EP1150709A2 - Antimikrobielle mittel, diagnostische reagenzien, und impfstoffe auf der grundlage von apicomplexanparasitenkomponenten - Google Patents

Antimikrobielle mittel, diagnostische reagenzien, und impfstoffe auf der grundlage von apicomplexanparasitenkomponenten

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Publication number
EP1150709A2
EP1150709A2 EP00928534A EP00928534A EP1150709A2 EP 1150709 A2 EP1150709 A2 EP 1150709A2 EP 00928534 A EP00928534 A EP 00928534A EP 00928534 A EP00928534 A EP 00928534A EP 1150709 A2 EP1150709 A2 EP 1150709A2
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EP
European Patent Office
Prior art keywords
gondii
parasite
synthase
apicomplexan
gene
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EP00928534A
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English (en)
French (fr)
Inventor
Rima W. Mcleod
Craig Roberts
Fiona Roberts
Jennifer Johnson
Michael Kirisits
David Ferguson
Russell Lyons
Ernest Mui
Doug Mack
Benjamin Samuel
Piotri Gornicki
Ellen Zuther
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Individual
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Publication of EP1150709A2 publication Critical patent/EP1150709A2/de
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphorfructokinase and not generally operative in animals or encoded by the plastic DNA, to develop compositions that interfere with Apicomplexan growth and survival.
  • Components of the pathways include enzymes, transit peptides and 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 components and their inhibitors.
  • a cDNA sequence that encodes chorismate synthase expressed at an early state of Apicomplexan development, is disclosed and may be altered to produce a "knockout" organism useful in vaccine production.
  • Toxoplasmosis is the major opportunistic brain infection in AIDS patients, causes loss of life, sight, hearing, cognitive and motor function in congenitally infected infants, and considerable morbidity and mortality in patients immunocompromised by cancer, transplantation, autoimmune disease and their attendant therapies.
  • Cryptosporidiosis is an unbeatable cause of diarrhea in AIDS patients and a cause of epidemics of gastrointestinal disease in immunocompetent hosts Eunciia infections of poultry lead to billions of dollars in losses to agricultural industries each year Other Apicomplexan infections, such as babesiosis, also cause substantial morbidity and mortality Although there are some methods for diagnosis and treatment of Apicomplexan caused diseases, some of these treatments are ineffective and often toxic to the subject being treated
  • 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 polymerase chain reaction (PCR) to detect a parasite gene
  • 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 polymerase chain reaction (PCR) to detect a parasite gene
  • Most of the assays utilize whole organisms or extracts of whole organisms rather than recombinant proteins or purified parasite components In many instances, the available assays have limited ability to differentiate whether an infection v.
  • the primary antimicrobial agents used to treat toxopljsmosis are py ⁇ methamine (a DHPR inhibitor) and sulfadiazme (a PAJBA antagonist)
  • the use of py ⁇ methamine is limited by bone marro toxicity which can be partially corrected by the concomitant administration of folinic acid T goiidn cannot utilize folinic acid but mammalian cells can Another problem is that py ⁇ methamine is potentially teratogenic in the first trimester of pregnancy
  • the use of sulfonamides is limited by allergy, ga_.tioinie_.un:. I intolerance, kidney stone formation and Ste ⁇ ens-Johnson syndrome
  • cm spiramvcm, c ⁇ n clanihroim cm and atovaquone Usefulness of these medicines for treatment of toxoplasmosis is limited by toxicities including allergy and antibiotic-associated diarrhea, (especially Clostndium difficile toxin associated colitis with clindamycin use) Lesser or uncertain efficacy of macrolides such as spiramycin, azithromycin, and clanthromycin also limits use of these antimicrobial agents 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.
  • T. gondii plastid DNA sequences were reported to have homologies to algal plastid DNA sequences.
  • the plastid membrane of T. gondii was reported to be composed of multiple membranes that appear morphologically similar to those of plant/algal chloroplasts. except for the presence of two additional membranes in the T. gondii plastid, suggesting that it may have been an ancient algal endosymbiont
  • Apicomplexan proteins such as tubulin calmodulin and enolase with certain plant-like feai i es also are found in animals, and therefore appear in the host as ell as the parasite.
  • herbicides have been reported to inhibit the growth of Apicomplexans.
  • 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 Plasmodi m falciparum (malaria) and Babesia microti.
  • 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 psbA or PPi phosphofructokinase) to de .
  • elop compositions that interfere with Apicomplexan growth and survival Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoiei s of these nucleotide sequences to w ich antibodies antisense molecules and oihei inhibitoi ⁇ aie dnected Dia g nostic and thci pentic rcasient and vaccines aie developed ba>ed on the components and their inhibitors Attenuation of live parasites through disruption of any ot these components or the components themselves provide vaccines protective against Apicomplexans
  • Transit peptides are used to identify other proteins and their organeUe targeting sequences that enter and exit from unique Apicomplexan organeUes
  • the identified components are potential for production of medicines, reagents and assays, and vaccines
  • the protein which includes the transit peptide is not necessa ⁇ ly an enzyme in a biochemical pathway
  • the methods and compositions of the present invention arise from the inventors' discover)' that metabolic pathways, and targeting signals similar to those found in plants and algae, especially, but not exclusively those encoded withm the nucleus, are present in Apicomplexan parasites
  • These plant-like pathways in Apicomplexan parasites are targetable b inhibitors, as measured by determining whether the inhibitors, either singly or in combination, are effective in inhibiting or killing Apicomplexan parasite,, m vitto and/or /// vivo
  • the present invention includes new methods and compositions to treat, diagnose and prevent human and vete ⁇ narv disease due to Apicomplexan infections
  • the invention is based on applications and manipulations of components of algal and higher plant-like metabolic pathways discov red in Apicomplexan parasites
  • Plant- like ' means that products ol the pathwa . s enz . mes and nucleotide sequences encoding enzv mes in uu pathwa ⁇ s are homologous or similar to products enz ⁇ mes and nucleotide s ⁇ _ iiccs know n in plants lurein plants include algae As used herem plant-like - • ⁇ udes m ⁇ aboln. pathw a . s.
