Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms inPlasmodium falciparum

Abstract

Epigenetic factors such as histone methylation control the developmental progression of malaria parasites during the complex life cycle in the human host. We investigated Plasmodium falciparum histone lysine methyltransferases as a potential target class for the development of novel antimalarials. We synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its derivative TM2-115, inhibited P. falciparum 3D7 parasites in culture with IC 50 values of ∼100 nM, values at least 22-fold more potent than their apparent IC 50 toward two human cell lines and one mouse cell line. These compounds irreversibly arrested parasite growth at all stages of the intraerythrocytic life cycle. Decrease in parasite viability (>40%) was seen after a 3-h incubation with 1 µM BIX-01294 and resulted in complete parasite killing after a 12-h incubation. Additionally, mice with patent Plasmodium berghei ANKA strain infection treated with a single dose (40 mg/kg) of TM2-115 had 18-fold reduced parasitemia the following day. Importantly, treatment of P. falciparum parasites in culture with BIX-01294 or TM2-115 resulted in significant reductions in histone H3K4me3 levels in a concentration-dependent and exposure time-dependent manner. Together, these results suggest that BIX-01294 and TM2-115 inhibit malaria parasite histone methyltransferases, resulting in rapid and irreversible parasite death. Our data position histone lysine methyltransferases as a previously unrecognized target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life-cycle stages.chromatin | global health | chemical genetics M alaria continues to claim >1 million lives annually, particularly in the vulnerable populations of pregnant women and children <5 y of age (1, 2). Although artemisinin-based combination therapies have helped rescue the world's antimalarial armamentarium, the parasite's ability to develop resistance continues to outpace our ability to control this devastating disease (3). Effective malaria drug discovery efforts must therefore include both the development of improved antimalarials from existing compounds and the discovery of new parasite drug targets and novel small-molecule inhibitors.Epigenetic control of gene regulation in Plasmodium falciparum, the main causative agent of human malaria, has received considerable attention originally due to the apparent lack of recognizable transcription factors in the parasite genome (4). Although the recent discovery of a family of apicomplexan AP2 transcription factors (5, 6) has partly fulfilled the search for more traditional transcription factors, epigenetic gene regulation, particularly at the level of histone posttranslational modifications, has proven to play a significant role in P. falciparum virulence gene regulation. For example, expression of variant surface antigen gene families (7) and ligands involved in parasite red bl...

Histone Methyltransferase Inhibitors Are Orally Bioavailable, Fast-Acting Molecules with Activity against Different Species Causing Malaria in Humans

“…1A) possess similar antimalarial efficacies against both drug-sensitive and drug-resistant laboratory strains of P. falciparum (8). The strains tested were resistant to longstanding antimalarials for which resistance has developed in the field, namely, chloroquine, mefloquine, and pyrimethamine.…”

“…We previously showed a rapid parasite-killing effect in treatment and washout experiments (8). A more robust analysis of the parasite-killing rate has since been developed, and this improved method was employed to better characterize the rate and extent of parasite killing by BIX-01294 and TM2-115 (3).…”

“…We previously demonstrated that our diaminoquinazoline compounds possess activity against malaria parasites in an animal model of infection after intraperitoneal administration (8). To explore the oral efficacy of BIX-01294 and TM2-115, we performed a 4-day Peters test, which examines the abilities of compounds to reduce or clear blood stage infection (31).…”

“…1A) cause rapid and irreversible parasite-killing activity in vitro throughout the intraerythrocytic asexual cycle (8). Importantly, the same class of molecule shows a "wakeup" effect on dormant liver stage malaria parasites (hypnozoites), suggesting an important role of HKMTs in this yet ill-defined biological liver stage (9).…”

An Epigenetic Antimalarial Resistance Mechanism Involving Parasite Genes Linked to Nutrient Uptake

“…Epigenetic histone modifications regulate cytoadherence, erythrocyte invasion, and developmental progression in malaria parasites (45)(46)(47), but were not known to produce drug resistance. We report this new role for epigenetic marks.…”