EP0918868A2 - 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
EP0918868A2
EP0918868A2 EP97937983A EP97937983A EP0918868A2 EP 0918868 A2 EP0918868 A2 EP 0918868A2 EP 97937983 A EP97937983 A EP 97937983A EP 97937983 A EP97937983 A EP 97937983A EP 0918868 A2 EP0918868 A2 EP 0918868A2
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EP
European Patent Office
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gaa
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German (de)
English (en)
Inventor
Rima L. W. Mcleod
Craig W. Roberts
Fiona Roberts
Jennifer J. Johnson
Laurens Mets
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MRJ TRUST
Arch Development Corp
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MRJ Trust
Arch Development Corp
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Application filed by MRJ Trust, Arch Development Corp filed Critical MRJ Trust
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphofructokinase and not generally operative in animals or encoded by the plastid DNA, to develop compositions that interfere with Apicomplexan
  • 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 directed. Diagnostic and therapeutic reagents and vaccines are developed based on the
  • Toxoplasmosis is the major opportunistic brain infection in AIDS patients, causes loss
  • Cryptosporidiosis is
  • the tests available to diagnose Apicomplexan infections include assays which isolate the parasite, or utilize light, phase, or fluorescence microscopy, ELISAs,
  • 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. gondu cannot utilize folinic acid but
  • Clostridium difficile toxin associated colitis with clindamycin use Clostridium difficile toxin associated colitis with clindamycin use). Lesser or uncertain efficacy of macrolides such as spiramycin, azithromycin, and clarithromycin also limits use of these antimicrobial agents. Atovaquone treatment of toxoplasmosis
  • Medicines used to treat malaria include quinine sulfate, pyrimethamine,
  • Toxicities of mefloquine include nausea, vomiting, diarrhea, dizziness, disturbed sense of balance, toxic psychosis and
  • Halofantrene has caused first degree heart block. It cannot be used for patients with cardiac conduction defects. Quinidine gluconate or dihydrochloride also can be hazardous. Parenteral quinine may lead to severe
  • T. gondu plastid DNA sequences were reported to have homologies to algal plastid DNA sequences
  • the plastid membrane of T. gondu was reported to be composed of
  • the herbicides which affect growth of Apicomplexans are known to affect plant microtubules or a plant photosynthetic protein.
  • a compound, salicylhydroxamic acid, (SHAM) had been found to inhibit Plasmodium falciparum
  • This invention relates uses of components of plant-like metabolic pathways (not
  • compositions that interfere with Apicomplexan growth and survival include enzymes,
  • Transit peptides are used to identify other proteins and their organelle targeting sequences that enter and exit from unique Apicomplexan organelles.
  • the identified components are potential for production of medicines, reagents and assays, and
  • 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
  • the present invention includes new methods and compositions to treat,
  • plant-like excludes metabolic pathways generally operative in animals and pathways involving psbA or phosphofructokinase and those encoded by the plastid genome
  • pathway The limits of a "pathway" are defined as they are generally known to those of skill in the art
  • Methods to detect plant counterparts in Apicomplexan include, a) immunoassays using antibodies directed to products and enzymes known in plants, b) hybridization assays
  • the Apicomplexan parasite is preferably selected from the
  • the pathway may utilize a component encoded by an Apicomplexan nuclear
  • Suitable metabolic pathways or components include a) synthesis of heme from glutamate and tRNA glu by the plant-like, heme synthesis (5 carbon) pathway (hereinafter the "heme synthesis pathway"), b) synthesis of C4 acids (succinate) by the breakdown of lipids into fatty acids
  • glyoxylate cycle acetyl CoA, and their use in the glyoxylate cycle
  • shikimate pathway phosphate by the shikimate pathway
  • histidine, threonine, lysine and methionine by the use of plant-like amino acid synthases;
  • auxin growth regulators from indoleacetic acid derived from
  • the interfering compositions are selected from the group consisting of enzyme inhibitors including competitors, inhibitors and competitive or toxic analogues of
  • compositions include gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG
  • Interfering with the operation of the metabolic pathway is also accomplished by introducing a plurality of compositions to the pathway, wherein each of the compositions singly interferes with the operation of the metabolic pathway.
