Abstract
Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.
Highlights
Malaria remains the dominant public health issue in many developing regions of the world, and it is the cause millions of deaths annually with highly mortality among children [1]
Alignment of the malarial Dihydroorotate dehydrogenase (DHODH) sequence onto this structure indicated that more than half of the residues that are within van der Waals distance of A77-1726 are variable (Fig. 1)
We previously demonstrated that P. falciparum DHODH is poorly inhibited by the potent human DHODH inhibitors redoxal, dichloroallyl lawsone, and A77-1726 analogs [15]
Summary
Malaria remains the dominant public health issue in many developing regions of the world, and it is the cause millions of deaths annually with highly mortality among children [1]. The redesign and synthetic optimization of existing anti-malarials extends their lifetime as useful drugs, there is a need to identify novel chemical scaffolds that inhibit existing and previously unexploited biological targets. We previously demonstrated that P. falciparum DHODH is poorly inhibited by the potent human DHODH inhibitors redoxal, dichloroallyl lawsone, and A77-1726 analogs [15]. 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. We report the use of high-throughput screening technology of a 220,000 small druglike molecule library to identify a number of potent and selective inhibitors of the P. falciparum DHODH. Two classes of molecules, based on biphenylamide and urea structural scaffolds, were characterized in detail
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