Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum

doi:10.1016/S0166-6851(96)02808-3

Molecular and Biochemical Parasitology

Volume 85, Issue 1, March 1997, Pages 25-40

https://doi.org/10.1016/S0166-6851(96)02808-3 Get rights and content

Abstract

Pyrimethamine and cycloguanil are competitive inhibitors of the Plasmodium enzyme dihydrofolate reductase (DHFR). They have been effective treatments for malaria, but rapid selection of populations of the parasite resistant to these drugs has compromised their effectiveness. Parasites resistant to either drug usually have point mutations in the dhfr gene, but the frequency of these mutations is unknown. To study drug resistance more effectively, we transferred the DHFR domain of the dhfr-thymidylate synthase gene from a drug-sensitive line of P. falciparum to a strain of the budding yeast, Saccharomyces cerevisiae, that lacks endogenous DHFR activity. Expression of the P. falciparum dhfr is controlled by the yeast dhfr 5′ and 3′ regulatory regions and the heterologous enzyme provided all of the functions of the yeast dhfr gene. These yeast were susceptible to pyrimethamine and cycloguanil at low concentrations that inhibit P. falciparum (IC₅₀ about 10⁻⁸ and 10⁻⁷ M, respectively). Yeast expressing constructs with dhfr alleles from pyrimethamine-resistant strains were resistant to both pyrimethamine and cycloguanil (IC₅₀>10⁻⁶ M); resistance of the yeast depended on the dhfr allele they expressed. The experimental drug WR99210 efficiently killed all three yeast strains (IC₅₀ about 10⁻⁸ M) but the pyr^R strains showed collateral hypersensitivity to drug. The yeast transformants carrying the drug-sensitive allele can now be screened quickly and quantitatively to identify new drugs or combinations of drugs and determine which drugs select resistant parasites least efficiently. Such compounds would be excellent candidates for development of treatments with a longer life in clinical practice.

Introduction

The selection of populations resistant to standard regimens of chemotherapy has led to a major public health problem in many regions where falciparum malaria is common. Currently, most of the drugs that have been used in the past to treat or prevent malaria are no longer effective 1, 2. It is critical to increase chemotherapeutic options by identifying new drugs and drug targets, and optimizing deployment of drugs that are already in use. Two drugs that have been used widely against malaria are pyrimethamine and proguanil (which is metabolized in humans into the active form, cycloguanil [1]). These are both competitive inhibitors of the enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) that controls a key step in thymidylate biosynthesis [3]. Drug screening studies have identified many other DHFR inhibitors that are effective in vitro against P. falciparum 4, 5, 6, but the rapid selection of resistance to pyrimethamine and cycloguanil has strongly discouraged further development. The assumption is that other drugs with a mode of action similar to pyrimethamine and cycloguanil would show equally strong selection for drug resistance, but it has not been possible to test this directly.

Although the gene that encodes DHFR has been strongly conserved during evolution, subtle differences in the active sites of bacterial, human and parasite enzymes have been exploited to develop DHFR inhibitors specific to particular pathogens 1, 3. For example, pyrimethamine and cycloguanil bind strongly to the P. falciparum DHFR active site, but not to that of humans [7]. In eukaryotes, resistance to inhibitors of DHFR develops in three major ways: changes in drug transport or accumulation, overexpression of the wild type enzyme or point mutations in the dhfr gene that reduce the binding affinity of the inhibitor 3, 8. In mammalian cells, the first two mechanisms predominate and in the kinetoplastid, Leishmania tropica, resistance results mostly from over-replication of the region that encodes the wild type enzyme [9]. Resistance due to overexpression of the enzyme has only been reported in a laboratory line of P. falciparum selected for pyrimethamine resistance [10]. In all other cases, resistance to DHFR inhibitors correlated with point mutations in the DHFR enzyme itself, both in lines selected in the lab 10, 11 and in field populations 12, 13, 14. Based on these studies, there is considerable agreement that resistance to pyrimethamine is associated with a mutation in the Ser¹⁰⁸ to Asn and that a further increase in resistance results from changes to Ile⁵¹, Arg⁵⁹ or Leu¹⁶⁴ 7, 12, 15, 16. Transfection into pyrimethamine-sensitive P. falciparum of a dhfr gene carrying the Asn¹⁰⁸ mutation has recently confirmed the importance of that mutation [17]. Similar studies of the dhfr coding region of parasites from cycloguanil resistant populations have identified positions Thr¹⁰⁸ and Val¹⁶ as possible mutations to cycloguanil resistance, but the correspondence between particular mutations and the cycloguanil-resistant phenotype (cyc^R) is considerably less clear for this drug 18, 19.

