Plasmodium falciparum is a single celled parasite that causes the most deadly of the 4 kinds of human malaria. There is currently no vaccine to prevent this disease, and about 200 million people suffer from malaria each year. In fact, malaria causes 2 million deaths each year, most of them children in sub-Saharan Africa. There is an acute need for effective chemotherapeutic agents for prophylaxis and treatment of falciparum malaria. Drugs that target dihydrofolate reductase (DHFR), a key enzyme in the synthesis of deoxythymidine, histidine, and methionine and sulfonamides that target dihydropteroate synthase (DHPS), required for the synthesis of folate have been extremely effective in the past . In most cases, combinations of these drugs have been used, because the drugs act synergistically. However, the incredibly rapid selection of resistant P. falciparum populations has made the drugs virtually useless in many regions. The parasites are resistant to the drugs because they carry alleles of the DHFR or DHPS genes that encode mutant forms of the target enzyme.
The P. falciparum parasites can be grown in the lab, but their culture is expensive and labor intensive. To simplify study of these target enzymes from P. falciparum, we have engineered a series of strains of the budding yeast, Saccharomyces cerevisiae. These yeast lack endogenous DHFR or DHPS, but can grow because we have transformed them with the P. falciparum version of one of these enzymes. This approach has allowed us to use the yeast system to study the function of the parasite enzymes in a simple, inexpensive way. Normally, yeast are insensitive to antimalaria drugs, but these engineered yeast strains are now sensitive to inhibitors of the P. falciparum enzymes. We are studying the mutations in the DHFR and DHPS genes that confer resistance to inhibitors of these enzymes. We have used this approach to identify new mutations that can encode drug-resistant enzymes, and to screen a panel of potential new inhibitors for their effectiveness against the parasite enzymes. In addition, we have used the polymerase chain reaction to amplify DHFR genes from blood samples collected between 1984 and 2001 in Kenya. Yeast strains that express these alleles have been engineered, and used to characterize the drug sensitivity profiles of the parasites that infected these patients. These data are being used to reconstruct the history of the selection for drug-resistant alleles that has occurred since the introduction of Fansidar into use in Kenya.
We have recently extend these studies to include the DHFR and DHPS genes from a number of other related pathogens (Plasmodium vivax, Cryptosporidium parvum, Toxoplasma gondii) and the bacterium that causes tuberculosis, Mycobacterium tuberculosis. We have two overall goals in our work. First, to use the basic techniques of genetics and molecular biology to understand the mechanism of inhibition of DHFR and DHPS by antifolate drugs. This will allow the design of alternative drugs that are effective against parasites that are resistant to currently available drugs. Second, to use this simple yeast system to understand the selection pressures that have resulted in mutations that confer drug resistance. Our work is highly collaborative. We have strong collaborations with colleagues at the Wellcome Trust Research Laboratory in Nairobi, Kenya, the National Institute for Medical Research, Amani-Tanga, Tanzania, the Liverpool School of Tropical Medicine, the London School of Hygiene and Tropical Medicine, and the University of Manchester Institute of Science and Technology, in the UK, Jacobus Pharmaceutical Company in Princeton, NJ, the Dana Farber Cancer Institute, Boston, MA and the CSIRO in Melbourne, Australia. We hope to apply our understanding of antifolate drugs to design deployment and use strategies that will slow the selection of drug-resistant parasites in the future.
Certain, L and Sibley, CH. 2007. Plasmodium falciparum: A novel method for analyzing haplotypes in mixed infections. Experimental Parasutology, 115:233-241.
Sandefur, CI, Wooden, JM, Quaye, IK, Sirawaraporn, W and Sibley, CH . 2007. Pyrimethamine-resistant dihydrofolate reductase enzymes of P. falciparum are not enzymatically compromised in vitro. Molecular and Biochemical Parasitology.154:1-5.
VN Hawkins, H Joshi, K Rungsihirunrat, K Na-Bangchang, CH Sibley: 2007 Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol 23: 213-222.
Sibley CH, Barnes KI and Plowe CV .2007. The rationale and plan for creating a World Antimalarial Resistance Drug Network (WARN). Malar J 6:118.
Price RN, Dorsey G, Ashley EA, Barnes KI, Baird JK, d'Alessandro U, Guerin PJ, Laufer MK, Naidoo I, Nosten F, Olliaro P, Plowe CV, Ringwald P, Sibley CH, Stepniewska K and White NJ .2007. World Antimalarial Resistance Network (WARN) I: Clinical efficacy of antimalarial therapy. Malar J 6:119.
Plowe CV, Roper C, Barnwell JW, Happi CT, Joshi HH, Mbacham W, Meshnick SR, Mugittu K, Naidoo I, Price RN, Shafer RW, Sibley CH, Sutherland CJ, Zimmerman PA and Rosenthal PJ. 2007. World Antimalarial Resistance Network (WARN) III: Molecular markers for drug resistant malaria. Malar J 6:121.
Bacon DJ, Jambou R, Fandeur T, Le Bras J, Wongsrichchanalai C, Fukuda MM, Ringwald P, Sibley CH and Kyle DE. 2007. World Antimalarial Resistance Network (WARN) II: In vitro antimalarial drug susceptibility. Malar J 6:120.
Barnes KI, Lindegardh N, Ogundahunsi O, Olliaro P, Plowe CV, Randrianarivelojosia M, Gbotosho GO, Watkins WM, Sibley CH and White NJ. 2007. World Antimalarial Resistance Network (WARN) IV: Clinical pharmacology. Malar J 6:122.
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