  • compositions used singly that is, exhibits a synergistic effect
  • Embodiments of a plurality of compositions include gabaculine and sulfadiazine
  • the interfering composition acts on a latent bradyzoite
  • a method of determining the effectiveness of a composition in reducing the 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
  • a composition which inhibits a specific life cycle stage of an Apicomplexan parasite by interfering with a plant-like metabolic pathway that utilizes a component encoded by a nuclear gene includes gabaculine; a composition including an enzyme in a
  • cycle stage of the parasite includes the enzymes alternative oxidase, and UDP glucose starch glycosyl transferase.
  • a composition comprising SHAM and 8-OH-quinoline
  • a method to identify a plant-like gene encoding a component of a plant-like metabolic pathway in an Apicomplexan parasite is a) obtaining a strain of E. coli that is deficient for a component of the metabolic pathway, said deficiency causing the strain
  • Another method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite with a gene probe,
  • 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 of the plant-like pathway, an antibody specific for an enzyme that is a component of a plant-like metabolic pathway in the parasite, or all or part of a nucleotide sequence that hybridizes to a nucleic acid encoding a component of the pathway
  • a diagnostic assay that identifies the presence of an Apicomplexan parasite or specific life-cycle stage of
  • the parasite may use the diagnostic reagents defined herein
  • a diagnostic reagent for identifying the presence of an Apicomplexan parasite includes an antibody specific for an enzyme that is part of a plant-like metabolic
  • the assay is directed towards a nucleotide sequence
  • appropriate antibody or nucleotide 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 parasite is manipulated, for example, deleted or modified
  • the gene is
  • the component of the pathway may be replaced by the presence of the product of an enzymatic reaction in tissue culture medium
  • vaccine strain can then be cultivated in vitro to make the vaccine A vaccine for protecting animals against infection by an Apicomplexan parasite
  • the vaccine may use a component of the pathway that is operative at a
  • a suitable component is the AroC gene from T. gondu 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 T. gondu
  • FIG. 1A diagrams the heme synthesis pathway
  • FIGS. IB and 1C show that
  • FIG. 2A-B shows unique lipid degradation in the glyoxylate cycle m T. gondu
  • FIG. 2 A 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. 4A is a schematic representation of the pathway for conversion of shikimate to chorismate in T. gondu.
  • 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 foreskin f ⁇ broblasts (HFF) after 4 days, as measured by tritiated thymidine uptake and
  • FIG. 4C shows product rescue of NPMG' s inhibitory effect
  • FIG. 5 is a schematic representation of interrelationships of metabolic pathways
  • FIG. 6 shows inhibitory effects ofNPMG, gabaculine, SHAM 8-OH-quinoline
  • FIG. 9 shows nucleotide and deduced amino acid sequences of T. gondii
  • chorismate synthase cDNA The asterisk indicates the stop codon.
  • chorismate synthase sequence which is homologous are aligned.
  • the amino acid sequence is given in one letter code * indicates an identical amino acid in the Wx Zea
  • transferase begins at amino acid number 1 and ends at amino acid number 72.
  • P. falc ⁇ arum AroC which corresponds to the novel internal sequence of the T. gondii AroC which includes the amino acids homologous to the maize protein, is as follows
  • plastid Components include enzymes, products, targetting
  • peptides nucleotide sequences encoding the enzymes or peptides, and promoters, as targets for specific inhibitors.
  • Use of these pathways provide a rational and novel framework to discover, characterize and develop medicines, diagnostic reagents and vaccines for Apicomplexan parasites.
