Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.

Malaria is one of the most significant causes of childhood mortality but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective towards DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200–400 mg. DSM265 was well tolerated in repeat dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood and liver-stage activity, and predicted long human half-life position DSM265 as a new potential drug combination partner for either single-dose treatment or once weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive on the parasite liver-stage

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-m widths were readily traversed by the 8 m-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2-to 4-m channels; some that passed through the 4-m channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-m channels, trophozoites mimicked ''pitting,'' a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-m channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-m capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential anti-malarials we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound towards clinical candidate status.

Continual exposure of malarial parasite populations to different drugs may have selected not only for resistance to individual drugs but also for genetic traits that favor initiation of resistance to novel unrelated antimalarials. To test this hypothesis, different Plasmodium falciparum clones having varying numbers of preexisting resistance mechanisms were treated with two new antimalarial agents: 5-f luoroorotate and atovaquone. All parasite populations were equally susceptible in small numbers. However, when large populations of these clones were challenged with either of the two compounds, significant variations in frequencies of resistance became apparent. On one extreme, clone D6 from West Africa, which was sensitive to all traditional antimalarial agents, failed to develop resistance under simple nonmutagenic conditions in vitro. In sharp contrast, the Indochina clone W2, which was known to be resistant to all traditional antimalarial drugs, independently acquired resistance to both new compounds as much as a 1,000 times more frequently than D6. Additional clones that were resistant to some (but not all) traditional antimalarial agents acquired resistance to atovaquone at high frequency, but not to 5-f luoroorotate. These findings were unexpected and surprising based on current views of the evolution of drug resistance in P. falciparum populations. Such new phenotypes, named accelerated resistance to multiple drugs (ARMD), raise important questions about the genetic and biochemical mechanisms related to the initiation of drug resistance in malarial parasites. Some potential mechanisms underlying ARMD phenotypes have public health implications that are ominous.

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...

Antimalarial drugs are key tools for the control and elimination of malaria. Recent decreases in the global malaria burden are likely due, in part, to the deployment of artemisinin-based combination therapies. Therefore, the emergence and potential spread of artemisinin-resistant parasites in southeast Asia and changes in sensitivities to artemisinin partner drugs have raised concerns. In recognition of this urgent threat, the International Centers of Excellence for Malaria Research (ICEMRs) are closely monitoring antimalarial drug efficacy and studying the mechanisms underlying drug resistance. At multiple sentinel sites of the global ICEMR network, research activities include clinical studies to track the efficacies of antimalarial drugs, ex vivo/in vitro assays to measure drug susceptibilities of parasite isolates, and characterization of resistance-mediating parasite polymorphisms. Taken together, these efforts offer an increasingly comprehensive assessment of the efficacies of antimalarial therapies, and enable us to predict the emergence of drug resistance and to guide local antimalarial drug policies. Here we briefly review worldwide antimalarial drug resistance concerns, summarize research activities of the ICEMRs related to drug resistance, and assess the global impacts of the ICEMR programs.