2.2 times higher (95% CI = 1.34 to 3.7) for each additional p fdhfr mutation, whereas there was no association with mutations in the p fdhps gene (P = 0.13). The p fdhfr 164-Leu polymorphism is prevalent in eastern Rwanda. Antimalarial treatments with currently available antifol-sulfa combinations are no longer effective in Rwanda because of high-level resistance.

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Molecular epidemiology of Plasmodium falciparum antifolate resistance in Vietnam: genotyping for resistance variants of dihydropteroate synthase and dihydrofolate reductase

International Journal of Antimicrobial Agents ◽

10.1016/s0924-8579(99)00061-8 ◽

1999 ◽

Vol 12 (3) ◽

pp. 203-211 ◽

Cited By ~ 10

Author(s):

C.M Masimirembwa ◽

N Phuong-dung ◽

B.Q Phuc ◽

L Duc-Dao ◽

N.D Sy ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Molecular Epidemiology ◽

Dihydrofolate Reductase ◽

Dihydropteroate Synthase ◽

Antifolate Resistance

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In Vitro Generation of Novel Pyrimethamine Resistance Mutations in the Toxoplasma gondiiDihydrofolate Reductase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1271-1277.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1271-1277 ◽

Cited By ~ 32

Author(s):

Mary G. Reynolds ◽

Jung Oh ◽

David S. Roos

Keyword(s):

Potential Role ◽

Opportunistic Infections ◽

Potent Inhibitor ◽

Random Mutagenesis ◽

Protozoan Parasite ◽

Amino Acid Residues ◽

Wild Type ◽

Resistance Mutations

ABSTRACT Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-tsprotein sequences. To assess the potential for pyrimethamine-resistantdhfr mutants to emerge, three drug-sensitive variants of the T. gondii dhfr-ts gene (the wild-type T. gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T. gondii parasites ordhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trp→Arg), 98 (Leu→Ser), and 134 (Leu→His).

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Trade-offs with stability modulate innate and mutationally acquired drug resistance in bacterial dihydrofolate reductase enzymes

Biochemical Journal ◽

10.1042/bcj20180249 ◽

2018 ◽

Vol 475 (12) ◽

pp. 2107-2125 ◽

Cited By ~ 1

Author(s):

Nishad Matange ◽

Swapnil Bodkhe ◽

Maitri Patel ◽

Pooja Shah

Keyword(s):

Drug Resistance ◽

Structural Stability ◽

Dihydrofolate Reductase ◽

Mutational Analysis ◽

Hydrophobic Interactions ◽

Wild Type ◽

Resistance Mutations ◽

Acquired Drug Resistance ◽

Trade Offs ◽

And Function

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In the present study, we show that trimethoprim (TMP)-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harboring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild-type, making them prone to aggregation and proteolysis. This destabilization is associated with a lower expression level, resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis, we show that perturbation of critical stabilizing hydrophobic interactions in wild-type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial dihydrofolate reductases (DHFRs). Mutational and computational analyses in EcDHFR and in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues modulates TMP resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to TMP. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.

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Antifolate resistance due to new and known Plasmodium falciparum dihydrofolate reductase mutations expressed in yeast

Molecular and Biochemical Parasitology ◽

10.1016/s0166-6851(98)00075-9 ◽

1998 ◽

Vol 94 (2) ◽

pp. 205-214 ◽

Cited By ~ 53

Author(s):

Joseph F Cortese ◽

Christopher V Plowe

Keyword(s):

Plasmodium Falciparum ◽

Dihydrofolate Reductase ◽

Antifolate Resistance

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Defining the Role of Mutations in Plasmodium vivax Dihydrofolate Reductase-Thymidylate Synthase Gene Using an Episomal Plasmodium falciparum Transfection System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00628-10 ◽

2010 ◽

Vol 54 (9) ◽

pp. 3927-3932 ◽

Cited By ~ 7

Author(s):

Alyson M. Auliff ◽

John H. Adams ◽

Michael T. O'Neil ◽

Qin Cheng

Keyword(s):

Plasmodium Vivax ◽

Dihydrofolate Reductase ◽

Thymidylate Synthase ◽

Resistance Index ◽

Rapid Screening ◽

Compensatory Mutation ◽

Wild Type ◽

Triple Mutant ◽

Antifolate Drugs ◽

Antifolate Resistance

ABSTRACT Plasmodium vivax resistance to antifolates is prevalent throughout Australasia and is caused by point mutations within the parasite dihydrofolate reductase (DHFR)-thymidylate synthase. Several unique mutations have been reported in P. vivax DHFR, and their roles in resistance to classic and novel antifolates are not entirely clear due, in part, to the inability to culture P. vivax in vitro. In this study, we use a homologous system to episomally express both wild-type and various mutant P. vivax dhfr (pvdhfr) alleles in an antifolate-sensitive line of P. falciparum and to assess their influences on the susceptibility of the recipient P. falciparum line to commonly used and new antifolate drugs. Although the wild-type pvdhfr-transfected P. falciparum line was as susceptible to antifolate drugs as the P. falciparum parent line, the single (117N), double (57L/117T and 58R/117T), and quadruple (57L/58R/61M/117T) mutant pvdhfr alleles conferred a marked reduction in their susceptibilities to antifolates. The resistance index increased with the number of mutations in these alleles, indicating that these mutations contribute to antifolate resistance directly. In contrast, the triple mutant allele (58R/61M/117T) significantly reversed the resistance to all antifolates, indicating that 61M may be a compensatory mutation. These findings help elucidate the mechanism of antifolate resistance and the effect of existing mutations in the parasite population on the current and new generation of antifolate drugs. It also demonstrates that the episomal transfection system has the potential to provide a rapid screening system for drug development and for studying drug resistance mechanisms in P. vivax.

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