  • compositions of this invention are tested singly or in a plurality, against Apicomplexan parasites alone or together, and in conjoint Apicomplexan, bacterial and fungal infections,
  • the general compositions of this invention are A. Inhibitory compounds based on
  • nucleic acids encoding proteins e.g., antisense, ribozymes
  • Vaccines live knockout, live mutated, components - genes, proteins,
  • HEME SYNTHESIS H ⁇ mL glutamate-1 -semialdehyde 3-am ⁇ no-2,3-d ⁇ hydrobenzo ⁇ c S AS R aminotransferase (GSAT) acid (Gabaculine) 4-am ⁇ no- 5-hexyno ⁇ c acid 4-am ⁇ no-5- fluoropentanoic acid 4- am ⁇ no-5-hexyno ⁇ c acid ( ⁇ acetylenic GABA), 2-am ⁇ no- 3-butano ⁇ c acid (vinyl glycine), 2-am ⁇ no-4- methoxy-trans-3-butano ⁇ c, -am ⁇ no-5-fluoropentano ⁇ c
  • AroB dehydroquinate synthase (5- dehydroquinate dyhdrolase)
  • AroC chorismate synthase 5- enolpyruvylshikimate 3- phosphate phospholyase
  • AroG chonsmate mutase (7-phospho- 2-dehydro-3-deoxy-arab ⁇ no- heptulate aldolase)
  • EPSP synthase inhibitor 4 refers to 3-(phosphonooxy)-4-hydroxy-5-[N-(phosphonomethyl-2- oxoethyl)am ⁇ no-1-cyclohexene-1-carboxyl ⁇ c acid (3 ⁇ , 4 ⁇ , 5 ⁇ ), compound with diethyl ethanamide EPSP synthase inhibitor 5 refers to shortened R phosphonate
  • the present invention includes new methods and
  • Apicomplexan parasite Toxoplasma gondu
  • Apicomplexan parasites appear to be phylogenetically related and have organelles and enzymes which are critical for their growth and
  • parasite burden refers to the amount (number) of parasites present as measured in vivo in tissues of an infected host
  • 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 within the plastid, glyoxosomes, cytoplasm or mitochondria. In addition, not suggested
  • 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, two clones designated W2 and D6 were inhibited).
  • the pathway is inhibited by a range of compounds including salicylhydroxamic acid, 8-hydroxyquinoline, Benzyhydroxamic
  • Cryptosporidium parvum, and Eimeria Inhibitors include N-(phosphonomethyl)
  • T. gondu chorismate synthase has features in common with other chorismate synthases
  • the unique features are novel sequences not shared with chorismate synthases from other organisms but with homology to an amyloplast/chloroplast transit sequence of Zea mays (sweet corn) A P. falciparum
  • cDNA sequence encoding chorismate synthase and its deduced amino acid sequence also provide information useful for developing novel antimicrobial agents
  • genomic sequences provide information about regulation of the gene (e g , unique promoter regions) and such unique regions enable targeting their regulatory elements with antisense
  • RASDGRTTSRHEEEVERG in the T. gondii AroC (chorismate synthase) gene has homology
  • glucose-starch-glycosyl transferase protein i e , MAAIATSQLVATRAGLGVPDASTFRRG
  • AroC chorismate synthase
  • branched chain amino acids valine, leucine and isoleucine
  • acetohydroxy acid 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 debranching enzymes and enzymes of lipid, terpene, giberellin and auxin synthesis, are
  • glucose starch glycosyl transferase pathway leads to a switch from amylose to amylopectin synthesis and thus the bradyzoite phenotype.
  • enzymes in parasite metabolic pathways that can be inhibited include: glutamyl-tRNA synthetase; glutamyl-
  • tRNA reductase prephenate dehydrogenase; aromatic acid aminotransferase (aromatic transaminase); cyclohexadienyl dehydrogenase; tryptophan synthase alpha 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); dehydroquinate dehydratase, shikimate dehydrogenase, 3-deoxy-d-arabino-
  • 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
  • inhibitors either alone or in combination and controls include the buffer for the enzyme alone
  • crystal structure is useful for characterizations of enzyme active
  • An aspect of the invention is identifying potential targets for therapeutic intervention by considering nuclear as well as organellar genes as part of the production of enzymes for unique plant-like pathways. For example, the protein synthesis of plant ⁇
  • Apicomplexan parasites whether or not the pathways involve the plastid. Consequently, Apicomplexan parasites are sensitive to inhibitors that block several of these unique pathways. Combined attack on multiple targets retards the emergence/selection of resistant organisms. Considering nuclear and organellar genes
  • An aspect of the invention is a plurality of inhibitors, singly or in combination,
  • inhibitors suitable for practice of the present invention include GSAT,
  • inhibitors of EPSP synthase or chorismate synthase which include competitive substrate analogues, transitional state inhibitors and direct active site inhibitors as well as other known compounds (Table I). Some pluralities of inhibitors produce synergistic effects.