The DHFR enzyme activity in P. falciparum is one domain of a bifunctional enzyme that includes thymidylate synthase (TS), an enzyme that catalyzes a subsequent step in the folate pathway, 3, 20. A construct that contains just the dhfr domain has been cloned and expressed in Escherichia coli, but the endogenous enzyme is still produced and the P. falciparum enzyme is mostly sequestered in inclusion bodies 7, 21, 22, 23. Therefore, while purification of the P. falciparum DHFR from the E. coli has been extremely useful for study of the biochemistry of the enzyme, the system does not allow the selection of drug resistant E. coli. Several groups have studied the development of pyrimethamine resistance (pyr^R) in laboratory populations of P. falciparum 10, 11, but the practical limitations of parasite growth in the lab prevent the determination of the frequency with which the resistance develops. Peters and his colleagues have also conducted extensive studies to select mutants resistant to antifolate drugs using P. bergei in mice [1], but these studies are extremely time consuming and expensive.

To circumvent these limitations, we have expressed the P. falciparum dhfr gene in the budding yeast Saccharomyces cerevisiae. This work is the first step to establish a biological system that will allow new anti-malaria drugs or combinations of drugs that target the dihydrofolate reductase enzyme to be screened quickly and quantitatively to determine their potential as antimalaria treatments and to measure the frequency with which colonies resistant to the drug arise.

Section snippets

Malaria and yeast strains

Three strains of Plasmodium falciparum were used to isolate DNA for this study. The strains SL/D6 [24] and Mikenga (Kenyan isolate, personal communication) were from Dr Wilbur Milhous (Experimental Therapeutics Division, Walter Reed Army Institute of Research, Silver Spring, MD) and were grown in vitro with a modification of the method of Trager [25]. Genomic DNA from the strain Honduras 1 [26] was provided by Dr R.F. Howard (Seattle Biomedical Research Institute, Seattle, WA). The two

The P. falciparum dhfr gene can supply the enzyme function in dhfr-yeast

To determine whether the P. falciparum dhfr gene could provide the enzyme function in S. cerevisiae that lack DHFR activity, we made the construct diagrammed in Fig. 1A. The 600 bp region from the 5′ promoter of the S. cerevisiae dhfr gene [32] was cloned 5′ of the dhfr domain from the P. falciparum dhfr-ts gene from D6 [31] and the translation termination and 3′ termination region from the S. cerevisiae dhfr gene were added 3′ of the Pf-dhfr-D6 coding region gene [32]. This hybrid gene was

Discussion

The experiments described here are the first step in establishing a system for estimating rapidly and quantitatively the potential of a DHFR inhibitor for selecting populations of P. falciparum resistant to the drug. We have demonstrated that the DHFR enzyme from P. falciparum can supply the enzyme function in dhfr^-S. cerevisiae. Furthermore, the sensitivity of the transformed yeast to antimalaria drugs that target DHFR depends only upon the allele they express. Thus, one can now treat yeast

Acknowledgements

The advice and encouragement of Barbara Garvik, Dr David Jacobus, Dr Wilbur Milhous and Dr Wallace Peters are gratefully acknowledged. Critical reading of the manuscript by Barbara Garvik, Kelly Hamilton, Victoria Brophy, Tuyen Vu, Eleanor Hankins, Somnath Mookherjee, Erik Jacobson and Alexis N'zila greatly improved the manuscript. Initial stages of this work were supported by the Royalties Research Fund of the University of Washington, and NIH grant GM 17709 to LHH. LHH is an ACS Research

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¹: Current address: Oregon Health Sciences University, Portland, Oregon, USA.

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