  • Methods to detect plant counterparts in Apicomplexan include a) immunoassays using antibodies directed to products and enzymes known in plants, b) hybridization assays using nucleotide probes that hybridize to specific sequences in plants, c) determining homologies of Apicomplexan nucleotide or protein sequences with plant nucleotide or protein sequences, and/or d) substrate tests for specific enzymatic activity
  • the "plant-like" pathways of the present invention are identified by: a) identification of metabolic pathways characteristic of plants but not generally present in animals, b) identification and characterization of Apicomplexan enzymes, nucleic acids and transit sequences as components similar or homologous to those in a), c) identification and development of compounds (inhibitors) which abrogate the effect of the components of the pathways in vitro and m vivo, singly or in a plurality, against one or more types of Apicomplexan parasites and in conjoint Apicomplexan, bacterial and fungal infections
  • 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 group that includes Toxoplasma, Plasmodium, Qyptosporidia, Ei eria, Babesta and Theilerict
  • the pathway may utilize a component encoded by an Apicomplexan nuclear
  • Suitable metabolic pathways or components include a) synthesis of heme from glutamate and tR A 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 and then acetyl CoA, and their use in the glyoxylate cycle (hereinafter the “giv oxylate cycle”).
  • n sv ntliesis of auxin growth regulators from indoleacetic acid derived from chorismate
  • o s ntliesis of isoprenoids (diterpenes 5 carbon units with some properties of lipi ⁇ .
  • 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 pathways; toxin conjugated antibodies or components of the pathways; antisense molecules; and inhibitors of transit peptides in an enzyme.
  • the interfering 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.
  • the plurality of compositions inhibits the parasite to a degree greater than the sum of 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; NPMG and cycloguanil (which inhibits Apicomplexan DHFR [TS]), and other inhibitors and competitors of interrelated cascades of plant-like enzymes. Wherein the effect of inhibitors together is greater than the sum of the effects of each alone, the synergistic combination retards the selection of emergence of resistant organisms and is more effective than the individual components alone.
  • the interfering composition acts on a latent bradyzoite form of the parasite, or multiple infecting Apicomplexan parasites simultaneously, or on conjoint infections with other pathogenic microorganisms which also utilize the plantlike metabolic pathway.
  • a method of determining the effectiveness of a composition in reducing the deleterious effects of an Apicomplexan m an animal include a) identifying a composition that inhibits growth or survival of an Apicomplexan parasite /// vitio by interfering with a plant-like metabolic pathway and b) determining a concentration of the 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 in the animal
  • Developing a lead compound that inhibits an Apicomplexan parasite is accomplished by a) identifying a plant-like metabolic pathway in an Apicomplexan parasite and b) identifying a composition that interferes with the operation of the pathway as a lead compound
  • 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 metabolic pathway in an Apicomplexan parasite that is selectively operative in a life- cycle stage of the parasite includes the enzymes alternative oxidase, and UDP glucose starch glycos l transferase
  • a composition comprising SHAM and 8-OH-qu ⁇ nol ⁇ ne inhibits the alternative oxidase in the latent bradyzoite form of an Apicomplexan parasite ⁇ method to identify a plant-like gene encoding a component of a plant-like metabolic pathw av in an Apicomplexan parasite is a) obtaining a strain of /J coli that is deficient for a component of the metabolic pathway, said deficiency causing the strain lo icquii e supplemented media for gi ⁇
  • Another method for identifying a plant-like gene product of a metabolic pathway in an ⁇ picomplexan parasite is a) contacting the parasite with a gene probe, and b) determining whether the probe has complexed with the parasite from which the identity of the gene product is inferred
  • 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) determining hether the gene is homologous to a plant gene which encodes a plant enzyme with the same function
  • a method for identifying a plant-like gene product in a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite or its enzyme with a substrate for the plant-like enzyme, b) measuring enz me activity, c) determining whether the enzyme is operative, and d) inhibiting activity of the enzyme in vitro with an inhibitor
  • the invention also relates to a diagnostic reagent for identifying the presence ot an ApicompleOii parasite in a subject, w here the subject includes a domestic or livestock an ' r ,al oi a human
  • the reagent may include all or a portion of a component ol the plam-'.ke pathw av, an a ⁇ iibodv.
  • 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 pathway.
  • 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 plant-like metabolic pathway that operates in an Apicomplexan parasite, and b) determining whether the antibody has complexed with the sample, from which the presence of the parasite is inferred Alternatively, the assay is directed towards a nucleotide sequence In both these cases, 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, domestic animals or a human against infection or adverse consequences of infection by an Apicomplexan parasite
  • the vaccine may be produced for an Apicomplexan parasite in w hich a gene encoding a component of a plant-like metabolic pathway in the parasite is manipulated, for example, deleted or modified When the gene is deleted oi modified in the live vaccine, the component of the pathway may be replaced bv the pi esence of the
  • the accine 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.
  • a method of treatment for an infection in a subject by an Apicomplexan parasite includes the following steps: a) obtaining an inhibitor of a plant-like metabolic pathway in an Apicomplexan parasite, and b) administering an effective amount of the inhibitor to the subject.
  • FIG. 1A-C illustrates the heme synthesis pathway and the effect of GSAT in T gondii
  • FIG. 1A diagrams the heme synthesis pathway
  • ALA synthase is also present in T gondii and constitutes an alternative pathway for heme sy ⁇ thesi.
  • FIG. 2 ⁇ -I3 shows unique lipid degradation in the glyo ylatc cycle in 7 g ⁇ ndii
  • FIG. 2 shows uptake of irn._a.ed uracil bv tachyzoites (RH strain) is inhibited bv 3-NPA (0 00s R, . mg G/ML) Note this inhibitor also effects succinate dehydrogenase, so its inhibitory effect does not unequivocally support presence of the glyoxylatp pathway
  • FIG. 3 A is a schematic representation of a pathway which demonstrates alternative oxidase as an alternative pathway for generation of energy in Apicomplexan parasites
  • FIG. 3B shows that uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by SHAM
  • FIG. 4A 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 foreskin fibroblasts (HFF) after 4 days, as measured by tritiated thymidine uptake and microscopic evaluation
  • FIG. 4A 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 foreskin fibroblasts (HFF
  • 4C shows product rescue of NPMG's inhibitory effect on EPSP svnthase by PABA
  • the effect of PABA on sulfadiazine is similar, but the effect on pv ⁇ methamine, as predicted reduces the enzyme to the levels that were present when media alone was utilized, as measured by the uracil uptake
  • FIG. 4D show s functional and enzymatic evidence for the shikimate pathway in / g ⁇ ndii with inhibition of EPSP synthase enzj mc activ ity by I mM glyosate Squares without glv phosate Circles, w ith glyphosate
  • FIG. 4C show s evidence for the shikimate path . av in /' fakipai ii with functional ev idence for the shikimate pathwav in /' luk tpai iini Glv phosate inhibition of in ⁇ ⁇ o growth of asexual erythrocv tic forms and PABA and folate antagonism of growth inhibition.