  • compositions may inhibit the operation of more than one pathway, thereby producing a strong effect and lessening the probability of resistance to the drug emerging because more than one mutation may be required;
  • 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 life cycle of the parasite, making them more selective e.g. against the latent phase.
  • compositions may inhibit other microorganisms (including other Apicomplexans.)
  • Apicomplexan An additional detail of the invention is that representative Apicomplexan
  • T. gondii are used for assaying candidate inhibitors.
  • the invention is directed at effects of inhibitors of the unique plant-like pathways in Apicomplexan,
  • Organisms used for the assays include T. gondii tachyzoites,
  • bradyzoites and a mutant that expresses 50% tachyzoite and 50% bradyzoite antigens.
  • ESP synthase enolpyruvylshikimate synthase
  • shikimate to chorismate which is a precursor for synthesis of folate, ubiquinone, and certain amino acids essential for survival
  • the invention provides a rational, conceptual basis for development of novel
  • 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
  • Inhibitors are effective against more than one parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in these pathways also are present in other parasite (e.g. T. gondu, P. falciparum and C. parvum) and enzymes in
  • 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 concurrently infect a host Because enzymes are utilized differentially in different tissues.
  • stage-specific inhibitors are within the scope of the invention.
  • Genes encoding the enzymes in Apicomplexans are identifiable The genes encoding
  • the parasites in which a gene is knocked out are a useful basis for an attenuated vaccine
  • the pathways and enzymes of the invention are useful to design related antimicrobial agents
  • 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
  • Enzymes of the invention are a novel basis for unique diagnostic
  • 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 These pathways or their
  • 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
  • Mutants with the gene "knocked out” also can be selected because when the parasites are grown in vitro they are grown
  • 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.
  • adjuvants include incorporation of the gene for alternative oxidase or chorismate synthase in a construct which has a CMV promoter and expresses the protein following intramuscular injection (i.e., a DNA vaccine). This type of construct,
  • T. gondii ESTs Woodington University T. gondii gene Sequencing project
  • T. gondii has alternative methods for synthesis of ALA.
  • E. coli mutants and yeast deficient in the enzyme are complemented with plasmid DNA from T. gondii cDNA expression libraries or the isolated gene or a
  • 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 using the probe to screen the relevant parasite libraries Genomic DNA is sequenced
  • Apicomplexan transit peptides are identified by their homology to known transit peptides in other species Attachment of reporter proteins to the wild type transit peptide, or deletion or mutations of the transit peptide or portion of the peptide or gene encoding it, and then characterization of targeting of these constructs
  • sequences of the transit peptide determine intracellular localization and site of function of proteins with this sequence Stage specificity of these enzymes is determined in vitro by using antibodies to stage-specific antigens in inhibitor-treated cultures, by Western
  • RT PCR Kerits, et al, 1996) and a DNA competitor as an internal standard to quantitate the amount of mRNA in parasite samples, by ELISA
  • HFF foreskin fibroblasts
  • isocitrate lyase isocitrate lyase, malate synthase, and alternative oxidase genes
  • malate synthase isocitrate lyase
