IN VITRO SELECTION OF PLASMODIUM FALCIPARUM LINES RESISTANT TO DIHYDROFOLATE-REDUCTASE INHIBITORS AND CROSS RESISTANCE STUDIES
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Abstract:
A cloned Plasmodium falciparum line was subjected to in vitro drug pressure, by employing a relapse protocol, to select progressively resistant falciparum lines to pyrimethamine and cycloguanil, the two dihydrofolatereductase (DHFR) inhibitor antimalarial drugs. The falciparum lines resistant to pyrimethamine were selected much faster than those resistant to cycloguanil. In 348 days of selection/cultivation, there was 2, 400-fold increase in IC50 value to pyrimethamine, whereas only about 75-fold decrease in sensitivity to cycloguanil was registered in 351 days. Pyrimethamine-resistant parasites acquired a degree of cross resistance to cycloguanil and methotrexate, another DHFR inhibitor, but did not show any cross resistance to some other groups of antimalarial drugs. The highly pyrimethamine-resistant line was not predisposed for faster selection to cycloguanil resistance. Resistance acquired to pyrimethamine was stable. The series of resistant lines obtained form a good material to study the ‘evolution’ of resistance more meaningfully at molecular level.Keywords:
Dihydrofolate reductase
Cross-resistance
Antifolate
Quinine
Pyrimethamine acts against malarial parasites by selectively inhibiting their dihydrofolate reductase-thymidylate synthase. Resistance to pyrimethamine in Plasmodium falciparum is due to point mutations in the DHFR domain, initially at residue 108 (S108N), with additional mutations imparting much greater resistance. Our previous work, the development of a simple rational drug design strategy to overcome such resistance, used suitable meta-substituents in the pyrimethamine framework to avoid the unfavorable steric clash with mutant side chains at position 108. Interestingly, the meta-chloro analog of pyrimethamine not only overcame the resistance due to S108N, but also that contributed by the more remote mutation, C59R. The present work improves on this by means of other meta-substituents. Against wild type DHFR, double mutant types A16V + S108T and C59R + S108T, and the highly pyrimethamine/cycloguanil-resistant quadruple-mutant form N51I + C59R + S108N + I164L, pyrimethamine itself gave Ki values of 1.5, 2.4, 72.3 and 859 nM, respectively. The meta-substituted analogs, especially the meta-bromo analog, were much more powerful inhibitors of these DHFRs, including the quadruple-mutant form (meta-bromo analog, Ki 5.1 nM). For comparison, the dihydropyrazine antifolate, WR99210, gave Ki values of 0.9, 3.2, 0.8 and 0.9 nM, respectively. Ki values were also measured against recombinant human DHFR, as were their activities against the growth of Plasmodium falciparum cultures bearing the double mutations (FCB and K1 strains) and quadruple mutation (V1/S) and the wild type (3D7). The meta-analogs were highly active against all of these, with the meta-bromo again being the strongest, having an IC50 of 37 nM against V1/S, compared to > 5000 nM for pyrimethamine itself and 1.1 nM for WR99210.
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In vitro pyrimethamine response of Plasmodium falciparum isolates and dihydrofolate reductase ( dhfr ) gene sequences were analyzed in 2004–2005 and compared with our previous data. Most isolates (n = 103, all dhfr mutants) had 50% inhibitory concentrations (IC 50 s) ≥ 119 nM, and six isolates had low IC 50 s (five wild-type or mixed dhfr , 0.04–1.37 nM; one triple mutant, 6.4 nM). Of 194 isolates, only 7 had the wild-type dhfr and 187 were mutants. The results of the two methods were highly concordant and indicated a significant increase ( P < 0.05) in the prevalence of mutant, pyrimethamine-resistant P. falciparum between 1994 and 2005. The addition of probenecid or sulfinpyrazone to pyrimethamine resulted in a slight-to-moderate decrease in the level of in vitro pyrimethamine resistance without rendering the parasites susceptible to pyrimethamine. Analysis of molecular markers may be useful for the long-term surveillance of antifolate-resistant malaria.
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Dihydrofolate reductase was obtained from Pneumocystis carinii isolated from heavily infected lungs of female Sprague-Dawley rats infected by transtracheal inoculation. The enzyme differed significantly from other forms of dihydrofolate reductase in response to KCl and to antifolate drugs. Dihydrofolate reductase from P. carinii was used to assess activity of analogs of pyrimethamine, methotrexate, and trimetrexate. One pyrimethamine analog was selective for P. carinii dihydrofolate reductase; potency was in the micromolar range. In contrast, 21 methotrexate analogs and 2 trimetrexate analogs were selective for P. carinii dihydrofolate reductase; potencies for these were in the nanomolar range.
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Plasmodium berghei
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Single and multiple mutations at residues 16, 51, 59, 108, and 164 of Plasmodium falciparum dihydrofolate reductase (pfDHFR) have been linked to antifolate resistance in malaria. We prepared and characterized all seven of the pfDHFR mutants found in nature, as well as six mutants not observed in nature. Mutations involving residues 51, 59, 108, or 164 conferred cross resistance to both the antifolates pyrimethamine and cycloguanil, whereas mutation of residue 16 specifically conferred resistance to cycloguanil. The antifolate resistance of enzyme mutants found in nature correlated with in vivo antifolate resistance; however, mutants not found in nature were either poorly resistant or had insufficient catalytic activity to support DNA synthesis. Thus, specific combinations of multiple mutations at target residues were selected in nature to optimize resistance. Further, the resistance of multiple mutants was more than the sum of the component single mutations, indicating that residues were selected for their synergistic as well as intrinsic effects on resistance. Pathways inferred for the evolution of pyrimethamine-resistant mutants suggested that all multiple mutants emerged from stepwise selection of the single mutant, S108N. Thus, we propose that drugs targeted to both the wild-type pfDHFR and S108N mutant would have a low propensity for developing resistance, and hence could provide effective antimalarial agents.
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Toxoplasma gondii RH was obtained in high yield from culture in RPMI medium on a line of Chinese hamster ovary cells lacking dihydrofolate reductase activity (ATCC 3952 dhfr-; American Type Culture Collection). Dihydrofolate reductase preparations from harvested organisms had specific activities of 22.9 +/- 2.1 nmol/min/mg. The 50% inhibitory concentrations against reference compounds were 0.014 microM for methotrexate, 0.24 microM for pyrimethamine, 2.7 microM for trimethoprim, and 0.010 microM for trimetrexate. The Km value for NADPH was 11 microM and followed Michaelis-Menten kinetics; the Km for dihydrofolate was ca. 11 microM, but substrate inhibition appeared to occur at high substrate concentrations. Dihydrofolate reductase from T. gondii was used to screen 130 compounds from the National Cancer Institute repository. Thirteen compounds were > 100-fold more potent than pyrimethamine toward T. gondii dihydrofolate reductase; six compounds with various potencies were 8 to 46 times as selective as pyrimethamine for the protozoal form of the enzyme over the mammalian form. Four trimetrexate analogs were more potent than trimetrexate, and two were significantly more selective. Representative compounds were also tested in a culture model of T. gondii employing uracil incorporation as an index of growth. One pyrimethamine analog was as effective as pyrimethamine in inhibiting T. gondii in culture (50% inhibitory concentration, 0.45 microM). Three other compounds were also effective at micromolar concentrations.
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Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC50s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance.
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Dihydropteroate synthase
Proguanil
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Dihydrofolate reductase
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Trimethoprim
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