  • 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 on Cryptosporidia. 3NPA also inhibited Cryptosporidia.
  • FIG. 7 shows the effects of gabaculine (20 mM) on growth of tachyzoites/bradyzoites (R5) in human foreskin fibroplasts, over 8 days as determined by uracil uptake. Note increased uptake of uracil by the 8th day.
  • FIG. 8 shows the effect of NPMG, pyrimethamine, and pyrimethamine plus NPMG on survival of mice following intraperitoneal infection with 500 tachyzoites of the RH strain of T. gondii. Dosage of NPMG was 200mg/kg/day and pyrimethamine was 12.5 mg/kg/day).
  • FIG. 9 shows nucleotide and deduced amino acid sequences of T. gondii chorismate synthase cDNA. The asterisk indicates the stop codon.
  • FIG. 10 shows results of CLUSTAL X alignments of the deduced amino acid sequences if the putative T. gondii, chorismate synthase with the corresponding sequences from Synechocystites, S. cerevisiae, S. lypocersicum, N. crassa and
  • FIG. 11 shows the transit sequences of Zea mays and T. gondii chorismate synthases.
  • the sequences of the transit peptide directing the transport of the wx+ protein into maize amyloplasts and chloroplasts and the portion of the T. gondii 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 mays and T. gondii sequences. • indicates homologous amino acids in the Wx Zea mays and T. gondii sequences.
  • the transit sequence in the Wx Zea mays protein begins at amino acid number I and ends at amino acid number
  • FIG. 13 shows a genomic sequence of T. gondii chorismate synthase.
  • FIG. 14 shows (A) a T. gondii cDNA chorismate synthase DNA construct which is useful to produce antibody or a vaccine; (B) a Western blot (arrows mark chorismate synthase).
  • FIG. 15 shows green fluorescent (gfp) protein expression in a stably transfected tachyzoite; this tachyzoite has a reporter construct, a chorismate synthase- gfp; gfp is cytoplasmic (grey) and a defined structure in the area of the plastid is the white dot; the nucleus is the larger red area; gfp is in the cytoplasm.
  • FIG. 16 shows life cycle stage associated expression and localization of chorismate synthase in T. gondii.
  • Tachyzoites (1) 15 days - - Double stained with tachyzoite surface antigen 1 (SAG1) (perimeter raised, top and bottom left) and DNA stain (DAPI) (bottom right) and chorismate synthase (top right, white); (2) Double stained with dense granule protein 4 (granular stain, top left), chorismate synthase (white); p30, lower right panel, (perimeter raised) rhoptry probe (raised grey, rhop); (3) Double stained chorismate synthase-punctate white, SAG1 (P30, perimeter raised).
  • SAG1 tachyzoite surface antigen 1
  • DAPI DNA stain
  • DAPI DNA stain
  • rhoptry probe raised grey, rhop
  • Bradyzoites (1) Abbreviations are the same as in A; Note diffuse granular appearing cytoplasmic staining of bradyzoite chorismate synthase (top
  • C Microgametes (Mi), Macrogametes (Ma); Note darker immunoperoxidase staining of these forms but not schizonts (s) in cat intestine.
  • D Chorismate synthase mRNA production in tachyzoites and with bradyzoites; Note SAG1 message for a tachyzoite protein, BAG 1-5 message for a bradyzoite protein and constitutively expressed mRNA for tubulin.
  • FIG. 17 shows: (a) schematic illustration of gly oxy late cycle, (b) inhibitors of isocitrate lyase (ICL), (c) T. gondii isocitrate lyase enzyme activity, (d) inhibition of ICL enzyme activity by 3NPA, and (e) inhibition of tachyzoites in tissue culture.
  • FIG. 18 shows a T. gondii isocitrate lyase (ICL) cDNA sequence.
  • FIG. 19 shows a T. gondii isocitrate lyase (ICL) amino acid sequence.
  • FIG. 20 shows (a) T.
  • gondii isocitrate lyase (ICL) binding pocket and active site inside box and (b) comparison with the published sequence of yeast isocitrate lyase with mutated lysine (K) which inactivated the enzyme (arrows).
  • FIG. 21 shows a T. gondii isocitrate lyase genomic DNA sequence (ICL).
  • FIG. 22 shows T. gondii isocitrate lyase in bradyzoites; Note brown areas in immunoperoxidase stain preparation.
  • FIG. 23 shows isocitrate lyase (a) in a western blot of tachyzoites (b) during stage conversion, and (c) mRNA during stage conversion. (Abbreviations are the same as in FIG. 16A and D legends).
