Malarial Dihydroorotate Dehydrogenase

Abstract

The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k cat 8 s ؊1 , K m app DHO (40 -80 M)). The substrate specificity of the ubiquinone cofactor (CoQ n ) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ n was a determinant of reaction efficiency; CoQ 4 , CoQ 6 and decylubiquinone (CoQ D ) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ 0 and vitamin K 3 ) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K m app . Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC 50 's ranging from 0.1-1.0 mM. The IC 50 values for inhibition of the malarial enzyme are 10 2 -10 4 -fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.Malaria afflicts between 500 and 900 million people worldwide and causes greater than 2 million deaths per year (1, 2), making this parasitic disease an enormous public health problem throughout the developing world. Currently, several medicinal therapies are available for use as prophylaxis and treatment for at-risk or infected individuals. However, widespread drug resistance against these agents (e.g. chloroquine (3), atovaquone (4), pyrimethamine (5), and sulfadoxine (6)), has compromised the effectiveness of these treatments resulting in the pressing need for the development of new anti-malarial compounds. The identification of targets that exploit the unique biology of the parasite is an essential step in the development of new therapeutics.Pyrimidines are essential metabolites in all cells. They are required not only for DNA and RNA biosynthesis, but also for the biosynthesis of phospholipids and glycoproteins. The de novo pyrimidine biosynthetic pathway is intact in most organisms, including Plasmodium (7-9). However, unlike mammalian cells, the human malaria parasite, Plasmodium falciparum, cannot salvage preformed pyrimidine bases or nucleosides and utilizes de n...

High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

“…Assay Methodology-The cloning and expression of recombinant P. falciparum and human DHODH were described previously (15). The standard colorimetric DHODH continuous assay that monitors 2,6-dichloroindophenol (DCIP) reduction was adapted to an end-point assay in 384-well plates for compatibility with the high-throughput screening format.…”

“…Thirdly, species-selective pyrazole-based inhibitors of Hylicobacter pylori and of Escherichia coli DHODH have been reported that were identified by highthroughput screening of chemical libraries (13,14). Finally, we previously demonstrated that P. falciparum DHODH is poorly inhibited by the potent human DHODH inhibitors redoxal, dichloroallyl lawsone, and A77-1726 analogs (15). Thus, these studies suggest it should be feasible to exploit active-site differences to identify inhibitors that exhibit a high degree of selectivity toward malarial DHODH.…”

Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

“…Fractions containing PfDHODH were pooled and concentrated to 20 mg/ml. The construction and purification of the H185A, F188A, F227A, and R265A mutant PfDHODH enzymes were described previously (12,19).…”

“…Enzyme Kinetic Analysis-Steady-state kinetic analysis was performed as described previously (12,19). To determine the k cat and K m of PfDHODH ⌬384 -413 in comparison with the wildtype enzyme the direct assay that follows the oxidation of DHO at 296 nM (⑀ ϭ 4.3 mM Ϫ1 cm Ϫ1 ) was used (Supplemental Table S1).…”

“…An inhibitor of human DHODH (hDHODH) (A77 1726 the active metabolite of leflunomide) is marketed for the treatment of rheumatoid arthritis, illustrating that DHODH is a druggable target (10,11). Finally, biochemical (12,13) and structural studies (14,15) suggested that the identification of species-selective inhibitors against this target was feasible.…”