Carol Hopkins Sibley

Pyrimethamine–sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the worlds burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

PCR and strain identification in Plasmodium falciparum

Anyone who has cultured Plasmodium falciparum is aware that confusion about the identity of commonly used strains, and inadvertent contamination of one strain with another have been persistent problems. These issues have been recently reviewed by Robson and colleagues. Jason Wooden, Susan Kyes and Carol Hopkins Sibley have recently adapted two methods that offer a quick, easy alternative for the identification of P. falciparum strains in the laboratory.

Dihydrofolate Reductase Mutations in Plasmodium vivax from Indonesia and Therapeutic Response to Sulfadoxine plus Pyrimethamine

BACKGROUND The target enzyme of pyrimethamine is dihydrofolate reductase (DHFR), but little is known about allelic variants of dhfr in Plasmodium vivax populations. Still less is known about associations between specific alleles and the failure of sulfadoxine/pyrimethamine (S/P) to clear the erythrocytic stages of P. vivax in vivo. METHODS We studied P. vivax dhfr mutations in 24 patients who received S/P therapy in Papua or Central Java, Indonesia, and we measured the resistance of the alleles in vitro in a dhfr yeast expression assay. RESULTS Fourteen (58%) of 24 patients had an inadequate therapeutic response. Two of 6 alleles that were identified were novel. One allele that expressed 4 point mutations (57L+58R+61M+117T) correlated with a high risk of therapeutic failure. The 9 patients infected by P. vivax carrying this allele proved 23 times more likely to experience early therapeutic failure, compared with patients infected by P. vivax carrying other alleles (P=.003; 95% confidence interval, 2-450). This allele also conferred high levels of pyrimethamine resistance in vitro. The experimental antifolate WR99210 inhibited the allele in this system. CONCLUSIONS The present study identified a strong correlation between specific mutations in P. vivax dhfr and S/P treatment failure. Our results suggest that WR99210 could provide effective therapy for S/P-resistant P. vivax.

Highly pyrimethamine-resistant alleles of dihydrofolate reductase in isolates of Plasmodium falciparum from Tanzania

In 2000 we used a sensitive technique to examine 9 isolates from malaria patients in Muheza, Tanzania who had failed treatment with sulfadoxine-pyrimethamine (SP). Three isolates carried, at low levels, the leucine to isoleucine change at amino acid 164 that is associated with clinical failure of SP. Numerous other highly resistant alleles were also observed.

A database of antimalarial drug resistance

A large investment is required to develop, license and deploy a new antimalarial drug. Too often, that investment has been rapidly devalued by the selection of parasite populations resistant to the drug action. To understand the mechanisms of selection, detailed information on the patterns of drug use in a variety of environments, and the geographic and temporal patterns of resistance is needed. Currently, there is no publically-accessible central database that contains information on the levels of resistance to antimalaria drugs.This paper outlines the resources that are available and the steps that might be taken to create a dynamic, open access database that would include current and historical data on clinical efficacy, in vitro responses and molecular markers related to drug resistance in Plasmodium falciparum and Plasmodium vivax. The goal is to include historical and current data on resistance to commonly used drugs, like chloroquine and sulfadoxine-pyrimethamine, and on the many combinations that are now being tested in different settings. The database will be accessible to all on the Web.The information in such a database will inform optimal utilization of current drugs and sustain the longest possible therapeutic life of newly introduced drugs and combinations. The database will protect the valuable investment represented by the development and deployment of novel therapies for malaria.

Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae

ABSTRACT There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinantC. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvumDHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.

Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa

ABSTRACT Competitive inhibitors of dihydrofolate reductase (DHFR) are used in chemotherapy or prophylaxis of many microbial pathogens, including the eukaryotic parasites Plasmodium falciparum andToxoplasma 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 cerevisiaewith 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.

Novel Approaches to Tackling Malarial Drug Resistance Using Yeast

Yeasts have a justified reputation as one of the worlds most versatile organisms. Bakers yeast continues to live up to this recognition by joining the war against malaria. Yeast can now be used to study antifolate drug resistance patterns that depend on the dihydrofolate reductase enzyme (DHFR) from the malaria parasite.

Molecular data on Plasmodium falciparum chloroquine and antifolate resistance: a public health tool

The authors are grateful to Pascal Ringwald for critically reading the manuscript and for his contributions.