  • FIG. 24 shows enzymatic, genetic, functional activity of Apicomplexan parasites and its inhibition and show T. gondii acetyl coA carboxylase is inhibited by - fop herbicides: (A) Acetyl coA carboxylase enzyme activity is inhibited by -fop herbicides;
  • sequences of Apicomplexan acetyl coA carboxylases sequences of acetyl coA carboxylase biotin carboxylase domains from apicomplexan parasites are as in Genbank Accession Numbers AF
  • a domain swap yeast with the T. gondii active site and recombinant enzymes made from a fragment of the T. gondii gene are amenable to high throughput screens;
  • This invention uses components of plant-like interrelated metabolic pathways that are essential for growth or survival of Apicomplexan parasites
  • the pathways are generally not operative in animals and do not include psbA or PPi phosphofructokinase and are not encoded in the 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 A. inhibitor ⁇ compounds based on a) targeting proteins bv i i ) substrate competition and transition state analogues ⁇ i) product competition (jii) alteration of active site directly or by modification of secondary structure or otherwise altering function of the active site
  • EPSP synthase inhibitor 4 refers to 3-(phos ⁇ honooxy)-4-hydroxy-5-[N-(phosphonomethyl-2- oxoethyl)am ⁇ nc-l -cyclohexene-1-carboxyl ⁇ c acid (3 ⁇ , 4 ⁇ , 5(5), compound with diethyl etha ⁇ amide EPSP synthase inhibitor 5 refers to shortened R pnosphonate
  • Tg Toxoplasma gondii
  • Pf Plasmodium falciparum
  • Cryptospondi ⁇ m. csrvum (Co) EPSP synthase b M-phosphonomethylglycine (NPMG), Tg and Pf chorismate synthase [AroC) cDNA and deduced a-r.mo acid sequences a novel sequence in the Tg chorismate synthase gene ( 4roC-fs) a portion of *.n ⁇ ch is homologous v/ith the plastid transit sequence of Zea mays (s -.ee; corn)
  • the Pf chorismate s ⁇ -ase (AroC) also has a corresponding novel and unique internal region Cp Emien.o bows (Eb) ge ⁇ ciic DNA which hybridizes with Tg AroC (chorismate synthase) Inh'cition of Tg in vitro
  • Tg Pf Cp SHAM and 8-hydroxyqu ⁇ nohne inhibited Tg Pf Cp in vitro, reactivity of Tg protein of -66Kd with 5 antibodies (monoclonal and polyclonal to VooOoo lily and 7 " brucei alternative oxidases) and reduction to monomer similar to VooOoo lily and T brucei alternative oxidases on a reducing gel, Identification of Tg cONA and genomic DNA PCR products using primers based on conserved sequences in other alternative oxidases which are probed and sequenced, Tg, Pf, 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, Pf, Cp in vitro by 3NPA
  • Enzymes invol . d in the synthesis of these essential amino acids include the following Lysine homocitrate synthase homocitrate dehydrase (Euglena fungi) aspartokinase aspartate semialdehyde dehyd'ogenase dihydropicolinate synthase, dihydropicolinate reductase J pipendeine - 2 6 - dicarbowlate Cansferase N - succinyl - ⁇ -keto- ⁇ aminopimelate transammase N - succinyl - L L ⁇ - ⁇ -diaminopimelate desuccinylase L L ⁇ - c diaminopimelate epimerase meso- ⁇ c diaminopimelate decarboxylase
  • Inhibitors of lysine synthesis include +2-4-Am ⁇ no-4-carboxybutyl azr ⁇ d ⁇ ne-2-carboxyl ⁇ c ac ⁇ d(3) (azi ⁇ di ⁇ o-diaminopimelate [DAP], aziDAP), N-Hydroxy DAP4, N-amino DAP5 4 methyelene DAP 6 3,4 didehydro DAP 4 methylene DAP 4 Methio ⁇ ine L-homose ⁇ ne acyltransferase, o-succinylhomose ⁇ ne sulfhydrolase, L-homocysteme transferase (to activate methio ⁇ ine - but not exclusively in plants S-adenosylmethonine [SAM] synthase, SAM-methyltransferase, SAM decarboxylase, S-adenosylhomocysteine hydrolase) Threonine L homose ⁇ n
  • Enzymes in the heme s>nthes ⁇ s [with a default ALA synthase pathway], shikimate pathwav alternative generation of energy and glyowlate cycle are exemplified (Table 1 A) and the others (Table I B) are suitable for the practice of the invention
  • Apicomplexan infections include those due to loxop/ ⁇ snici gondn (toxopl ismosis), Pl ⁇ vnodt ⁇ (malaria), Ci )pl ⁇ spo ⁇ idi ⁇ (cry ptosporidiosis) Luna t ⁇ (ennciO s) Ba/icsia (babesiosis) Iheil na (theile ⁇ osis) Neo ⁇ oia uiiiiiiuni and others ⁇ -, ⁇ p omplcxan parasite n
  • vitro assays include product rescue (i.e., complete or. partial abrogation of the effect of an inhibitor by providing the product of the reaction and thus bypassing the need for the enzyme which catalyzes the reaction.
  • the assays are based on inhibition of the parasite i.e. restriction of growth, multiplication or survival of the parasite.
  • Another measure of infection is "parasite burden” which refers to the amount (number) of parasites present as measured in vivo in tissues of an infected host.
  • Another measure of infection is destruction of host tissues by the parasites. Inhibitors reduce parasite burden and destruction of host tissues caused by the parasites.
  • the inhibitors must not be toxic or carcinogenic to the parasites' host and for / ' // 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, lyoxosomes, cytoplasm or mitochondria.
  • some enzymes used in these pathways arc encoded by ge e w ithin the nucleus Plant- ke pathways detected in Apicomplexan parasites include a) the 5-carbon heme biosynthesis pathway that utilizes glutamate as a carbon skeleton for synthesis and requires the unique enzyme glutamate- 1 -semialdehyde ammotransferase, b) the mobilization of lipids in the glyoxylate cycle which is a unique pathway that includes the 5 enzymes isocitrate lyase and malate synthase, c) the generation of energy by an alternative pathway which includes a unique alternative oxidase and/or other unique pathways and enzymes for generating energy in the mitochondria or plastid, and, d)
  • the shikimate pathway includes the 10 enzyme 3-phospho-5-e ⁇ olpyruvylshikimate (EPSP) synthase, chorismate synthase, and chorismate lyase, as well as a number of enzymes unique to plants, fungi, bacteria, and mycobacteria, but not to animals. Inhibitors of some of these enzymes also provide information about the functioning and targeting of the enzymes
  • the heme synthesis pathway invol. es enzymes encoded in the nucleus and 1 imported to the plastid. This pathway is present in Apicomplexans including T. gondii, P. falciparum, and Cryptosporidia parvum.
