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WO1998003661A9 - Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan - Google Patents

Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan

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Publication number
WO1998003661A9
WO1998003661A9 PCT/US1997/012497 US9712497W WO9803661A9 WO 1998003661 A9 WO1998003661 A9 WO 1998003661A9 US 9712497 W US9712497 W US 9712497W WO 9803661 A9 WO9803661 A9 WO 9803661A9
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PCT/US1997/012497
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WO1998003661A3 (fr
WO1998003661A2 (fr
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Priority to EP97937983A priority Critical patent/EP0918868A2/fr
Priority to AU40411/97A priority patent/AU4041197A/en
Publication of WO1998003661A2 publication Critical patent/WO1998003661A2/fr
Publication of WO1998003661A9 publication Critical patent/WO1998003661A9/fr
Publication of WO1998003661A3 publication Critical patent/WO1998003661A3/fr

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  • This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphofructokinase and not generally operative in animals or
  • Components of the pathways include enzymes, transit peptides
  • nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences to which antibodies, antisense molecules and other inhibitors are
  • Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors.
  • Toxoplasmosis is the major opportunistic brain infection in ATDS patients, causes loss
  • the tests available to diagnose Apicomplexan infections include assays which isolate the parasite, or utilize light, phase, or fluorescence microscopy, ELISAs, agglutination of parasites or parasite components to detect antibodies to parasites, or
  • PCR polymerase chain reaction
  • the available assays have limited ability to differentiate whether an infection was acquired remotely or recently, and are limited in their capacity to diagnose infection at the outpatient or field setting.
  • the primary antimicrobial agents used to treat toxoplasmosis are:
  • pyrimethamine is limited by bone marrow toxicity which can be partially corrected by the concomitant administration of folinic acid.
  • T. gondii cannot utilize folinic acid but
  • mammalian cells can. Another problem is that pyrimethamine is potentially teratogenic in the first trimester of pregnancy. The use of sulfonamides is limited by allergy,
  • Atovaquone treatment of toxoplasmosis may be associated with lack of efficacy and/or recrudescent disease. There are no medicines known to eradicate the latent, bradyzoite stage of T. gondii, which is very
  • Medicines used to treat malaria include quinine sulfate, pyrimethamine,
  • Toxicities of mefloquine include nausea, vomiting, diarrhea, dizziness, disturbed sense of balance, toxic psychosis and
  • dihydrochloride also can be hazardous. Parenteral quinine may lead to severe
  • the plastid membrane of T. gondii was reported to be composed of
  • the herbicides which affect growth of Apicomplexans are known to affect plant microtubules or a plant photo synthetic protein.
  • a compound, salicylhydroxamic acid, (SHAM) had been found to inhibit Plasmodium falciparum (malaria) and Babesia microti.
  • toxic molecules are newly discovered strategies employed to interrupt cellular functions and can be utilized to rationally develop novel antimicrobial compounds, but they have not yet been utilized to design medicines effective against Apicomplexans.
  • This invention relates uses of components of plant-like metabolic pathways (not usually associated with animals, not encoded in the plastid genome, and not including
  • Components of the pathways include enzymes,
  • Transit peptides are used to identify other proteins and their organelle targeting
  • the protein which includes the transit peptide is not necessarily an enzyme in
  • Apicomplexan parasites are present in Apicomplexan parasites. These plant-like pathways in Apicomplexan parasites are targetable by inhibitors, as measured by determining whether the inhibitors, either singly or in combination, are effective in inhibiting or killing Apicomplexan parasites in vitro and/or in vivo.
  • the present invention includes new methods and compositions to treat,
  • the invention is based on applications and manipulations of components of algal and
  • enzymes in the pathways are homologous or similar to products, enzymes and
  • Plant-like excludes metabolic pathways generally operative in animals and pathways
  • nucleotide probes that hybridize to specific sequences in plants, c) determining homologies of Apicomplexan nucleotide or protein sequences with plant nucleotide or
  • plant-like pathways of the present invention are identified by a) identification of metabolic pathways characteristic of plants but not generally present in animals,
  • a method for inhibiting an Apicomplexan parasite includes selecting the metabolic pathway of the present invention and interfering with the operation of the pathway in the parasite
  • the Apicomplexan parasite is preferably selected from the
  • Theileria may utilize a component encoded by an Apicomplexan nuclear
  • Suitable metabolic pathways or components include
  • shikimate pathway phosphate by the shikimate pathway
  • auxin growth regulators from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid derived from indoleacetic acid
  • the interfering compositions are selected from the group consisting of enzyme
  • inhibitors including competitors; inhibitors and competitive or toxic analogues of substrates, transition state analogues, and products; antibodies to components of the
  • compositions include gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG. Interfering with the operation of the metabolic pathway is also accomplished by
  • each of the compositions singly interferes with the operation of the metabolic pathway.
