Malaria is a devastating infection caused by protozoa of the genus Plasmodium. Drug resistance is widespread, no new chemical class of antimalarials has been introduced into clinical practice since 1996 and there is a recent rise of parasite strains with reduced sensitivity to the newest drugs. We screened nearly 2 million compounds in GlaxoSmithKline's chemical library for inhibitors of P. falciparum, of which 13,533 were confirmed to inhibit parasite growth by at least 80% at 2 microM concentration. More than 8,000 also showed potent activity against the multidrug resistant strain Dd2. Most (82%) compounds originate from internal company projects and are new to the malaria community. Analyses using historic assay data suggest several novel mechanisms of antimalarial action, such as inhibition of protein kinases and host-pathogen interaction related targets. Chemical structures and associated data are hereby made public to encourage additional drug lead identification efforts and further research into this disease.

Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication.

In response to heat stress, Bacillus subtilis activates the transcription of well over 100 different genes. Many of these genes are members of a general stress response regulon controlled by the secondary sigma factor, B , while others are under control of the HrcA or CtsR heat shock regulators. We have used DNA microarrays to monitor the global transcriptional response to heat shock. We find strong induction of known B -dependent genes with a characteristic rapid induction followed by a return to near prestimulus levels. The HrcA and CtsR regulons are also induced, but with somewhat slower kinetics. Analysis of DNA sequences proximal to newly identified heat-induced genes leads us to propose ϳ70 additional members of the B regulon. We have also identified numerous heat-induced genes that are not members of known heat shock regulons. Notably, we observe very strong induction of arginine biosynthesis and transport operons. Induction of several genes was confirmed by quantitative reverse transcriptase PCR. In addition, the transcriptional responses measured by microarray hybridization compare favorably with the numerous previous studies of heat shock in this organism. Since many different conditions elicit both specific and general stress responses, knowledge of the heat-induced general stress response reported here will be helpful for interpreting future microarray studies of other stress responses.DNA microarray technology provides a powerful tool for the analysis of global transcriptional responses elicited by various physical and chemical stresses. One challenge in this sort of analysis is to distinguish stress-specific responses from more general stress responses. For example, in Bacillus subtilis, many different stresses (including heat shock, osmotic stress, and energy stress) activate the large general stress response controlled by the B transcription factor (20,44,45). While others have used two-dimensional protein gels to classify cellular stress responses (55, 58), DNA-based methods have several advantages: they can be rapidly adapted to new organisms, they provide greater coverage of the genome, and data processing is comparatively easy to automate. Ultimately, it may be possible to integrate both technologies, at least for well-studied model organisms (19,41,56).We have initiated a series of studies to characterize the global transcriptional responses of B. subtilis, a model grampositive microorganism. Here, we document the heat-induced general stress response. Heat shock was chosen for this initial study since it is arguably the best-studied stress response in this organism and includes activation of the large general stress response under the control of B (20, 44). In addition, a subset of antibiotics that inhibit translation have been reported to induce heat shock genes in other organisms (57). It is anticipated that knowledge of transcriptional responses to antimicrobial compounds will be useful for both antibacterial discovery and characterization (47).Analysis of the transcriptional profile of B...

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.