  • Inhibitors of the enzyme GSAT in the pathway include gabaculine (3-ami ⁇ o-2,3-dihydro benzoic acid), 4-am ⁇ no-5-hexano ⁇ c acid, and 4-ammo-5-fluropentano ⁇ c acid
  • the late cycle reported to be present in plants, fungi, and algae is also — ⁇ -' present in / gondii
  • the cycle uses lipids and converts them to C4 acids through a•s of biochemical reactions
  • One of the last steps in this series of reactions is dependent on the ⁇ >ociU le lyase enzyme and anothei on the malate .svntliase enzs mos liilnbiloi s ol ire>e enzymes include 3- ⁇ troptop ⁇ on ⁇ c acid and itaconic acid
  • the alternative respiratory pathway present in a range of organisms including some bacteria, plants, algae and certain protozoans (trypanosomes), is present in T.
  • Enzymes involved in the synthesis of chorismate including those which convert shikimate to chorismate, and enzymes which generate folate, aromatic amino acids and ubiquinone from chorismate in plants, are present in T. gondii, Plasntodium falciparum. Ciyptosporidium pan'itm, and Eimeria. Inhibitors include N-(phosphonomethyl) glycine (glyphosate, sulfosate and others). A full-length T. gondii cDNA sequence encoding a chorismate synthase from this pathway and the deduced amino acid sequence provide information useful in developing novel antimicrobial agents. The T.
  • gondii chorismate synthase has features in common with other chorismate synthases and entirely unique features as well.
  • 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.
  • the 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
  • a part of the novel internal sequence i c., SCS FSESAASTI KHERDGSAATLSRE RASDGRTTSRHEEEVEPG) in the 7 " . gondii AroC (chorismate synthase) gene has homology with the chloroplast/ amyloplast targeting sequence of Zea mays (sweet corn) wx (UDP.
  • glucose-starch-glycosyl transferase protein
  • MAALATSQLVATRAGLGVPDAST ⁇ RRG AAQG RGAP ⁇ s a u-TLSMRTSA ⁇ AAPRHQQQARRGGRFPS wc glucose-starch-glycosyl transferase protein
  • This transit sequence provides a novel way to target T. gondii enzymes that move from the cytoplasm into the plastid and is generally applicable to targeting any subcellular organeUe.
  • the P. falciparum 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 part of other pathways in Apicomplexan parasites. Down modulation of the UDP- glucose starch glycosyl transferase pathway leads to a switch from amylose to amylopectin synthesis and thus the bradyzoite phenotype.
  • 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 phenol 2-monooxygenase
  • catechol 1,2-deoxygenase catechol 1,2-deoxygenase (phenol hydroxylase), cyclohexadienyl dehydratase
  • 4-hydroxybenzoate octaprenyltransferase 3-oxtaprenyl-4- hydroxybenzoate carboxylyase
  • dehydroquinate synthase (5-dehydroquinate hydrolase)
  • chorismate synthase 5-enolpyruv
  • Recombinant protein produced by constructs with genes encoding these 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 nativ e (from v aried life-cycle stages) proteins are used and the expressed protein is a souice of ihe euzv inc, and ihe enzyme assay is carried out in the presence and absence of the inhibitor-, eithci alone or in combination and contiols include the buffer for the enzyme alone.
  • the crystal structure is useful for characterizations of enzyme active sitc(s), secondary structure, transit sequence, substrate and product interactions.
  • the design of additional inhibitors is carried out using published methods such as modifyins substrates as had been done with inhibitors of EPSP synthase as well as high through put screening of av ailable compounds.
  • synergistic inhibitors /// vitro are gabaculine, (heme synthesis) and SHAM (alternative energy generation); NPMG and SHAM; NPMG and sulfadiazine; and NPMG and pyrimethamine: Gabaculine and sulfadiazine are an additive . combination / ' // vitro.
  • 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.
  • the protein synthesis of plantlike proteins that is also demonstrated in Apicomplexan parasites suggests not only conservation of plastid genes but also conservation of nuclear genes which encode enzymes that act inside or outside the plastid, from an ancestor that is common to Apicomplexan parasites and algae.
  • Many vital metabolic pathways of algae (often shared with their evolutionary relatives, higher plants) also are conserved in the 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 has the dual advantage of rapidly identifying conservation of specific pathways and simultaneously identifying both target sites and lead compounds for therapeutic dru ⁇ development.
  • 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, thereby producing a strong effect and lessening the probability of resistance to the drug emerging because more than one mutation may be required; 2. some 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 5 some compositions may inhibit other microorganisms (including other
  • representati e Apicomplexan parasites notably / ' . gondii
  • the inveiuion is directed at effects of inhibitors of the unique plant-like pathways in Apicomplexan, alone and in combination.
  • Organisms used for the assays include T.
  • gondii tachyzoites, bradyzoites and a mutant that expresses 50% tachyzoite and 50% bradyzoite antigens Unique plant enzymes and pathways that were found to be inhibited by compounds shown to inhibit plant pathways in Apicomplexans include: (1) glutamate- 1 semialdehyde amino transferase, an enzyme important in heme synthesis, (2) isocitrate lyase, an enzyme important in utilization of lipids, (3) alternative oxidase enzyme complex, enzymes important in energy production and (4) 3-phospho-5- enolpyruvylshikimate synthase (EPSP synthase), an enzyme important in conversion of shikimate to chorismate which is a precursor for synthesis of folate, ubiquinone, and certain amino acids essential for survival.
  • glutamate- 1 semialdehyde amino transferase an enzyme important in heme synthesis
  • isocitrate lyase an enzyme
  • the invention provides a rational, conceptual basis for development of novel classes of antimicrobial agents that inhibit Apicomplexan parasites, unique diagnostic reagents, and attenuated vaccines.
  • the inhibitors provide lead compounds for the development of antimicrobial agents.
  • conserveed 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 related protein inhibitors, intracellular antibodies, antisense DNA. and ribozymes
  • Inhibitors are effective against more than one parasite (e.g. T. gondii, P falciparum and C. parvum) and enzymes in these pathways also are present in other bacterial and fungal pathogens such as Pneumocysits cariiin, ⁇ cohacteruim luherciilosi ni Siapliylo occus aiireii.s. and Henuiphilits influenza, but not animals T va. . inhibitor of these pathways affect susceptible microorganisms which concurrently infect a host. Because enzymes are utilized differentially in different parasite life-cycle stages, stage-specific inhibitors are within the. scope of the invention Genes encoding the enzymes in Apicomplexans are identifiable.