  • the plurality of compositions inhibits the parasite to a degree greater than the
  • compositions used singly that is, exhibits a synergistic effect.
  • Embodiments of a plurality of compositions include gabaculine and sulfadiazine
  • NPMG and sulfadiazine SHAM and gabaculine
  • NPMG and pyrimethamine SHAM and gabaculine
  • NPMG and pyrimethamine which inhibits Apicomplexan DHFR [TS])
  • TS Apicomplexan DHFR
  • the interfering composition acts on a latent bradyzoite
  • deleterious effects of an Apicomplexan in an animal include: a) identifying a composition that inhibits growth or survival of an Apicomplexan parasite in vitro by
  • composition in an animal model that is non-toxic and effective in reducing the survival of the parasite in the animal host and/or the deleterious effects of the parasite
  • Developing a lead compound that inhibits an Apicomplexan parasite is accomplished by a) identifying a plant-like metabolic pathway in an Apicomplexan
  • a nuclear gene includes gabaculine; a composition including an enzyme in a
  • a composition comprising SHAM and 8-OH-quinoline
  • metabolic pathway in an Apicomplexan parasite is a) obtaining a strain of E. coli that is
  • pathway in an Apicomplexan parasite is a) contacting the parasite with a gene probe; and b) determining whether the probe has complexed with the parasite from which the
  • a method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite also includes: a) cloning and sequencing the gene; and b)
  • Apicomplexan parasite is a) contacting the parasite or its enzyme with a substrate for
  • the invention also relates to a diagnostic reagent for identifying the presence of
  • an Apicomplexan parasite in a subject where the subject includes a domestic or
  • the reagent may include all or a portion of a component
  • a diagnostic assay hybridizes to a nucleic acid encoding a component of the pathway.
  • a diagnostic reagent for identifying the presence of an Apicomplexan parasite may use the diagnostic reagents defined herein.
  • a diagnostic reagent for identifying the presence of an Apicomplexan parasite may use the diagnostic reagents defined herein.
  • a diagnostic assay for the presence of an Apicomplexan parasite in a biological sample includes: a) contacting the sample with an antibody selective for a product of a
  • the assay is directed towards a nucleotide sequence.
  • appropriate antibody or nucleotide is provided.
  • sequences are selected to distinguish infections by different Apicomplexans.
  • An aspect of the invention is a vaccine for protecting livestock animals
  • the vaccine may be produced for an Apicomplexan
  • the component of the pathway may be replaced
  • vaccine strain can then be cultivated in vitro to make the vaccine.
  • the vaccine may use a component of the pathway that is operative at a particular life stage of the parasite.
  • a suitable component is the AroC gene from T. gondii or P. falciparum.
  • parasite includes the following steps: a) obtaining an inhibitor of a plant-like metabolic
  • FIG. 1A-C illustrates the heme synthesis pathway and the effect of GSAT in
  • FIG. 1A diagrams the heme synthesis pathway.
  • FIGS. IB and 1C show that
  • ALA synthase is also present in T. gondii and constitutes an alternative pathway for
  • FIG. 2A-B shows unique lipid degradation in the glyoxylate cycle in T. gondii.
  • FIG. 2A is a schematic representation of the glyoxylate cycle.
  • FIG. 2B shows
  • FIG. 3A is a schematic representation of a pathway which demonstrates alternative oxidase as an alternative pathway for generation of energy in Apicomplexan
  • FIG. 3B shows that uptake of tritiated uracil by tachyzoites (RH strain) is
  • FIG. 4 A is a schematic representation of the pathway for conversion of shikimate to chorismate in T. gondii..
  • the inhibitor of EPSP synthase is NMPG.
  • FIG. 4B shows uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by
  • NPMG Toxicity of NPMG was assessed by its ability to prevent growth of human
  • HFF foreskin fibroblasts
  • FIG. 4C shows product rescue of NPMG' s inhibitory effect
  • FIG. 5 is a schematic representation of interrelationships of metabolic pathways in Apicomplexan parasites.
  • FIG. 6 shows inhibitory effects of NPMG, gabaculine, SHAM 8-OH-quinoline
  • FIG. 7 shows the effects of gabaculine (20 mM) on growth of tachyzoites/bradyzoites (R5) in human foreskin fibroblasts, over 8 days as determined by uracil uptake Note increased uptake of uracil by the 8 th day
  • FIG. 8 shows the effect of NPMG, pyrimethamine, and pyrimethamine plus
  • FIG. 9 shows nucleotide (SEQ ID NO 1) and deduced amino acid (SEQ ID NO 1)
  • FIG. 10 shows results of CLUSTAL X alignments of the deduced amino acid sequences of the putative T. gondii, chorismate synthase (SEQ ID NO 2) with the corresponding sequences from Synechocystis (SEQ ID NO 38), S. cerevisiae (SEQ ID NO 39), S. lycopersicum (SEQ ID NO 40), N. crassa (SEQ ID NO 41) and H.