  • alternative oxidase genes Such genes are used as
  • FIG. 1A compares heme biosynthesis in plants, algae and bacteria with heme biosynthesis in mammals
  • ALA is produced in the plastid by
  • the pathway in animals involves the condensation of
  • 3-amino 2,3-dihydroxybenzoic acid inhibits GSA in the heme synthesis pathway
  • HFF human foreskin fibroblasts
  • Non-toxic doses were used in parasite growth inhibition assays
  • In vitro parasite growth inhibition assays included confluent monolayers of HFF infected with
  • Toxoplasma organisms were grown in human foreskin fibroblasts alone and in
  • T. gondii growth is measured by ability of the parasites to incorporate
  • 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
  • FIG. 7 demonstrates the ability of gabaculine to inhibit the growth of the
  • CPM counts/minute
  • 3-amino-2,3-dihydrobenzoic acid is an inhibitor of the 5 carbon heme synthesis pathway present in Apicomplexan parasites. Heme synthesis occurs by a different pathway in mammalian cells and is therefore unaffected by 3-
  • Tachy- BradyIFA result on culture day used for of zoite zoite IFA culture Control Control 0 2 6
  • FIG. 2A illustrates how the
  • 3-NPA an inhibitor of isocitrate lyase
  • Salicylhydroxamic acid (SHAM) and 8-hydroxyquinoline are inhibitors of the alternative oxidase and are also effective against T. gondu, presumably by inhibiting the alternative pathway of respiration Salicylhydroxamic acid and 8-hydroxyquinoline inhibit the alternative oxidase of T. gondii tachyzoites Since alternative oxidative respiration does not occur in mammals, this makes antimicrobial compounds targeting this pathway therapeutic candidates
  • 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 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.
  • enolpyruvylshikimate (EPSP) synthase and thus an inhibitor of shikimate to chorismate
  • FIG. 4B demonstrates the ability of NPMG (a specific inhibitor of EPSP-synthase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period.
  • T. gondii growth is
  • 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 with intracellular antibodies are used as medicines to inhibit the parasite.
  • GSAT heme synthesis pathway
  • FIG. 6 demonstrates the effect ofNPMG, 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 dehydrogenase in Apicomplexan parasites (FIG. 2B, T. gondii) and also inhibited P. falciparum and C. parvum.
  • the inhibitor of isocitrate lyase is 3-nitropropionic acid (concentration ranging from 0.005 to 5mg/ml in vitro, and 5 to 50 mg/kg/day in vivo). Mutants [Yale Stock
  • E. coli strains E. coli strains
  • DV 21A01 aceA
  • the plastid is a likely site for the alternative pathway of respiration
  • 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 sequencing of the Tg EST zyllc05.rl clone obtained from the "Toxoplasma EST Project at Washington University” and of P. falciparum EST czap PFD d2.1 clone
  • gondii sequence has substantial homology with tomato and several other chorismate synthases and a region of the 71 gondii protein has 30% identity and 45% homology with the transit sequence of Zea mays (sweet com).
  • 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
  • the plant-like acetyl coA decarboxylase is inhibited by a number of inhibitors
  • auxins and Giberellins The known auxin mimics and Giberellin synthesis and Giberellin inhibitors
  • Glufosinate inhibits Apicomplexan glutamine/glutamate synthesis because the critical enzyme is plant-like. x. Transit Sequence
  • the transit sequence is conjugated with toxic molecules such as ricins and used
  • Example 5 The Combined Effects of lnhibitors 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
  • EPSP-synthase indirectly affects ubiquinone production and, thus, the electron transport chain.
  • heme is required for the production of cytochromes in the electron transport chain.
