The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection.

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis. IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.

Selenium-containing analogues of WC-9 are extremely potent inhibitors of Trypanosoma cruzi proliferation.

The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis, which is the most prevalent parasitic disease in the Americas. The present chemotherapy to control this illness is still deficient particularly in the chronic stage of the disease. The ergosterol biosynthesis pathway has received much attention as a molecular target for the development of new drugs for Chagas disease. Especially, inhibitors of the enzymatic activity of squalene synthase were shown to be effective compounds on T. cruzi proliferation in in vitro assays. In the present study we designed, synthesized and evaluated the effect of a number of isosteric analogues of WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, on T. cruzi growth in tissue culture cells. The selenium-containing derivatives turned out to be extremely potent inhibitors of T. cruzi growth. Certainly, 3-phenoxyphenoxyethyl, 4-phenoxyphenoxyethyl, 4-(3-fluorophenoxy)phenoxyethyl, 3-(3-fluorophenoxy)phenoxyethyl selenocyanates and (±)-5-phenoxy-2-(selenocyanatomethyl)-2,3-dihydrobenzofuran arose as relevant members of this family of compounds, which exhibited effective ED50 values of 0.084 µM, 0.11 µM, 0.083, µM, 0.085, and 0.075 µM, respectively. The results indicate that compounds bearing the selenocyanate moiety are at least two orders of magnitude more potent than the corresponding skeleton counterpart bearing the thiocyanate group. Surprisingly, these compounds exhibited excellent selectively index values ranging from 900 to 1800 making these molecules promising candidates as antiparasitic agents.

Synergistic Activity between Statins and Bisphosphonates against Acute Experimental Toxoplasmosis.

Bisphosphonates are widely used for the treatment of bone disorders. These drugs also inhibit the growth of a variety of protozoan parasites, such as Toxoplasma gondii, the etiologic agent of toxoplasmosis. The target of the most potent bisphosphonates is the isoprenoid biosynthesis pathway enzyme farnesyl diphosphate synthase (FPPS). Based on our previous work on the inhibitory effect of sulfur-containing linear bisphosphonates against T. gondii, we investigated the potential synergistic interaction between one of these derivatives, 1-[(n-heptylthio)ethyl]-1,1-bisphosphonate (C7S), and statins, which are potent inhibitors of the host 3-hydroxy-3-methyl glutaryl-coenzyme A reductase (3-HMG-CoA reductase). C7S showed high activity against the T. gondii bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS), which catalyzes the formation of FPP and GGPP (50% inhibitory concentration [IC50] = 31 ± 0.01 nM [mean ± standard deviation]), and modest effect against the human FPPS (IC50 = 1.3 ± 0.5 µM). We tested combinations of C7S with statins against the in vitro replication of T. gondii We also treated mice infected with a lethal dose of T. gondii with similar combinations. We found strong synergistic activities when using low doses of C7S, which were stronger in vivo than when tested in vitro We also investigated the synergism of several commercially available bisphosphonates with statins both in vitro and in vivo Our results provide evidence that it is possible to develop drug combinations that act synergistically by inhibiting host and parasite enzymes in vitro and in vivo.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii.

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite's lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.

Acidocalcisomes.

Acidocalcisomes are acidic organelles containing calcium and a high concentration of phosphorus in the form of pyrophosphate (PP(i)) and polyphosphate (poly P). Organelles with these characteristics have been found from bacteria to human cells implying an early appearance and persistence over evolutionary time or their appearance by convergent evolution. Acidification of the organelles is driven by the presence of vacuolar proton pumps, one of which, the vacuolar proton pyrophosphatase, is absent in animals, where it is substituted by a vacuolar proton ATPase. A number of other pumps, antiporters, and channels have been described in acidocalcisomes of different species and are responsible for their internal content. Enzymes involved in the synthesis and degradation of PP(i) and poly P are present within the organelle. Acidocalcisomes function as storage sites for cations and phosphorus, and participate in PP(i) and poly P metabolism, calcium homeostasis, maintenance of intracellular pH, and osmoregulation. Experiments in which the acidocalcisome Ca(2+)-ATPase of different parasites were downregulated or eliminated, or acidocalcisome Ca(2+) was depleted revealed the importance of this store in Ca(2+) signaling needed for host invasion and virulence. Acidocalcisomes interact with other organelles in a number of organisms suggesting their association with the endosomal/lysosomal pathway, and are considered part of the lysosome-related group of organelles.

The acidocalcisome as a target for chemotherapeutic agents in protozoan parasites.

Acidocalcisomes are acidic organelles rich in calcium and phosphorus that have been conserved from bacteria to man. In parasitic protozoa acidocalcisomes possess enzymes that are absent or different from their mammalian counterparts and could be potential targets for chemotherapy, such as the vacuolar proton translocating pyrophosphatase, and the soluble inorganic pyrophosphatase, both of which are inhibited by pyrophosphate analogs (bisphosphonates). In addition, a number of drugs, including bisphosphonates, and diamidines appear to accumulate in these organelles and/or induce an increase in their numbers. The mechanism of action of bisphosphonates, however, is by inhibition of the isoprenoid pathway and more specifically the prenyl diphosphate synthases.

Acidocalcisomes in Apicomplexan parasites.

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.

Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes.

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.