Introduction

Drug resistance has become a major problem in malaria-endemic areas. New drugs with novel mechanisms of action are required. Malaria is a major world health problem with 300 million people infected and 2–3 million deaths annually. There are 4 species of malaria which infect man and Plasmodium falciparum is responsible for the most fatalities. The parasite has 14 chromosomes constituting approximately 25 Mb of DNA. The AT content of the DNA is very high, approximately 80%, which makes expression of recombinant malarial proteins in other organisms, such as E. coli, quite difficult. Malarial genes often lack introns which means that genes can be cloned by PCR directly from genomic DNA. Sequencing of the genome in the Malarial Genome Project started in 1996 with contributions from the Sanger Centre, The Institute for Genomic Research (TIGR) and Stanford University; and was completed in October 2002 (1). The annotated sequence of the malarial genome is available at http://plasmodb.org.

The malarial parasite can only synthesise pyrimidine nucleotides via the de novo pathway (2) whereas the human patient has an alternative salvage pathway for pyrimidine nucleotides. Inhibitors of de novo pyrimidine biosynthesis may therefore have selective toxicity against the parasite. The suitability of the de novo pyrimidine pathway as an antimalarial target is demonstrated by the successful drug, atovaquone (3), which inhibits the malarial electron transport chain at Complex III and indirectly inhibits dihydroorotate dehydrogenase (reaction 4). We have designed and synthesised several dihydropyrimidine analogues which are potent inhibitors of DHOase. Two good examples are 2-oxo-1,2,3,6-tetrahydro-pyrimidine-4-6-dicarboxylate (HDDP, Ki = 0.74 µM) and 6-L-thiodihydroorotate (TDHO, Ki = 0.85 µM) (4). We showed that exposure of P. falciparum in erythrocytic culture to TDHO or atovaquone induces accumulation of N-carbamyl-L-aspartate (CA-asp), and CA-asp and L-dihydroorotate (DHO), respectively (2), with depletion of dTTP but not dCTP (5), resulting in inhibition of DNA synthesis and antimalarial activity. Combination chemotherapy with several potent inhibitors of the pyrimidine pathway would overcome metabolic resistance (6), which results from accumulation of the substrate for an inhibited enzyme to levels sufficient to out-compete the inhibitor. HDDP and TDHO are effective inhibitors of DHOase, but more potent inhibitors will be required for clinical antimalarial activity. The cloning and expression of malarial enzymes described here is a first step toward obtaining three-dimensional structures, and designing and synthesizing a second generation of more potent inhibitors.