  • the genes encoding the enzymes are effectively knocked out in these parasites by conventional techniques "Knockout" mutants and reconstitution of the missing genes of the parasite demonstrate the importance of gene products to the varying life-cycle stages of the parasite which are identified using antibodies to proteins and ability to form cysts / ' // vivo which define the life cycle stages.
  • the parasites in which a gene is knocked out are a useful basis for an attenuated vaccine.
  • the genes encoding the enzymes or parts of them (e.g., a novel targeting sequence) or the proteins themselves alone or with adjuvants comprise a useful basis for a vaccine.
  • the pathways and enzymes of the invention are useful to design related antimicrobial agents.
  • sequences and definition of the active sites of these enzymes, and pathways, and organeUe (e.g., plastid) targeting sequences provide even more specific novel and unique targets for rational design of antimicrobial agents effective against Apicomplexan parasites
  • 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 riboz mes that function as inhibitors are useful, novel antimicrobial agents Enzymes of the invention are a novel basis for unique diagnostic tests Because some of these pathwa s are important in dormant parasites, or in selecting the dormant oi active life c cle Mages thcv are especially important as aiuimi ⁇ obial agent targets for life cycle stages of the parasite for which no effective anlimicrobial agents are known or as diagnostic reagents which ascertain the duration
  • Identification of the pathways in Apicomplexan parasites provides additional enzyme targets present in these pathways which are not present in or are differentially expressed in animal cells. Identification of the interrelatedness of these pathways with each other provides the basis for the development and demonstration of combinations of inhibitors which together have an effect which is .greater than the expected additive effect (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. These pathways or their products are interrelated.
  • the enzymes or DNA which encodes them are targeted by using two or more inhibitors leading to an additive or synergistic effect.
  • examples include the additive effect of gabaculine and sulfadiazine and the synergistic effects of NPMG and sulfadiazine and NPMG and pyrimethamine.
  • 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 diagnostic les; not only for presence of a parasite, but also for identification of the stage of the parasite
  • Genes encoding enzymes in pathw ays of ihe present invention are "knocked out" using le.v. ⁇ iques known in the an ⁇ parasite with a gene knocked out is said to be attenuated either because the gene expression of the enzyme is stage specific so the parasite cannot become latent, or because the knocked out enzyme is essential for parasite survival.
  • the importance of an enzyme's functions in various life-cycle stages is determined using a mutant-knockout-complementation system. In the former case, 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 immunity without persistence of latent organisms.
  • Mutants with the gene "knocked out” also can be selected because when the parasites are grown /// vitro they are grown in the presence of product of the enzymatic reaction to allow their survival. However, such attenuated parasites do not persist in vivo in the absence of the product and, consequently they are useful as vaccines, for example, in livestock animals.
  • 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 adjuvants Specific examples are 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 but with genes not identified or described as plant-like, has been used as in a vaccines that protect against bacterial and protozoal infections Plant-like pathways in Apicomplexans were inhibited /// vitro An
  • Apicomplexan glvoxylate cycle was analyzed to determine the sensitivity of tachyzoites and bradyzoites to glyoxylate cycle inhibitors Specifically, Apicomplexans have isocitrate lyase and malate synthase which present a unique pathway for lipid metabolism that is targeted by inhibitors Apicomplexan alternative oxidase is targeted, as evidenced by effects of inhibitors of alternative oxidase on this pathway and its expression and immunolocalization in tachyzoites and bradyzoites, Apicomplexan parasites have a metabo cally active EPSP synthase enzyme involved in conversion of shikimate to chorismate These four metabolic pathways, i e , heme synthesis, shikimate pathwav.
  • 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 modification (e.g , removing a transit sequence) of the isolated gene which allows the production of a functional protein in the E. coli or yeast, demonstrating that the gene encoding the enzyme is functional.
  • Neospora, and Enneria are identified when relevant plant or T. gondii 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 and identifies unique promoters which are targeted Unique parts of the genes were identified in the sequences and provide additional antimicrobial agent targets, diagnostic reagents and vaccine components or bases for v accines
  • Clade and bootstrap analyses (Kohler et al , 1997) establish the phvlogenetic origin of novel, sequenced, parasite genes and this indicates other related antimicrobial agent targets based on components molecules, and pathways of phv loge ⁇ eticallv related organisms
  • Gene products are expies ed and utilized l t enzyme assa . s and lot screening novel inhibitors lot making antibodics for isolation of native protein, for x-ray crystallography which resolves enzyme structures and thus establishes structure-function
  • V ⁇ Y V ⁇ Y; IS cetermined by observing effects of the inhibiioi s on dilleient I : cvcle stages in aculclv . > chronicallv infected mice and bv determining whethei a p 1 -no w ith the gene knoc- ec out can form cvsts in ⁇ n o
  • Useful techniques to develop diagnostic reagents for detection of these proteins or nucleic acids include ELISAs, Western blots, and specific nucleotides used as probes.
  • New elements include use of longer culture times (e.g., extending the duration of the assay to > 6 days is also a unique and useful aspect of this invention, because it allows demonstration of antimicrobial effect for compounds which have to accumulate prior to exerting their effect), use of Me49 PTg and R5 strains / ' // vitro, employing synergistic combinations of NPMG and low dosage pyrimethamine in vivo, and assays of parasitemia in vivo using competitive PCR.
  • results using the assay systems are shown in FIGS. 4, 6-8
  • toxicity of a candidate inhibitor was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) after 4 days and after S days as measured by tritiated 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
  • Example 2 Detection of Plant-like Pathw ays in Apicomplexans
  • Apicomplexan parasites were found to contain at least four metabolic pathways previously thought to be unique to plants, algae, bacteria, dinoflagellates, and fungi Specifically , the presence of a unique heme synthesis pathway, an alternative oxidase pathway, a glyoxylate cycle and a pathway necessary for the biosynthesis of chorismate and its metabolites were explored Growth of the parasite, T. gondii, depends upon these pathways To examine T.
