Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.

Twenty six peroxides belonging to bridged 1,2,4,5-tetraoxanes, bridged 1,2,4-trioxolanes (ozonides), and tricyclic monoperoxides were evaluated for their in vitro antimalarial activity against Plasmodium falciparum (3D7) and for their cytotoxic activities against immortalized human normal fibroblast (CCD19Lu), liver (LO ), and lung (BEAS-2B) cell lines as well as human liver (HepG2) and lung (A549) cancer-cell lines. Synthetic ozonides were shown to have the highest cytotoxicity on HepG2 (IC =0.19-0.59 μm), and some of these compounds selectively targeted liver cancer (selectivity index values for compounds 13 a and 14 a are 20 and 28, respectively) at levels that, in some cases, were higher than those of paclitaxel, artemisinin, and artesunic acid. In contrast some ozonides showed only moderate antimalarial activity against the chloroquine-sensitive 3D7 strain of P. falciparum (IC from 2.76 to 24.2 μm; 12 b, IC =2.76 μm; 13 a, IC =20.14 μm; 14 a, IC =6.32 μm). These results suggest that these derivatives have divergent mechanisms of action against cancer cells and malaria-infected cells. A cyclic voltammetry study of the peroxides was performed, but most of the compounds did not show direct correlation in oxidative capacity-activity. Our findings offer a new source of antimalarial and anticancer agents through structural modification of peroxide compounds.

This article discloses a new horizon for the application of peroxides in medical chemistry. Stable cyclic peroxides are demonstrated to have cytotoxic activity against cancer cells; in addition a mechanism of cytotoxic action is proposed. Synthetic bridged 1,2,4,5‐tetraoxanes and ozonides were effective against HepG2 cancer cells and some ozonides selectively targeted liver cancer cells (the selectivity indexes for compounds 11 b and 12 a are 8 and 5, respectively). In some cases, tetraoxanes and ozonides were more selective than paclitaxel, artemisinin, and artesunic acid. Annexin V flow‐cytometry analysis revealed that the active ozonides 22 a and 23 a induced cell death of HepG2 by apoptosis. Further study showed that compounds 22 a and 23 a exhibited a strong inhibitory effect on P‐glycoprotein (P‐gp/ABCB5)‐overexpressing HepG2 cancer cells. ABCB5 is a key player in the multidrug‐resistant phenotype of liver cancer. Peroxides failed to demonstrate a direct correlation between oxidative potential and their biological activity. To our knowledge this is the first time that peroxide diastereoisomers have been found to show stereospecific antimalarial action against the chloroquine‐sensitive 3D7 strain of Plasmodium falciparum. Stereoisomeric ozonide 12 b is 11 times more active than stereoisomeric ozonide 12 a (IC50=5.81 vs 65.18 μm). Current findings mean that ozonides merit further investigation as potential therapeutic agents for drug‐resistant hepatocellular carcinoma.