Tartrolon E, a secondary metabolite of a marine symbiotic bacterium, is a potent inhibitor of asexual and sexual Plasmodium falciparum

ABSTRACT Due to the spread of resistance to front-line artemisinin derivatives worldwide, there is a need for new antimalarials. Tartrolon E (TrtE), a secondary metabolite of a symbiotic bacterium of marine bivalve mollusks, is a promising antimalarial because it inhibits the growth of sexual and asexual blood stages of Plasmodium falciparum at sub-nanomolar levels. The potency of TrtE warrants further investigation into its mechanism of action, cytotoxicity, and ease with which parasites may evolve resistance to it.

artemisinin have intensified the need for discovery of novel inhibitors that target unique pathways (2,3).
Natural products make up more than half of the FDA-approved drugs over the last 40 years and have long been an important source of and inspiration for antimicrobials due to their structural diversity (4,5).Some of the most efficacious and widely used antibiotics are polyketides, a large, diverse class of natural products that include the tetracyclines (6) and the macrolides azithromycin and erythromycin (7), which all possess antimalarial properties.Two of the most successful antimalarials in history are the natural products quinine and artemisinin, which have complex mechanisms of action and require multifactorial processes for the acquisition of resistance (8).Looking forward, the ocean is an underexplored frontier for natural product discovery, and marine organisms have been shown to produce a wide variety of unique chemical scaffolds with biomedi cal potential (9).
Tartrolon E (TrtE) (Fig. 1) is a secondary metabolite macrolide polyketide with a central complexed tetraborate.It was isolated from Teredinibacter turnerae, an intracellu lar endosymbiotic gammaproteobacteria of marine wood-boring bivalve mollusks of the family Teredinidae (shipworms) (10).Recently, we showed the potent inhibition of a diverse range of apicomplexan parasites including Plasmodium, Toxoplasma, and Cryptosporidium by TrtE (11).This was consistent with the hypothesis that T. turnerae may produce TrtE to protect mollusks against gregarines, the most ancestral organism of the apicomplexan phylum (12).Here, we describe the bioactivity of TrtE on asexual and sexual blood stages of Plasmodium falciparum.
Historically, the best antimalarials are highly potent, tolerable, and efficacious across multiple steps of the parasite life cycle (13).In parasite proliferation assays (14), TrtE exhibited sub-nanomolar potency against the asexual blood stages of P. falciparum (Pf) (3D7 EC 50 = 105 pm), responsible for the clinical manifestations of malaria disease.In vitro inhibition of parasite growth by TrtE was approximately equivalent to that of dihydroartemisinin (DHA; 3D7 EC 50 = 129 pm), the active metabolite of the front-line artemisinin (Fig. 2).
A diverse panel of P. falciparum clones, collected from four continents with vary ing genetic backgrounds and well-characterized susceptibilities to novel and existing antimalarials, was treated with TrtE and DHA in standard growth inhibition assays.All clones were inhibited by TrtE at sub-nanomolar concentrations, including two clones from Western Cambodia that were collected in a province with clinical artemisinin resistance and that display in vitro resistance to artemisinin in ring-stage survival assays (RSAs) (BEI, Didier Menard) (Table 1).The potency of TrtE is comparable to DHA across all parasite lines tested, with both compounds displaying slightly lower proliferation inhibition to the Cambodian cell lines.Based on these data, the preliminary indications are that the mechanism of action utilized by TrtE is orthogonal to those of all commonly used antimalarials except DHA.Additional studies are needed to determine whether there is any similarity in mechanism between TrtE and DHA.Gametocytes of Pf NF54, the competent gametocyte-producing clone, were treated with TrtE as previously described (15,16).In three independent experiments, TrtE inhibited the progression of Pf NF54 late-stage gametocytes (stage III to stage V) with an average EC 50 of 140 nM, which is superior to the gametocytocidal antimalarials pyronaridine (4.26 µM) and primaquine (>40 µM) (17) (Fig. 3).To validate the in vitro gametocytocidal activity of TrtE, mosquito infectivity assays were conducted.In four independent experiments, treatment with 100 nM TrtE, the approximate gametocidal EC 50 , significantly inhibited mosquito infection by P. falciparum (P = 0.016) (Fig. 4).Oocyst development in mosquitoes represents a significant bottleneck in the life cycle of Plasmodium spp.where transmission can be efficiently interrupted (18).In preliminary experiments to evaluate cytotoxicity to mammalian cells, the murine leukemia L1210 cell line and the human liver cell line HepG2 were grown under identical conditions alongside Pf 3D7.These were treated with TrtE, the chemotherapeutic methotrexate, and DSM265, an inhibitor of parasite dihydroorotate dehydrogenase (19).In vitro cytotoxicity assays demonstrated that TrtE inhibits L1210 growth at sub-micro molar concentrations, though the compound was 2,000-fold more toxic to parasites (Table 2).HepG2 was approximately threefold more sensitive to TrtE compared to L1210 cells (HepG2 EC 50 0.07 nM vs L1210 EC 50 0.23 nM).Previous toxicity selectivity indices (1302-2633) were comparable (11).A wide range of cellular effects of TrtE is consis tent with other macrolides.In the future, selectivity may be improved with chemical modification.
TrtE may be considered for further development because of its high potency across multiple stages of the malaria parasite life cycle.Preliminary in vitro single-step selection experiments indicate that pressure from TrtE presents high hurdles to resistance for asexual parasites (data not shown).TrtE is a complex molecule that requires significant effort to extract and would be challenging to synthesize; however, further investigation may disclose a minimum scaffold responsible for the activity.In addition to further defining windows of parasite-host cytotoxicity, high priorities include the characteriza tion of the kinetics of TrtE-mediated killing and identification of the cellular target(s) of TrtE using forward genetic approaches.

FIG 4
FIG 4 TrtE inhibition of oocyst development in mosquitoes.Late-stage Pf NF54 gametocytes were incubated with 100 nM TrtE, the approximate gametocidal EC 50 , or left untreated.Following incubation and development to stage V, the cultures were fed to female Anopheles dirus mosquitoes using standard membrane feeding techniques.After 9 days, the mosquitoes were dissected, and oocysts were stained and counted.The infection rate was measured as the ratio of mosquitoes with one or more oocysts to the number of dissected mosquitoes.* P < 0.05.

TABLE 2
Cellular sensitivity of TrtE a The selectivity index (EC50 of HepG2 or L1210/EC50 3D7) of TrtE was compared to the antimalarial DSM265 (phase IIa clinical trial) and the FDA-approved anti-arthritic, anti-cancer drug methotrexate. a