  • FIG. 11 shows the transit sequences of Zea mays and T. gondu chorismate
  • the transit sequence in the Wx Zea mays protein begins at amino acid number 1 and ends at amino acid number 72
  • the portion (amino acids 359 to 430) of P. falciparum AroC which corresponds to the
  • T. gondii AroC novel internal sequence of the T. gondii AroC which includes the amino acids homologous to the maize protein, is as follows
  • the first (single) arrow indicates the processing site of Zea
  • FIG. 12 shows P. falciparum chorismate synthase cDNA (SEQ ID NO 3) and deduced amino acid (SEQ ID NO 4) sequences
  • This invention uses components of plant-like interrelated metabolic pathways
  • plastid Components include enzymes, products, targetting
  • peptides nucleotide sequences encoding the enzymes or peptides, and promoters, as - 19 -
  • Medicines, diagnostic reagents and vaccines are based upon interrelated plant- like enzyme cascades involved in the synthesis or metabolism or catabolism of
  • compositions of this invention are:
  • Vaccines live knockout, live mutated, components - genes, proteins,
  • EPSP synthase inhibitor 4 refers to 3-(phosphonooxy)-4-hydroxy-5-[N-(phosphonomethyl-2- oxoethyl)am ⁇ no-1-cyclohexene-1 -carboxylic acid (3ct, 4 ⁇ , 5 ⁇ ), compound with diethyl ethanamide EPSP synthase inhibitor 5 refers to shortened R phosphonate
  • Tg protein of ⁇ 66Kd reactivity of Tg protein of ⁇ 66Kd with 5 antibodies (monoclonal and polyclonal to VooDoo lily and T brucei alternative oxidases) and reduction to monomer similar to VooDoo lily and T brucei alternative oxidases on a reducing gel
  • Identification of Tg cDNA and genomic DNA PCR products using primers based on conserved sequences in other alternative oxidases which are probed and sequenced Tg, FT, Cp inhibited by high concentrations of gabaculine Reactivity of Tg protein of ⁇ 40Kd with 3 antibodies to GSAT (polyclonal ⁇ soybean, barley and synechococcus GSATs and not preimmune sera) Reactivity of Cp protein of ⁇ 40Kd with ⁇ barley GSAT Inhibition of Tg, FT, Cp in vitro by 3NPA, Reactivity of Tg protein with polyclonal antibodies to cotton malate synthase and cotton
  • Enzymes in the heme synthesis [with a default ALA synthase pathway], shikimate pathway, alternative generation of energy and glyoxylate cycle are
  • the present invention includes new methods and compositions to treat, diagnose and prevent human and veterinary disease due to
  • Apicomplexan parasites Apicomplexan infections include those due to Toxoplasma gondii (toxoplasmosis), Plasmodia (malaria), Cryptosporidia (cryptosporidiosis),
  • Eimeria eimeriosis
  • Babesia babesiosis
  • Theileria theileriosis
  • Neospora caninum
  • Apicomplexan parasite Toxoplasma gondii
  • Apicomplexan parasites appear to be phylogenetically related and have organelles and enzymes which are critical for their growth and
  • the assays are based on inhibition of the parasite i.e. restriction of growth, multiplication or survival of the
  • parasite burden which refers to the amount
  • the inhibitors must not be toxic or carcinogenic to the parasites' host and for in vitro assays not be toxic to cells in culture.
  • Enzymes of the newly detected plant-like pathways provide novel, unique and useful targets for antimicrobial therapy. These unique pathways and enzymes are
  • Plant-like pathways detected in Apicomplexan parasites include a) the 5-carbon
  • the shikimate pathway includes the - 31 -
  • ESP enzyme 3-phospho-5-enolpyruvylshikimate
  • Inhibitors of some of these enzymes also provide information about the functioning and targeting of the enzymes.
  • the heme synthesis pathway involves enzymes encoded in the nucleus and
  • pathway include gabaculine (3-amino-2,3-dihydro benzoic acid), 4-amino-5-hexanoic acid, and 4-amino-5-fluropentanoic acid.
  • the glyoxylate cycle reported to be present in plants, fungi, and algae, is also present in T gondii.
  • the cycle uses lipids and converts them to C4 acids through a
  • Inhibitors of these enzymes include 3-nitropropionic acid and itaconic acid.
