Yeast as a Model System to Study Drugs Effective against Apicomplexan Proteins

Sibley, Carol Hopkins; Brophy, Victoria H.; Cheesman, Sandie; Hamilton, Kelly Lee; Hankins, Eleanor G.; Wooden, Jason M.; Kilbey, Brian J.

doi:10.1006/meth.1997.0511

Cited by 37 publications

(49 citation statements)

References 111 publications

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“…The assay was performed in 96-well plates in the manner described previously. 46 The degree of growth of each culture was ascertained by the absorbance at 650 nm after 24 hr. Inhibition curves were calculated in each case by plotting the percentage of growth of treated cultures (relative to the solvent control), versus the drug concentration of the treated cultures.…”

Section: Methodsmentioning

confidence: 99%

“…Spoke assays were performed in the manner described previously. 46 Briefly, clones of interest and control strains were patched radially onto complete plates, and allowed to grow for 3-4 days. These patches were then double replica plated onto two complete plates.…”

Section: Methodsmentioning

confidence: 99%

“…deficient yeast strain was transformed with the S108N or SI08N ϩ N51I Pf-DHFR alleles, each strain showed antifolate sensitivity that paralleled the sensitivity of the original P. falciparum strain. 45,46 Thus, the ability of transgenic yeast bearing Pf-DHFR alleles to grow in the presence of DHFR inhibitors can function as a surrogate test of drug sensitivity in the P. falciparum strain from which the DHFR allele was isolated.…”

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confidence: 99%

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Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Mookherjee¹,

Howard²,

Nzila-Mouanda³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. As resistance to chloroquine spreads in sub-Saharan Africa, pyrimethamine plus sulfadoxine (PSD) is increasingly used as a first-line treatment for falciparum malaria. Populations of Plasmodium falciparum (Pf) resistant to PSD have been selected quickly in other regions. The resistance is strongly correlated with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), the two targets of the drug. It is critical to identify drug-resistant Pf-DHFR alleles that are present at a low frequency in these populations since alleles that confer drug resistance will be quickly selected by PSD use. It is difficult to identify these rare alleles by standard molecular techniques. We have designed a yeast expression system that facilitates the identification and rapid analysis of Pf-DHFR alleles that confer PSD resistance, even when they are present at very low frequency in polyclonal patient samples. We analyzed samples from patients in Kilifi, Kenya collected between 1992 and 1995. We determined the prevalence of the drug-sensitive and drug-resistant alleles in patient samples analyzed in parallel by an allelespecific enzyme digestion (ASED) assay. We identified a pyrimethamine-resistant allele (S108N) present at a frequency of Ͻ 1% in a sample that was scored as only S108 by ASED. In addition, a novel pyrimethamine-resistant allele (I164M) was isolated twice, once each from two different patient samples. This approach will allow determination of the prevalence of Pf-DHFR alleles that confer pyrimethamine resistance in particular regions, and the rapid identification of novel alleles that confer drug resistance.Dihydrofolate reductase (DHFR) catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential step in the production of methyl groups for biosynthesis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Mookherjee¹,

Howard²,

Nzila-Mouanda³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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“…DHFR inhibitors function principally as competitive inhibitors of the enzyme. We have shown that the sensitivity of our engineered yeast strains to DHFR inhibitors depends on the interaction of the drug and the heterologous DHFR enzyme (4,44,48). For example, point mutations within the coding region of the P. falciparum DHFR gene can render the enzyme resistant to pyrimethamine.…”

