Discovery of novel antimalarial compounds enabled by QSAR-based virtual screening.

Quantitative structure-activity relationship (QSAR) models have been developed for a data set of 3133 compounds defined as either active or inactive against P. falciparum. Because the data set was strongly biased toward inactive compounds, different sampling approaches were employed to balance the ratio of actives versus inactives, and models were rigorously validated using both internal and external validation approaches. The balanced accuracy for assessing the antimalarial activities of 70 external compounds was between 87% and 100% depending on the approach used to balance the data set. Virtual screening of the ChemBridge database using QSAR models identified 176 putative antimalarial compounds that were submitted for experimental validation, along with 42 putative inactives as negative controls. Twenty five (14.2%) computational hits were found to have antimalarial activities with minimal cytotoxicity to mammalian cells, while all 42 putative inactives were confirmed experimentally. Structural inspection of confirmed active hits revealed novel chemical scaffolds, which could be employed as starting points to discover novel antimalarial agents.

Synthesis of artemiside and its effects in combination with conventional drugs against severe murine malaria.

This research describes the use of novel antimalarial combinations of the new artemisinin derivative artemiside, a 10-alkylamino artemisinin. It is a stable, highly crystalline compound that is economically prepared from dihydroartemisinin in a one-step process. Artemiside activity was more pronounced than that of any antimalarial drug in use, both in Plasmodium falciparum culture and in vivo in a murine malaria model depicting cerebral malaria (CM). In vitro high-throughput testing of artemiside combinations revealed a large number of conventional antimalarial drugs with which it was additive. Following monotherapy in mice, individual drugs reduced parasitemias to nondetectable levels. However, after a period of latency, parasites again were seen and eventually all mice became terminally ill. Treatment with individual drugs did not prevent CM in mice with recrudescent malaria, except for piperaquine at high concentrations. Even when CM was prevented, the mice developed later of severe anemia. In contrast, most of the mice treated with drug combinations survived. A combination of artemiside and mefloquine or piperaquine may confer an optimal result because of the longer half life of both conventional drugs. The use of artemiside combinations revealed a significant safety margin of the effective artemiside doses. Likewise, a combination of 1.3 mg/kg of body weight artemiside and 10 mg/kg piperaquine administered for 3 days from the seventh day postinfection was completely curative. It appears possible to increase drug concentrations in the combination therapy without reaching toxic levels. Using the drug combinations as little as 1 day before the expected death of control animals, we could prevent further parasite development and death due to CM or anemic malaria. Earlier treatment may prevent cognitive dysfunctions which might occur after recovery from CM.

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 µM) and achieves "artemisinin-like" kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

PfCHA is a mitochondrial divalent cation/H+ antiporter in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum is capable of adapting to vastly different extracellular Ca(2+) environments while maintaining tight control of its intracellular Ca(2+) concentration. The mechanisms underpinning Ca(2+) homeostasis in this important pathogen are only partly understood. Here we have functionally expressed the putative Ca(2+)/H(+) antiporter PfCHA in Xenopus laevis oocytes. Our data suggest that PfCHA mediates H(+)-coupled Ca(2+) and Mn(2+) exchange. The apparent dissociation constant K(M) for Ca(2+) of 2.2 +/- 0.7 mM and the maximal velocity V(max) of 0.6 +/- 0.1 nmol per oocyte per hour are consistent with PfCHA being a low-affinity high-capacity Ca(2+) carrier. In the parasite, PfCHA was found to localize to the mitochondrion. Physiological studies conducted with live parasitized erythrocytes, and using Fluo-4 and Rhod-2 to monitor cytoplasmic and mitochondrial Ca(2+) dynamics, suggest that the mitochondrion serves as a dynamic Ca(2+) store and that PfCHA functions as a Ca(2+) efflux system expelling excess Ca(2+) from the mitochondrion. PfCHA lacks appreciable homologies to the human mitochondrial Ca(2+)/H(+) exchanger and might represent an evolutionary divergent class of mitochondrial cation antiporter, which, in turn, might provide novel opportunities for intervention.