  • the alternative respiratory pathway present in a range of organisms including
  • T. gondii some bacteria, plants, algae and certain protozoans (trypanosomes), is present in T. gondii, Cryptosporidia parvum, and Plasmodium falciparum (in the latter parasite,
  • Enzymes involved in the synthesis of chorismate including those which convert
  • T. gondii Plasmodium falciparum, Ciyptosporidium parvum, and Eimeria Inhibitors include N-
  • T. gondii chorismate synthase has features in common with other amino acid sequence
  • chorismate synthases and entirely unique features as well
  • the unique features are novel sequences not shared with chorismate synthases from other organisms but with
  • a P. falciparum cDNA sequence encoding chorismate synthase and its deduced amino acid sequence also provide information useful for developing novel antimicrobial
  • genomic sequences provide information about regulation of the gene (e g , unique promoter regions) and such unique regions enable targeting their regulatory
  • RASDGRTTSRHEEEVERG (SEQ ID NO 43) in the T. gondii AroC (chorismate synthase)
  • wx UDP glucose-starch-glycosyl transferase protein
  • the P. falciparum AroC (chorismate synthase) has a corresponding novel internal sequence.
  • synthase is the first enzyme in the branched chain amino acid synthesis pathway, inhibited by sulfonylureas and imidazolinones, as well as the synthesis of other "essential" amino acids, such as histidine, methionine, lysine and threonine.
  • Starch synthesis including starch synthases, the UDP -glucose-starch glycosyl transferase, and
  • glucose starch glycosyl transferase pathway leads to a switch from amylose to amylopectin synthesis and thus the bradyzoite phenotype.
  • metabolic pathways that can be inhibited include: glutamyl-tRNA synthetase; glutamyl- tRNA reductase; prephenate dehydrogenase; aromatic acid aminotransferase (aromatic
  • transaminase transaminase
  • cyclohexadienyl dehydrogenase tryptophan synthase alpha subunit
  • tryptophan synthase beta subunit tryptophan synthase beta subunit
  • indole-3 -glycerol phosphate synthase anthranilate
  • isomerase (indoleglycerol phosphate synthase); anthranilate phosphoribosyltransferase; anthranilate synthase component I; phosphoribosyl
  • anthranilate isomerase anthranilate synthase component II; prephenate dehydratase
  • chorismate synthase (5-enolpyruvylshikimate 3-phosphate phosph-lyase); - 34 -
  • dehydroquinate dehydratase dehydroquinate dehydratase; shikimate dehydrogenase; 3-deoxy-d-arabino- heptuloonate 7 phosphate synthase; chorismate mutase (7-phospho-2-dehydro-3-
  • deoxy-arabino-heptulate aldolase 3-deoxy-d-arabino-heptuloonate 7 phosphate synthase; shikimate 3 -phospho transferase (shikimate kinase); UDP glucose starch
  • glycosyl transferase Q enzymes; acetohydroxy acid synthase; glutamate- 1- semialdehyde 2, 1 -aminotransferase; chorismate lyase; malate synthase; isocitrate lyase;
  • 3-enolpyruvylshikimate phosphate synthase (3-phosphoshikimate-l carboxyvinyltransferase).
  • enzymes in E coli or in other expression systems is useful for producing antibodies and obtaining a crystal structure Native enzyme is isolated. The expressed and native proteins are used to design and test new inhibitors in enzyme assays. Expressed and
  • the crystal structure is useful for characterizations of enzyme active
  • Gabaculine and sulfadiazine are an additive combination in vitro.
  • An aspect of the invention is identifying potential targets for therapeutic
  • An aspect of the invention is a plurality of inhibitors, singly or in combination, directed against enzymes and/or genes encoding a different metabolic pathway.
  • inhibitors suitable for practice of the present invention include GSAT,
  • compositions may inhibit the operation of more than one pathway
  • compositions may inhibit more than one step in a pathway
  • compositions may have synergistic effects, producing more effective drugs.
  • compositions may target pathways operative exclusively during a
  • compositions may inhibit other microorganisms (including other Apicomplexans.)
  • Apicomplexan An additional detail of the invention is that representative Apicomplexan
  • T. gondii a parasite that is used for assaying candidate inhibitors.
  • the invention is
  • Organisms used for the assays include T gondii tachyzoites,
  • bradyzoites and a mutant that expresses 50% tachyzoite and 50% bradyzoite antigens.
  • lyase an enzyme important in utilization of lipids
  • alternative oxidase enzyme - 37 -
  • enolpyruvylshikimate synthase (EPSP synthase), an enzyme important in conversion of shikimate to chorismate which is a precursor for synthesis of folate, ubiquinone, and
  • the inhibitors provide lead compounds for the development of antimicrobial agents.
  • conserved enzyme active sites or parts of the molecules or genes that encode the protein which are targeted by the inhibitors provide the basis for development of new but related ways to target the enzymes, such as
  • Inhibitors are effective against more than one parasite (e.g. T. gondii, P.
  • bacterial and fungal pathogens such as Pneumocystis carinii, Mycobacterium tuberculosism Staphylococcus aureus, and Hemophilus influenza, but not animals.
  • inhibitors of these pathways affect susceptible microorganisms which
  • stage-specific inhibitors are within the scope of the invention.
