Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED 50 values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

Murine models of Plasmodium falciparum malaria may become crucial tools in drug discovery. Here we show that non-myelodepleted NOD-scid IL2R␥ null mice engrafted with human erythrocytes support an infectious burden up to tenfold higher than that supported by engrafted NOD-scid ␤2microglobulin null mice. The new model was validated for drug discovery and was used to assess the therapeutic efficacy of 4-pyridones, selective inhibitors of P. falciparum cytochrome bc 1 .Malaria is caused by the erythrocytic stages of protozoan parasites of the genus Plasmodium. Among the species pathogenic for humans, Plasmodium falciparum is responsible for 300 to 500 million cases of malaria and over a million deaths annually, particularly in developing countries. The development of new antimalarial medicines and vaccines is a key part of the global strategy for malaria eradication (6).P. falciparum almost exclusively infects human erythrocytes (hE). As a result, candidate drugs and vaccines in early stages of preclinical development are usually tested in vivo by measuring their therapeutic efficacy against rodent-adapted plasmodial species and by assessing the antiparasitic response of non-human immune systems, respectively (11). To overcome the host specificity issue, two conceptually different murine models of erythrocytic stages of P. falciparum malaria have been developed. The first one requires chemical in vivo depletion of phagocytic cells from immunodeficient mice engrafted with hE in order to allow the growth of P. falciparum after intraperitoneal (i.p.) infection (2, 8). However, its variable kinetics of parasitemia and, particularly, the use of toxic reagents, which might affect the efficacy of antimalarials or effector cells, have limited its use in drug discovery (5). Recently, a new P. falciparum murine model that does not require in vivo myeloablative treatment of mice and is suitable for drug discovery was described (1). In this new model, NOD-scid mice genetically deficient in beta-2 microglobulin (␤2 m tm1Unc , abbreviated as ␤2 m null ) engrafted with hE (HM-␤2 m null ) are infected intravenously with P. falciparum strains selected in vivo for their competence to grow reproducibly in hE-engrafted immunodeficient mice (1).The NOD-scid ␤2 m null mouse strain retains residual NK cell activity as well as other innate immune functions and shows a high incidence of early thymic lymphomas, which dramatically diminish their life span (4). These characteristics may be a serious problem for addressing long-term pharmacokinetic/ pharmacodynamic (PK/PD) studies because of the relatively low total parasite burden per mouse achievable (1) and the short life span of NOD-scid ␤2 m null mice (4). Interestingly, NOD-scid strains carrying a null mutation of the interleukin 2 (IL-2) receptor ␥ chain (IL2R␥ tm1Wjll , abbreviated as IL2R␥ null ) have been developed (10). These murine strains lack fully mature NK cells and show additional defects in their innate immune system that explain their greater ability to support the engraftme...

Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (!0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD scidb2m-/-engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount TM tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparuminfected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation. MALARIA is caused by the erythrocytic stages of protozoa of the genus Plasmodium, which colonize and destroy host's erythrocytes (1). To counter this disease, murine models of malaria are essential tools for research (2), particularly for drug discovery (3). In addition to the standard rodent experimental systems, different murine models of P. falciparum malaria are currently available (4-6). These are of special interest for drug discovery because, with the exception of human subjects, these are the only experimental systems available that allow the evaluation in vivo of the real human pathogen growing inside human erythrocytes (hE) previously engrafted into immunodeficient mice. Not surprisingly, the peripheral blood of these chimeric mice [humanized mice (HM)] is a complex mixture of murine erythrocytes (mE) and hE, in which the hematological effects of massive transfusions of hE and their elimination from peripheral blood may have important effects. Hence, the specific and quantitative measurement of different erythrocytic subpopulations is crucial in HM models, particularly when these models are used to establish the relationship between the amount of an antimalarial drug in blood and the effect on parasitemia through experimental pharmacokinetic and pharmacodynamic studies (PK/PD). In this kind of

BackgroundThe emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs.MethodsParasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined.ResultsThe parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium–high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture ‘Malaria Box’.ConclusionThe quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium–high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.