  • 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)
  • NPMG and sulfadiazine combine in a synergistic manner
  • Candidate inhibitors are administered to animals by daily intraperitoneal
  • NPMG is administered at a dose of 1 OOmg/kg/day
  • Cyst burden and pathology in the brains of inhibitor-treated and control mice are compared using methods described previously (Roberts, Cruickshank and Alexander,
  • Cyst numbers present in a suspension of brain are enumerated, or cyst numbers in formalin fixed paraffin embedded sections are quantitated
  • T. gondii (Me49 strain) Inhibitors are administered to groups of mice from day 30 post infection to day 50 post infection 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 B 1 gene
  • m vivo is the synergistic effect ofNPMG and low dosage pyrimethamine NPMG is an inhibitor of infection and promotes survival of mice infected with the virulent RH strain of T. gondii when utilized in conjunction with a low dose of pyrimethamine, whereas neither low dosage pyrimethamine nor NPMG alone are protective Sulfadiazine reduced manifestations of infection in vivo SHAM affects parasitemia and number of
  • FIG. 8 demonstrates the ability ofNPMG 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
  • NPMG pyrimethamine
  • PYR NPMG pyrimethamine
  • Percent survival is marked on the Y-axis and days post infection on the X-axis
  • mice treated with pyrimethamine and NPMG survived until day 9 post infection and 33 percent survived until the conclusion of the treatment (day 30 post infection) After the withdrawal of treatment, all of these mice survived until the conclusion of the experiment (day 60 post infection)
  • T. gondu was studied by electron microscopy and was found to have a plastid
  • Tachyzoites of the RH strain are stained with antibodies to tachyzoites and bradyzoites Tachyzoites of the RH strain are grown in the peritoneum of ND4 mice for 3 days Tachyzoites are
  • Bradyzoites are isolated as described herein in the Material and Methods The tachyzoites are pelleted by centrifugation and the pellet is fixed in 2 5% glutaraldehyde Cysts and bradyzoites are purified from the brains of
  • barley GSAT indicate whether the enzyme is present or absent in both the tachyzoite and bradyzoite life cycle stages and localizes the enzyme in the
  • marker for tachyzoite stage specific antigens are used for immunostaining of parasites to establish stage of the parasite.
  • Transgenic parasites with bradyzoite genes with reporter genes are also useful for such studies.
  • tachyzoites and bradyzoites are compared using standard inhibition experiments in
  • Tachyzoites use conventional and alternative pathways of respiration whereas the bradyzoite stage relies on alternative respiration Inhibitors of conventional respiration favor tachyzoite to bradyzoite
  • stages d) Synergy studies, gabaculine treatment Synergy studies with gabaculine are of particular interest because heme is used in the conventional oxidase pathway If there is synergy, iron influences stage switch ⁇
  • Bradyzoites and tachyzoites also are compared directly for the relative amounts
  • chorismate synthase genes isocitrate lyase genes, and malate synthase genes are 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 Cryptosporidia, Eimeria, and Malaria are detected in the same manner as the T. gondii
  • T. gondii alternative oxidase is identified by screening T. gondii cDNA expression libraries using the 7D3
  • tachyzoite cDNA library and placed in the Genbank database was found to encode a protein with homology to tomato chorismate synthase.
  • the EST was obtained, cloned
  • isocitrate lyase (cotton), UDP glucose starch glycosyl transferase (sweet com), and acetohydroxy acid synthase (sweet co ) 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 determine whether other Apicomplexans have genes homologous to those in T. gondii.
  • mR A isolated from T. gondii tachyzoites of the Me49 strain of T. gondii using the Uni-ZAP XR cDNA library system.
  • the titer of the amplified library is 1-2 X l ⁇ '7ml.
  • Other cDNA libraries also are utilized.
  • 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.
  • Genomic DNA is examined by Southern blot analysis for the
  • 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 probes, hybridization and chemiluminescence detection Alternatively, the Boehnnger
  • tion 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
  • PCR An alternative approach for identifying genes encoding enzymes of the present invention is by using PCR with primers selected on the basis of homologies already demonstrated between plant protein sequences for the relevant gene For example, for the gsa gene, 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 designed from conserved regions in alternative oxidase sequences from plant species
  • genes with other genes e.g. gsas, sequences are compared against those in Genbank
  • T. gondii sequences are searched for homologies to the known plant genes gsa,
  • Plasmodia also are compared for homologies in the same manner
  • hemL GE1377
  • RP523 RP523
  • SASX41B hemA from D. Soil. This strategy has been successful for cloning gsa genes from plants and algae (Avissar and Beale, 1990; Elliott et al, 1990; Grimm, 1990; Sangwan and O'Brian, 1993).
  • the hemA gene encodes glutamate-tRNA
  • reductase an enzyme important in the C5-pathway for heme synthesis.