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confidence: 99%

Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa

Lau

Ferlan

Brophy

et al. 2001

Antimicrob Agents Chemother

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Competitive inhibitors of dihydrofolate reductase (DHFR) are used in chemotherapy or prophylaxis of many microbial pathogens, including the eukaryotic parasites Plasmodium falciparum and Toxoplasma gondii. Unfortunately, point mutations in the DHFR gene can confer resistance to inhibitors specific to these pathogens. We have developed a rapid system for testing inhibitors of DHFRs from a variety of parasites. We replaced the DHFR gene from the budding yeast Saccharomyces cerevisiae with the DHFR-coding region from humans, P. falciparum, T. gondii, Pneumocystis carinii, and bovine or human-derived Cryptosporidium parvum. We studied 84 dicyclic and tricyclic 2,4-diaminopyrimidine derivatives in this heterologous system and identified those most effective against the DHFR enzymes from each of the pathogens. Among these compounds, six tetrahydroquinazolines were effective inhibitors of every strain tested, but they also inhibited the human DHFR and were not selective for the parasites. However, two quinazolines and four tetrahydroquinazolines were both potent and selective inhibitors of the P. falciparum DHFR. These compounds show promise for development as antimalarial drugs.The treatment of diseases caused by eukaryotic pathogens is particularly difficult because of the similarity between their cell biology and that of their human host. The selection for pathogens resistant to currently effective drugs and the increase in immunocompromised individuals have added urgency to the search for new therapies directed specifically against these pathogens. One fruitful avenue for identification of chemotherapeutic drugs is to screen compounds that have already been synthesized in order to identify those that might be active against these increasingly important pathogens. We have adopted this strategy and screened a large library of compounds that are directed against the enzyme dihydrofolate reductase (DHFR) (EC 1.5.1.3). DHFR is a central enzyme in nucleic acid and amino acid synthesis in all cells, but the active sites of enzymes from different organisms show subtle differences that allow the identification of inhibitors specific for a particular species (3,(16)(17)(18)24). For example, pyrimethamine is a selective inhibitor that is effective in the nanomolar range against the DHFRs from Plasmodium falciparum and Toxoplasma gondii, but the human enzyme is relatively insensitive to the drug (8,14,24). Thus, pyrimethamine has been used in malaria and toxoplasmosis therapy for many years (9, 49).We have designed an easy and inexpensive system to test in budding yeast (Saccharomyces cerevisiae) potential DHFR inhibitors against the enzymes from a variety of parasites. Function of the endogenous dfr1 gene was eliminated from the yeast (15), and the defect was complemented by expression of a heterologous DHFR gene from P. falciparum, T. gondii, Pneumocystis carinii, Cryptosporidium parvum, or humans (4). DHFR inhibitors function principally as competitive inhibitors of the enzyme. We have shown that the sensitivity of our engi...

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“…The drug sensitivity assays were performed as described previously to obtain quantitative measures of drug sensitivity (24). Briefly, the growth of the yeast in this assay depends upon the antifolate resistance of the dhfr allele expressed (35).…”

Section: Vol 49 2005 De Novo Synthesis Of Novel Antifolate Antimalamentioning

confidence: 99%

2,4-Diaminopteridine-Based Compounds as Precursors for De Novo Synthesis of Antifolates: a Novel Class of Antimalarials

Nduati

Hunt

Kamau

et al. 2005

Antimicrob Agents Chemother

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We have tested the hypothesis that 2,4-diamino-6-hydroxymethyl-pteridine (DAP), 2,4-diaminopteroic acid (DAPA), and 2,4 diamino-N10-methyl-pteroic acid (DAMPA) could be converted into aminopterin (from DAP and DAPA) and methotrexate (from DAMPA), both of which are potent inhibitors of dihydrofolate reductase, a proven drug target for Plasmodium falciparum. DAP, DAPA, and DAMPA inhibited parasite growth in the micromolar range; DAMPA was the most active, with 50% inhibitory concentrations in vitro of 446 ng/ml against the antifolate-sensitive strain and 812 ng/ml against the highly resistant strain under physiological folate conditions. DAMPA potentiates the activity of the sulfone dapsone, an inhibitor of dihydropteroate synthase, but not that of chlorcycloguanil, a known inhibitor of dihydrofolate reductase (DHFR). Experiments with a Saccharomyces cerevisiae strain dependent upon the P. falciparum DHFR enzyme showed that DHFR is a target of DAMPA in that system. We hypothesize that DAMPA is converted to methotrexate by the parasite dihydrofolate synthase, which explains the synergy of DAMPA with dapsone but not with chlorcycloguanil. This de novo synthesis will not occur in the host, since it lacks the complete folate pathway. If this hypothesis holds true, the de novo synthesis of the toxic compounds could be used as a framework for the search for novel potent antimalarial antifolates.Chemotherapy remains one of the most important tools for the management of falciparum malaria. However, malaria control is hampered by the emergence and spread of parasites resistant to almost all available antimalarial drugs. This situation is critical in Africa as a result of the spread of resistance to the combination sulfadoxine-pyrimethamine, an inexpensive treatment widely used in African countries (9, 16-18, 25, 28). As an alternative, a number of combinations with artemisinins are being recommended and implemented, but questions about the cost and the adequacy of the supply of artemisinins and the intrinsic ability of Plasmodium falciparum to select drug-resistant parasite populations underline the need to identify novel agents.

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Yeast as a Model System to Study Drugs Effective against Apicomplexan Proteins

Cited by 37 publications

References 111 publications

Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa

2,4-Diaminopteridine-Based Compounds as Precursors for De Novo Synthesis of Antifolates: a Novel Class of Antimalarials

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