  • the parasites in which a gene is knocked out are a useful
  • sequences and definition of the active sites of these enzymes, and pathways, and organelle (e.g., plastid) targeting sequences are useful to design related antimicrobial agents.
  • organelle e.g., plastid
  • proteins which interact with the enzyme and interfere with the function of the enzyme's active site or are competitive substrates or products or intracellular antibodies (i.e., with a gene encoding the Fab portion of an antibody that targets the protein the antibody recognizes), or
  • antisense nucleic acid or targeted ribozymes that function as inhibitors are useful, novel
  • Enzymes of the invention are a novel basis for unique diagnostic
  • synergistic i.e., synergistic
  • the meaning of synergism is that compound A has effect A',
  • compound B has effect B', compounds A+ B have an effect greater than A'+ B'.
  • Synergism is characteristic of inhibitors of these pathways because an initial pathway affected by an inhibitor often provides a product used as a substrate for another pathway so the inhibition of the first enzyme is amplified.
  • One or more of the inhibitors preferentially affect one of the life cycle stages of Apicomplexan parasites.
  • Some enzymes are preferentially used by specific stages of the parasites. Detection of an enzyme of this type or a nucleic acid encoding it offers a novel
  • the attenuated parasite is useful as a vaccine because the "knocked out" gene is critical for the parasite to establish latency. Its administration to livestock animals results in
  • genes that encode the protein also are used in DNA constructs to produce proteins themselves or the proteins or peptides are used in immunized animals. These constructs are used to elicit an immune response and are used for vaccines alone or with
  • chorismate synthase in a construct which has a CMV promoter and expresses the protein following intramuscular injection (i.e., a DNA vaccine).
  • a construct which has a CMV promoter and expresses the protein following intramuscular injection (i.e., a DNA vaccine).
  • T. gondii ESTs Woodington University T. gondii gene Sequencing project
  • T. gondii has alternative methods for synthesis of ALA.
  • T. gondii exemplified in T. gondii.
  • product inhibition studies were used in vitro. For example, growth of T. gondii is sensitive to
  • NPMG that inhibits the synthesis of folic acid via the shikimate pathway. Because mammalian hosts lack the entire shikimate pathway, it is unlikely that the parasites can
  • Genes are identified by search of available expressed sequence tags (ESTs, i.e., short,
  • E. coli mutants and yeast deficient in the enzyme are complemented with
  • genes are used as probes to DNA obtained from these organisms and the genes are identified either by cloning and sequencing the DNA recognized by the probe or by
  • Genomic DNA is sequenced
  • Stage specificity of these enzymes is determined in vitro by using antibodies to stage-specific antigens in inhibitor-treated cultures, by Western or Northern analyses (detection), by enzyme assays using selected parasite life cycle
  • New elements include use of longer culture times (e.g., extending the duration of the assay to > 6 days is also a
  • Me49 PTg and R5 strains in vitro, employing synergistic combinations of NPMG and low dosage pyrimethamine in vivo, and assays of parasitemia in vivo using
  • Parasites present as tachyzoites present as tachyzoites (RH, Ptg, a clone derived from the
  • Me49 strain that can be stage switched by culture conditions (Bohne et al, 1993; Soete et al, 1994; Tomovo and Boothroyd, 1995; Weiss et al, 1992) are suitable for
  • Me49, Ptg, and R5 mutant are unique aspects of the methods used in these assays in
  • HFF foreskin fibroblasts
  • thymidine uptake and microscopic evaluation Confluent monolayers of HFF were infected with tachyzoites or bradyzoites. Inhibitor was added one hour later. Non- toxic doses were used in parasite growth inhibition assays. Parasite growth was measured by ability to incorporate tritiated uracil during the last 18 hours of culture.
  • T. gondii tachyzoites were tested to see if they were sensitive in vitro to inhibition by
  • bradyzoites specific inhibitors of target pathways. Then bradyzoites are tested. Positive results for
  • the inhibitors effective in vitro were screened for activity in vivo in mice.
  • An example of an effective combination in vivo is NPMG and low dosage pyrimethamine.
  • isocitrate lyase isocitrate lyase, malate synthase, and alternative oxidase genes. Such genes are used as
  • FIG. 1A compares heme biosynthesis in plants, algae and bacteria with heme
  • ALA is produced in the plastid by
  • the pathway in animals involves the condensation of
  • heme biosynthesis 3-amino 2,3-dihydroxybenzoic acid inhibits GSA in the heme synthesis pathway
  • HFF human foreskin fibroblasts
  • Toxoplasma organisms were grown in human foreskin fibroblasts alone and in
  • T. gondii was measured by the ability of T. gondii to incorporate tritiated uracil This compound was effective at inhibiting the growth of T. gondii at the 20mM
  • FIG. IB demonstrates the ability of gabaculine (a specific inhibitor of GSA aminotransferase) to restrict the growth of T. gondii in an in vitro assay over a 4
  • medium medium (medium) produced a CPM of around 45,000 If no T. gondii were added to
  • FIG. IC demonstrates the ability of gabaculine to inhibit the growth of T gondii over 8 days in culture. T. gondii growth is measured by ability of the
  • CPM counts/minute
  • T. gondii utilizes the 5-carbon ALA synthesis pathway.