  • Mutant bacteria are made competent to take up DNA with CaCl 2 treatment and are transformed with plasmids from the cDNA library. Briefly, chilled bacteria
  • the M9 (Ausubel et al, 1987) medium contains 0.2% glycerol as the
  • Nonselective medium contains 25 ⁇ g/ml ⁇ -
  • bacteria can take up DNA by electroporation. Chilled bacteria are prepared by a repetition of
  • nonselective medium is compared to growth on nonselective medium, which contains 25 :g/ml ⁇ -
  • mutants used for complementation are as follows:
  • DV21 AOl aceA which lacks isocitrate lyase
  • DV21 A05 aceB which lacks malate synthase
  • T. gondii bradyzoites use unique alternative oxidases
  • Alternative oxidases are necessary and sufficient for bradyzoite survival Methods to characterize plant alternative oxidases are as described (Hill, 1976, Kumar and Soil, 1992; Lambers, 1994; Li et al, 1996, Mclntosh, 1994)
  • cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent from their effects on the host cell mitochondria SHAM, an inhibitor of the alternative respiratory
  • SHAM concentrations are 0 25 to 2 mg/ml in vitro and 200 mg/kg/day in vivo
  • RT-PCR is as performed using the protocol of Hill (Chaudhuri et al ,
  • AOX alternative oxidase gene
  • cob on the alternative oxidase should be antimycin-insensitive and SHAM-sensitive Clones recovered are tested for complementation of hemL, hemB and hemA mutants Growth is tested for inhibitor sensitivity Sequences of cDNA clones that provide
  • the Escherichia cob strain XL 1 -Blue was prepared for infection with the 7. gondii phage library according to Stratagene manufacturer's protocol The RH
  • tachyzoite library in the ⁇ -ZAP vector system was titred, and 10 6 pfu are added to the
  • IPTG-soaked nitrocellulose filters are placed on the plates after the short incubation period, and the growth of the plaques is allowed to proceed for an equivalent period of time Filters are blocked in BLOTTO overnight Screening is
  • plaques are grown for 8 hours post-infection, and placed at 45°C for 2 hours to overnight. Nitrocellulose filters are placed on the plates,
  • Blots are hybridized to 32 P-labeled probe overnight. Low stringency washes, containing 5X SSC and 0.1% SDS are performed twice at room temperature, and high stringency washes with 0.2X SSC and 0.1% SDS are performed at a temperature
  • Identification of the genes is accomplished by screening an Apicomplexan cDNA library with the antibody to homologous enzymes from plants or other
  • microorganisms or probes which recognize the genes which encode them and/or complementation of mutant bacteria lacking the enzyme with Apicomplexan DNA.
  • Mutant-Knockouts The alternative mitochondrial oxidase pathway is the preferred oxidative pathway for bradyzoites and is likely to be important for their
  • cytochrome oxidase can be active and mutants are recoverable.
  • the AOX-null strains may be hypersensitive to GSAT inhibitors, both in vitro and in vivo. The ability of the
  • AOX-null strains to switch stages both in vitro and in vivo is determined.
  • the AOX- null strains are examined for stage specific antigens. Virulence and ability to form cysts are assessed in vivo in C3H HeJ mice as described herein.
  • constructs and organisms with the genes knocked out are cultured under conditions that would ordinarily yield a bradyzoite phenotype. These are used to determine whether expression of the "knocked out" gene is critical for bradyzoite antigen expression and the bradyzoite phenotype.
  • Example 11 Production, Testing, and Use of Vaccines against Apicomplexa "Knock out" organisms (e.g., lacking GSAT, or alternative oxidase or EPSP-
  • the knock-out vaccine strain in some cases is cultivated in tissue culture because components which are deficient are provided by a single product or a plurality of products. DNA constructs and proteins are produced and
  • Parasite burden refers to quantitation of numbers of parasites using PCR for the Bl T. gondii
  • Vaccines are useful to prevent infections of livestock animals and humans. Standard methods of vaccine development are used when substantial prevention of infection is achieved in murine
  • T. gondii has the enzymes necessary to synthesize folates.
