A Plasmodium falciparum dihydroorotate dehydrogenase ( PfDHODH) inhibitor that is potent ( KI = 15 nM) and species-selective (>5000-fold over the human enzyme) was identified by high-throughput screening. The substituted triazolopyrimidine and its structural analogues were produced by an inexpensive three-step synthesis, and the series showed good association between PfDHODH inhibition and parasite toxicity. This study has identified the first nanomolar PfDHODH inhibitor with potent antimalarial activity in whole cells (EC50 = 79 nM).

Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into speciesselective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.The human malaria parasite is endemic in 87 countries putting 2.5 billion people in the poorest nations of the tropics at risk for the disease (1, 2). Despite intensive efforts to control malaria through combination drug therapy and insect control programs, malaria remains one of the largest global health problems. The most severe form of the disease is caused by Plasmodium falciparum, which kills 1-2 million people yearly, primarily children and pregnant woman. Effective vaccines have not been developed, and chemotherapy remains the mainstay of both treatment and prevention of the disease. Unfortunately widespread drug resistance to almost every known antimalarial agent has compromised the effectiveness of malaria control programs (3). The introduction of artemisinin combination chemotherapy has provided new treatment options to combat drug-resistant parasites (4). However, recent reports by the World Health Organization suggest that resistance to artemisinin is developing along the Thai-Cambodian border, underscoring the need for a continual pipeline of new drug development to combat this disease.The malaria parasite relies exclusively on de novo pyrimidine biosynthesis to supply precursors for DNA and RNA biosynthesis (5, 6). In contrast, the human host cells contain the enzymatic machinery for both de novo pyrimidine biosynthesis and for salvage of preformed pyrimidine bases and nucleosides. The lack of a redundant mechanism to acquire pyrimidines in malaria...

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 ureabased 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.

Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi-infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.