  • FIG. 7 demonstrates the ability of gabaculine to inhibit the growth of the
  • T. gondii growth is measured by their ability to
  • CPM counts/minute
  • the X-axis represents days post infection. Parasite growth was evident in the cultures where no drug was added (medium) over the entire time course. Parasite growth was - 50 -
  • T. gondii cyst-like structures are selected by
  • gabaculine for tachyzoite and bradyzoite specific antigens demonstrates that gabaculine selects bradyzoites.
  • Table 2 is a schematic representation of experiments designed to
  • 3-amino-2,3-dihydrobenzoic acid is an inhibitor of the
  • Tachy- BradyIFA result on culture day used for of zoite zoite IFA culture Control Control 0 2 6
  • IFA immunofluorescent assay.
  • SAG1 is surface antigen 1.
  • BSAG is bradyzoite surface antigen 1.
  • BAG5 is bradyzoite antigen 5.
  • ⁇ indicates no specific fluorescence of the organism;
  • ⁇ indicates specific surface fluorescence of the organism due to presence of the antigen recognized by the antibody (e.g., ⁇ SAG1 or ⁇ BSAG);
  • £ indicates specific internal fluorescence in the organism due to presence of the antigen within the parasite recognized by the antibody (e.g., ⁇ BAG ⁇ ).
  • 3-Nitropropionic acid is an inhibitor of isocitrate lyase in the degradation of
  • FIG. 2A illustrates how the
  • glyoxylate cycle manufactures C4 acids.
  • Acetyl CoA a byproduct of lipid breakdown combines with oxaloacetate to form citrate.
  • succinate is formed.
  • Glyoxalate the byproduct of this reaction is combined with a further molecule of acetyl CoA by the action of malate
  • FIG. 2B demonstrates the ability of
  • 3-NPA an inhibitor of isocitrate lyase
  • T. gondii degrades lipids using isocitrate lyase.
  • T. gondii growth is measured by their ability to incorporate
  • tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis.
  • CPM counts/minute
  • 3-NPA inhibits the glyoxylate cycle (isocitrate lyase) and/or succinate dehydrogenase in Apicomplexan parasites. - 53 -
  • FIG. 3A describes the
  • the pathway provides a source of energy and is preferred for conditions with
  • Toxoplasma gondii utilizes the alternative oxidase for respiration.
  • FIG. 3B demonstrates the ability of SHAM (a specific inhibitor of alternative oxidase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period.
  • T. gondii growth is measured by their ability to
  • CPM counts/minute
  • the X-axis describes how the T. gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of around 54,000. If no T. gondii were
  • Salicylhydroxamic acid (SHAM) and 8-hydroxyquinoline are inhibitors of the alternative oxidase and are also effective against T. gondii, presumably by inhibiting the alternative pathway of respiration.
  • Salicylhydroxamic acid and 8-hydroxyquinoline are inhibitors of the alternative oxidase and are also effective against T. gondii, presumably by inhibiting the alternative pathway of respiration.
  • the shikimate pathway is common to plants, fungi and certain other
  • FIG. 4A details the events that result in the production of tetrahydrofolate, aromatic
  • chorismate is formed through the sequential action of a number of enzymes including
  • EPSP-synthase and chorismate synthase EPSP-synthase and chorismate synthase. EPSP-synthase is inhibited by NPMG.
  • Chorismate is further processed to yield tetrahydrofolate or ubiquinone by a further
  • Toxoplasma gondii utilizes the shikimate pathway for synthesis of folic acid, ubiquinone and aromatic amino acids.
  • T. gondii growth is a specific inhibitor of EPSP-synthase
  • the X-axis describes how the T. gondii cultures
  • FIG. 4C the ordinate shows uptake of tritiated uracil into T. gondii nucleic
  • Imidazolinones and sulfonylureas inhibit acetohydroxy acid synthase in
  • Antisense, ribozymes, catalytic antibodies (Pace et al.., 1992; Cate et al., 1996; Charbonnier 1997; Askari et al, 1996) conjugation with toxic compounds allow targeting of parasite molecules using transit sequences.
  • Catalytic antibodies also are designed to destroy the transit sequence. These antisense compounds or ribozymes or toxic molecules targeted to transit sequences
  • FIG. 6 demonstrates the effect of NPMG, gabaculine, SHAM and 8-hydroxyquinoline and 3-NPA on Cryptosporidia in vitro.