  • T. gondii uses PABA.
  • chorismate is not only an essential substrate for the synthesis of folate, but it is required for the synthesis of ubiquinone and certain aromatic amino acids
  • the shikimate pathway may occur both inside and outside of the plastid
  • EPSP synthase exists in two forms in
  • Euglena one associated with the plastid of those grown in the light and the other found in the cytosol of those grown in the dark Apicomplexan parasites utilize the shikimate pathway for folate synthesis
  • chorismate synthase that has considerable homology with chorismate synthases from other organisms, provides information useful in developing novel antimicrobial agents
  • Chorismate synthases differs from other known chorismate synthases in length Chorismate synthases from other organisms range in length from 357-432 amino acids.
  • the larger size of the T. gondii protein is due to an internal region that has no counterpart in other known
  • T. gondii chorismate synthase sequence was used in a search with the BLAST program An EST from a Plasmodium falciparum cDNA library was located
  • the nucleotide sequence of the cDNA which encodes a putative T. gondu chorismate synthase and the amino acid sequence deduced from it is shown in FIG. 9.
  • the deduced amino acid sequence of putative T. gondii chorismate synthase has
  • chorismate synthases from diverse organisms including Solanum lycospersicum (tomato), Synechocystis species, Hemophilus influenza,
  • T. gondu chorismate synthase gene A homologous P. falciparum EST (FIG. 11) was identified. It was sequenced This provided additional evidence that at least a component of the shikimate pathway also was present in P. falciparum
  • Phagemid DNA was excised by simultaneously infecting XLl-Blue cells with the phage stock and VCS-M13 helper phage Purified phagemids were used to infect XL 1 -blue cells Infected XLl-Blue cells were grown in LB media and plasmid DNA purified using Qiagen maxi-prep kits The cDNA insert was excised using EcoR I and Xho I restriction enzymes and found to be approximately 2 4KB Initial sequencing of the 5 prime end of the insert's plus strand and its translation, revealed 30% homology with
  • fragments were electrophoresed through a 1% agarose gel and fragments visualized by
  • Plasmids containing the restriction fragments were used to transform DH5 ⁇ competent cells. Plasmid DNA was purified using Qiagen maxi-prep
  • restriction enzyme fragments To facilitate sequencing of a region on the same strand
  • primers were designed from an
  • primers were designed to be the complement
  • T gondii chorismate synthase A useful reporter protein for a chimeric construct is ⁇
  • Antisense, ribozyme or intracellular antibodies directed against the transit sequence nucleic acid or translated protein are useful as medicines.
  • the amino acid or nucleic acid which encodes the transit sequence are the bases for diagnostic reagents and
  • antisense nucleic acids intracellular antibodies which target a key parasite protein, and creation of constructs with accompanying molecules which are lethal to the parasites (Roush, 1997; Mahal et al, 1997). This transit sequence also is useful because it
  • the customized primers utilized were:

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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.
EP97937983A 1996-07-19 1997-07-18 Agents antimicrobiens, reactifs de diagnostic et vaccins a base de composants specifiques du parasite apicomplexan Withdrawn EP0918868A2 (fr)

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US6653531B1 (en) 1998-07-21 2003-11-25 E. I. Du Pont De Nemours And Company Chorismate synthase from plants
US6911331B2 (en) 1998-07-21 2005-06-28 E. I. Du Pont De Nemours And Company Chorismate biosynthesis enzymes
AU5218799A (en) * 1998-07-21 2000-02-14 E.I. Du Pont De Nemours And Company Chorismate biosynthesis enzymes
CA2377131A1 (fr) * 1999-05-04 2000-11-09 Rima W. Mcleod Agents anti-microbiens, reactifs diagnostiques et vaccins a base de constituants specifiques du parasite apicomplexan
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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
US5614551A (en) * 1994-01-24 1997-03-25 The Johns Hopkins University Inhibitors of fatty acid synthesis as antimicrobial agents

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