The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust highthroughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of Ϸ1.7 million compounds, we identified a diverse collection of Ϸ6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 M). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.antifolates ͉ cheminformatics ͉ high-throughput screening ͉ Plasmodium falciparum

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.

The antiplasmodial activity of a series of spirotetrahydro beta-carbolines is described. Racemic spiroazepineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in vitro IC(50) of 90 nM. Structure-activity relationships for the optimization of 1 to compound 20a (IC(50) = 0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved in four of five mice with three daily doses of 30 mg/kg.

A fundamental problem in systems biology and whole genome sequence analysis is how to infer functions for the many uncharacterized proteins that are identified, whether they are conserved across organisms of different phyla or are phylum-specific. This problem is especially acute in pathogens, such as malaria parasites, where genetic and biochemical investigations are likely to be more difficult. Here we perform comparative expression analysis on Plasmodium parasite life cycle data derived from P. falciparum blood, sporozoite, zygote and ookinete stages, and P. yoelii mosquito oocyst and salivary gland sporozoites, blood and liver stages and show that type II fatty acid biosynthesis genes are upregulated in liver and insect stages relative to asexual blood stages. We also show that some universally uncharacterized genes with orthologs in Plasmodium species, Saccharomyces cerevisiae and humans show coordinated transcription patterns in large collections of human and yeast expression data and that the function of the uncharacterized genes can sometimes be predicted based on the expression patterns across these diverse organisms. We also use a comprehensive and unbiased literature mining method to predict which uncharacterized parasite-specific genes are likely to have roles in processes such as gliding motility, host-cell interactions, sporozoite stage, or rhoptry function. These analyses, together with protein-protein interaction data, provide probabilistic models that predict the function of 926 uncharacterized malaria genes and also suggest that malaria parasites may provide a simple model system for the study of some human processes. These data also provide a foundation for further studies of transcriptional regulation in malaria parasites.