  • concentrations used were: SHAM (0.2% ETOH was added) 100, 10, 1, 0.1 ⁇ g/ml; 8-hydroxyquinoline 100, 10, 1, 0.1 ⁇ g/ml; NPMG 4.5, 0.45, 0.045 ⁇ g/ml;
  • paromomycin and ethanol ⁇ 100; SHAM 100 ⁇ g/ml ⁇ 400; SHAM
  • 3-NPA inhibited the glyoxylate cycle (isocitrate lyase) and/or succinate
  • the inhibitor of isocitrate lyase is 3-nitropropionic acid (concentration ranging
  • E. coli strains E. coli strains
  • DV 21A01 aceA
  • the plastid is a likely site for the alternative pathway of respiration
  • Presence of a product of the enzymatic reaction in the pathways of the present invention abrogates the effect of the inhibitor on a specific enzyme because the product no longer has to be made by enzyme catalysis of a substrate.
  • addition of the product proves the specificity of the effect of the inhibitor on the enzyme.
  • the product rescue assays are performed with PABA.
  • the mutants for complementation are as follows: E. coli, AroA; E. coli, AroC;
  • chorismate synthase was obtained following restriction digestion and primer-based
  • gondii sequence has substantial homology with tomato and several other chorismate - 62 -
  • Inhibitors 4 and 5 are Inhibitors 4 and 5, sulfosate (Marzabadi et al., 1996). Other inhibitors of enzymes in this pathway also have been developed by others and provide a paradigm for the
  • Acetohydroxy acid synthase is an enzyme present in plants but not animals and is inhibited by imindazolinones and sulfonylureas in Apicomplexan parasites. Inhibitors of histidine synthesis restrict growth of Apicomplexan parasites.
  • the plant-like acetyl coA decarboxylase is inhibited by a number of inhibitors
  • auxin mimics and Giberellin synthesis and Giberellin inhibitors inhibit Apicomplexan parasites' growth. - 63 -
  • Glufosinate inhibits Apicomplexan glutamine/glutamate synthesis because the
  • Transit Sequence The transit sequence is conjugated with toxic molecules such as ricins and used
  • ribozymes or the use of catalytic antibodies prevent translation of DNA to protein or catalyze the destruction of mature protein. This interferes with functioning of the molecule and thus the parasite's growth and survival.
  • Example 5 The Combined Effects of Inhibitors of Apicomplexan Parasites The effect of enzymes in pathways "in parallel” are additive and in “series” are more
  • the shikimate pathway produces 3,4-dihydroxybenzoate which is converted to ubiquinone, an essential component of the electron transport
  • NPMG an inhibitor of EPSP-synthase
  • NPMG and sulphadiazine (a competitive PABA analogue) which act at different points of the folate
  • sulphadizine (a competitive PABA analogue) which act on different pathways, are - 64 -
  • gabaculine and SHAM are a predicted synergistic
  • Table 4 demonstrates the additive inhibitory effect of sulphadiazine and gabaculine on the growth of T. gondii over 4 days in culture.
  • T. gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM). Cultures that were grown in medium (medium) produced a CPM of about 36,000. If no T. gondii were added to the cultures (no RH),
  • NPMG and sulfadiazine combine in a synergistic manner.
  • Candidate inhibitors are administered to animals by daily intraperitoneal
  • NPMG is administered at a dose of lOOmg/kg/day.
  • mice intraperitoneally to BALB/c mice. Cumulative mortality is followed in groups of mice given inhibitor compared to untreated controls.
  • Cyst numbers present in a suspension of brain are enumerated, or cyst numbers in formalin
  • T. gondii (Me49 strain). Inhibitors are administered to groups of mice from day 30
  • Cyst burden, mortality and pathology are compared in treated and control mice on days 30 and 50 post infection and in mice that
  • T. gondii B 1 gene is amplified by PCR in the presence of a construct which produces a product slightly smaller than the wild type Bl gene.
  • wild type gene For example, presence of a greater amount of the wild type gene will result in lesser use of the competitor.
  • NPMG is an
  • SHAM affects parasitemia and number of
  • FIG. 8 demonstrates the ability of NPMG and pyrimethamine administered in combination to protect mice from an otherwise lethal challenge with the virulent RH strain of T. gondii.
  • Mice were infected intraperitoneally with 500 tachyzoites and left untreated (control) or treated by the addition of pyrimethamine (PYR), NPMG (NPMG) or both pyrimethamine and NPMG (PYR/NPMG) to their drinking water.
  • PYR pyrimethamine
  • NPMG NPMG
  • PYR/NPMG both pyrimethamine and NPMG
  • Percent survival is marked on the Y-axis and days post infection on the X-axis.
  • T. gondii was studied by electron microscopy and was found to have a plastid.