  • FIG. 1 A compares heme biosynthesis in plants, algae and bacteria with heme biosynthesis in mammals
  • ALA is produced in the plastid by the action of GSA aminotra ⁇ sferase on glutamate 1 -sem ⁇ aldehyde
  • GSA aminotra ⁇ sferase on glutamate 1 -sem ⁇ aldehyde
  • ALA is formed through the condensation of glycine and succinyl CoA ALA is subsequently converted to heme In one dinoflagellate and T. gondii both pathways are present
  • Inhibitors of plant heme synthesis pathway restrict the growth of Toxoplasma gondii in vino
  • the synthesis of ⁇ -ammolevuhnic acid (ALA) the common precursor for heme bios nthesis occurs in the plastid of plants algae and Apicomplexan parasites by the 5-carbon pathway and ALA synthesis occurs by a difleicnt pathw av in animals
  • the pathw av in animals involves the condensation ol gl cine and succm l CoA The data in TIG.
  • HFF human foreskin fibroblasts
  • Toxoplasma organisms were grown in human foreskin fibroblasts alone and in the presence of different concentrations of gabaculine (3-amino-2,3-dihydrobenzoic acid). Growth was measured by the ability of T. gondii to incorporate tritiated uracil This compound was effective at inhibiting the growth of T. v ndii at the 20mM concentration.
  • FIG. IB demonstrates the ability of gabaculine (a specific inhibitor of GSA aminolransferase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period 7 ' .
  • gondii growth is measured by ability of the parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis
  • 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 45,000 If no / ' . gondii were added to ihe cultures ( ⁇ i ⁇ RH). a C M of around 2,00'J was observed Py ⁇ meihamine (0 I ⁇ iu ml) and suiphadiazinc ( 12 5 uu/ml) added to cultures resulted m a inai kcd reduction in CPM compared with untreated cultures. At a dose of 5 mM gabaculine restricted around 50% of CPM and at a dose of 20 mM it almost completely inhibited parasite growth, with counts of about 5,000 CPM.
  • FIG. IC demonstrates the ability of gabaculine to inhibit the growth of T. gondii over S days in culture.
  • T. gondii growth is measured by ability of the parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis.
  • 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 restricted in cultures with 20 mM gabaculine (gabaculine) over the 8 day time course.
  • parasite growth was restricted in cultures with pyrimethamine and sulphadiazine (P/S) over the 8 day time course.
  • FIG. 7 demonstrates the ability of gabaculine to inhibit the growth of the mutant R5 strain of T. gondii over S days in culture This mutant strain is atovaquone resistant and cossesscs certain characteristics of the tachyzoite stage and certain characteristics of the bradyzoite stage T.
  • gondii growth is measured by their ability to mcoi poi te tr.: ⁇ ated uracil and is expressed as counts/minute (CPM) on the Y-axis
  • CPM counts/minute
  • the X-axis represents days post infection Parasite grow th was evident in the cultures w hei e no dru_ '- as added (medium) ov er the enure time c ⁇ ui se Pai asite gi owih w as restricted in cultures with 20mM gabaculine (gabaculine) over the first 6 days of culture, after w hich a marked increase in parasite growth was detected. Furthermore groups of proliferating organisms which resembled tissue cysts were observed in similarly treated cultures.
  • T. gondii cystrlike structures are selected by gabaculine treatment of cultures.
  • 3-amino-2,3-dihydrobenzoic acid is an inhibitor of t -' 5 carbon heme synthesis pathway present in Apicomplexan parasites Heme synthesis occurs b ⁇ a c.ftei ent pathw ay m mammalian cells and is thercfoi e unaffected by 3 - amino-2, 3-d " • di Economicszoic acid Table 2. Gabacul'ne treatment of cultures selects bradyzoites.
  • IFA immunof.jc-oscent assay.
  • SAG1 is surface antigen 1.
  • BSAG is bradyzoite surface antigen 1.
  • BAGS is bradyzoite antigen 5.
  • CD Indicates no specific fluorescence of fe crga ⁇ ism;
  • Q indicates specific surface lluorescenco of the organism due lo presence of the antigen recognized by the antibody (e.g , ⁇ SAGI or ⁇ BSAG);
  • C ⁇ Indicates specific Internal f .crescence in the organism duo to presence ol the antigen within the parasite recognized by r . s .'.ib ⁇ dy (e g , ⁇ BAG5)
  • 3-Nitropropionic acid is an inhibitor of isocitrate lyase in the degradation of lipid to C4 and inhibits replication of T. gondii in vitro.
  • FIG. 2A illustrates how the glyoxylatc 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 synthase. Malate is then converted to oxaloacetate, thus completing the cycle.
  • FIG. 2B demonstrates the ability of 3-NPA (an inhibitor of isocitrate lyase) to restrict the growth of T. gondii in an / ' // vitro assay over a 4 day period. This result indicates it is likely that T. gondii degrades lipids using isocitrate lyase.
  • 7 * . 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
  • gondii were added to the cultures (no RH), a CPM of about 2,000 was observed.
  • FIG. 3A describes the electron transpo ⁇ respiratory chain that normally occurs on the inner membrane of mitochondria
  • NADH and succinate produced by the action of the citric acid cycle diffuse to the electron transport chain.
  • free energy is released This free energy yields the potential for the phosphorylation of ADP to ATP.
  • respiration branches from the conventional pathway at ubiquinone and donates released electrons directly to water in a single four electron step
  • An important feature of this pathway is that it does not contribute to transmembrane potential and thus free energy available for the phosphorylation of ADP to ATP
  • the pathway provides a source of energy and is preferred for conditions with relatively low ATP demands
  • a key enz me in this pathway is an alternative oxidase ' that is cyanide insensitive and does not require heme 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 ;// vii/o assay over a 4 day period
  • gondii were added to the cultures (no RH), a CPM of around 1 ,000 was observed Pyrimethamine (0 1 ⁇ g/ml) and sulphadiazine (12.5 ⁇ g/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures
  • Salicylhydroxamic acid (SHAM) and 8-hydroxyquinoli ⁇ e are inhibitors of the alternative oxidase and are also effective against T. gondii, presumably by inhibiting the alternative pathway of respiration Salicylhydroxamic acid and S-hydroxyquinoline inhibit the alternative oxidase of 7 " . gondii tachyzoites Since alternative oxidative respiration does not occur in mammals, this makes antimicrobial compounds targeting this pathway therapeutic candidates v Effect of NPMG The shikimate pathway is common to plants, fungi and certain other microorganisms and Apicomplexan parasites, but it is not present in mammalian cells FIG.