  • Bradyzoites are isolated as described herein in the
  • tachyzoites are pelleted by centrifugation and the pellet is fixed in 2.5% glutaraldehyde. Cysts and bradyzoites are purified from the brains of
  • Immunoelectronmicroscopy is as described by Sibley and Krahenbuhl (1988) using gold particles of different sizes with antibodies to the enzymes to identify the enzyme localization in different organelles which are identified morphologically.
  • tachyzoites and bradyzoites are compared using standard inhibition experiments in
  • Tachyzoites use conventional and
  • Bradyzoites and tachyzoites also are compared directly for the relative amounts of alternative oxidase, using northern blot analyses, enzyme assays of parasites,
  • GSAT lyase
  • chorismate synthase genes isocitrate lyase genes, and malate synthase genes are
  • soybean gsa identified by probing, and then sequenced.
  • the cDNA clone of soybean gsa is labeled for chemiluminescent detection (ECL) or 32 P detection to identify
  • T. gondii homologous gsa sequences in T. gondii. Probes are used on a membrane containing the genomic DNA of T. gondii and soybean (positive control). When T. gondii genes are isolated, they are used to probe other Apicomplexan DNA. Thus, the gsa genes of T. gondii.
  • T gondii alternative oxidase is identified by screening T. gondii cDNA expression libraries using the 7D3
  • This gene is used to detect the
  • Probes for gsa (soybean) alternative oxidase (soybean and tobacco), isocitrate lyase (cotton), UDP glucose starch glycosyl transferase (sweet corn), and acetohydroxy acid synthase (sweet corn) also are used to screen for clone, and
  • homologous genes encoding plant enzymes are used to compare with these sequences to determine whether they are identified in the libraries and if so to determine whether
  • the enzymes are encoded in the nucleus or plastid.
  • probes are generated to
  • the titer of the amplified library is 1-2 X 10 10 /ml.
  • the phagemids were excised with R408 or VCS-M13 helper phage and
  • bradyzoite, and Me49 tachyzoite libraries also are suitable, as are other tachyzoite and bradyzoite libraries prepared by Stratagene.
  • cDNA expression libraries are probed with DNA from the genomes of: a) Toxoplasma gondii;
  • Genomic DNA is examined by Southern blot analysis for the pathways.
  • Genomic DNA is extracted from Apicomplexan parasites by
  • DNA(5-10 ⁇ g) is digested with
  • restriction enzymes electrophoresed through 1% Agarose and transferred to a nylon
  • the ECL (Amersham) random prime system is used for labeling of DNA - 76 -
  • Hybridization with the 32 P-labeled probe is carried out in [1M NaCl, 20 mM NaH 2 PO4 pH 7.0, 1% SDS, 40% formamide, 10% dextran sulfate, 5 mg/ml dry milk, 100 ⁇ g/ml salmon
  • Probes are prepared from T. gondii cDNA clones obtained and characterized as described in Example 9. If lack of overall sequence conservation limits
  • polymerase chain reaction technology is used to amplify homologous sequences from a T. gondii cDNA library or T. gondii genomic
  • Neurospora crassa alternative oxidase gene has been isolated using degenerate primers

Abstract

La présente invention concerne l'utilisation de composants d'une voie métabolique de type végétal qui ne comportent pas de psbA ou PPi phosphofructokinase et généralement inopérants chez les animaux, ou qui sont codés par l'ADN plastidial, de façon à faire obstacle à la croissance et la survie de l'Apicomplexan. Les composants de ces voies sont notamment des enzymes, des peptides de transit et des séquences nucléotides codant pour les enzymes et peptides, voire des précurseurs de ces séquences nucléotides, et contre lesquels sont dirigés des anticorps, des molécules antisens et d'autres inhibiteurs.
PCT/US1997/012497 1996-07-19 1997-07-18 Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan WO1998003661A2 (fr)

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US6911331B2 (en) 1998-07-21 2005-06-28 E. I. Du Pont De Nemours And Company Chorismate biosynthesis enzymes
US6653531B1 (en) 1998-07-21 2003-11-25 E. I. Du Pont De Nemours And Company Chorismate synthase from plants
EP1098984A2 (fr) * 1998-07-21 2001-05-16 E.I. Du Pont De Nemours And Company Enzymes impliquees dans la biosynthese du chorismate
AU767117B2 (en) * 1999-05-04 2003-10-30 David Ferguson Anti-microbial agents, diagnostic reagents, and vaccines based on unique apicomplexan parasite components
DE102006024757A1 (de) * 2006-05-27 2007-12-06 Aesculap Ag & Co. Kg Chirurgischer Obturator
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US5068253A (en) * 1990-07-11 1991-11-26 The United States Of America As Represented By The Department Of Health And Human Services Treatment of a microbial infection with drugs containing para-acetamidobenzoic acid
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US5459063A (en) * 1993-10-14 1995-10-17 The University Of Pennsylvania Plasmodium falciparum ribonucleotide reductase DNA
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