  • 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 t.i ⁇ er pi ocessed to yield tetrah rofolate or ubiquinone bv a further se ⁇ e - ol ' e ⁇ i7v ⁇ - ie reactions T his pathwav has noi been described in mammals w ind ai dependent ⁇ r ⁇ diet loi folate and thereloie loi lurah iofoiaie p duclion ' I his pathway is required for the synthesis of certain aromatic amino acids and aromatic precursors of folic acid and ubiquinone. It is likely that Toxoplasma gondii utilizes the shikimate pathway for synthesis of folic acid, ubiquinone and aromatic amino acids.
  • N-(phosphonomethyl) glycine an inhibitor of 3-phospho-5- enolpyruvylshikimate (EPSP) synthase and thus an inhibitor of shikimate to chorismate conversion, affects the pathway (Table 1). the ability of this compound to inhibit the growth of T. gondii was examined by the assay described m Example 1.
  • 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 pe ⁇ od.
  • T. gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis.
  • the X-axis describes how the T. gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of around 72,000. If no T. gondii were added to the cultures (no RH), a CPM of around 2,000 was observed.
  • 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
  • GSAT heme synthesis pathway
  • FIG. 6 demonstrates the effect of NPMG, gabaculine, SHAM and 8-hydroxyquinoline and 3-NPA on Cryptosporidia in vitro.
  • the level of infection of each well was determined and analyzed by an immunofluorescence assay at 48 hours using an antibody to C parvum sporozoites made in rabbits at a concentration of 0.1%. Fluorescein-conjugated goat anli-rabbit antibody was used at a concentration of 1%.
  • 95% CI count was the mean parasite count per field when 16 fields counted at lOx magnification ⁇ s.d. of the mean The approximate 95% CI counts were as follows' media and ethanol - 1200; paromomycin (PRM) and ethanol - 100.
  • 3-NPA inhibited the glyoxylate cycle (isocitrate lyase) and/or succinate dehydrogenase in Apicomplexan parasites (FIG. 2B, T. gondii) and also inhibited R. falciparum and C. parvum.
  • the inhibitor of isocitrate lyase is 3-nitropropion ⁇ c acid (concentration ranging from 0 005 to 5 mg/ml in vitro, and 5 to 50 mg/kg day in vivo)
  • Mutants [Yale Stock Center] used for complementation are as follows E. coli strains, DV 21 A01 (aceA which lacks isocitrate lyase) and DV21 A05 (aceB which lacks malate synthase) Plant gene sequences suitable for comparison are those described by Kahn el al.
  • a biochemical assay for isocitrate lyase activity is the method of Kahn el al ( 1977)
  • the polyclonal antibodies to cotton malate sy nthase ana coiion isociti ate I se w ich hy bi idize to / gondii proteins of approximately 60 kd are used to identify these enzymes in other Apicomplexan parasites.
  • NPMG inhibited the shikimate pathwav m Apicomplexan paiasites (FIG. B. / go ⁇ nl ⁇ . idi ⁇ )
  • 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 Thus, addition of the product proves the specificity of the effect of the inhibitor on the enzyme.
  • the addition of PABA abrogates the exogenous effect of NPMG which is an inhibitor of EPSP synthase (FIG. 4B, T. gondii). Because PABA ablates the effect of the inhibitor NPMG on EPSP synthase, the presence of the shikimate pathway in Apicomplexan parasites is demonstrated.
  • the full length nucleotide sequence of chorismate sy nthase was obtained follow ing restriction digestion and primer-based sequencing of the Tg EST zyllcOS r l clone obtained from the "Toxoplasma EST Piojcct at Washington University" and of I'. falcipai iim EST czap PFD d2 1 clone obtained fi om the malaria ESI project I) Chakrabarti, Florida
  • the ' oxoplasmu gondii sequence has substantial homology wiih tomato and sev eral othci chorismate synthases and a region of the T.
  • gondii protein has 30% identity and 45% homology with the transit sequence of Zea mays (sweet corn).
  • Other inhibitors of EPSP synthase 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 rational synthesis of competitive substrate inhibitors of Apicomplexan parasites. v. Branched Chain Amino Acid and Other Essential
  • 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. vi. Starch (Amylose/Aniylopcctiu) Synthesis and Degradation
  • the plant-like acetyl coA decarboxylase is inhibited by a number of inhibitors shown in Table I B. Linoleic acid and linoleneic acid synthases are inhibited by newly designed competitive substrates. viii. Auxins and GibcrcIIins The know n auxin mimics and Gibercllin synthesis and Gibcrellin inhibitors inhibit Apicomplexan parasites' growth iv. Glutamiiic/Gliitamate Synthesis
  • Glufosinate inhibits Apicomplexan glutamine/glutamate. synthesis because the critical enzyme is plant-like. ⁇ .
  • Transit Sequence The transit sequence is conjugated with toxic molecules such as ricins and used to disrupt plastid function in Apicomplexans. Other strategies, such as antisense, 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.
  • FIG. 5 demonstrates the inter-relationship of the shikimate pathway and heme synthesis with the electron transport chain
  • the shikimate pathway produces 3,4-dihydroxybenzoate w hich is conv erted to ubiquinone, an essential component of the electron transport chain
  • NPMG an inhibitor of EPSP-synthase
  • heme is required for the production of cytochromes in the electron transport chain
  • inhibition of heme production gabaculine also indirectly affects the conventional electron transport chain
  • NPMG and sulphadiazine ( a competitive PABA analogue) w hich act at different points of the folate sv iuhesis paihw av are pi cdicted to be sv nei gistic, w hereas the effects of gabaculine and ulpludt/me l competitive PABA analogue) winch act on different pathways, ai e prcdicted to be additive.
  • 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 RU). a CPM of about 2,000 was observed. Pyrimethamine (0.1 ⁇ g/ml) and sulphadiazine (12.5 ⁇ g ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures.
  • T. gondii The growth of T. gondii was inhibited by about 60% in cultures treated with 5 mM gabaculine (gabaculine).
  • this dose of sulphadiazine was used in combination with 5 mM gabaculine, as expected, the combined effect of gabaculine plus sulfadiazine is additive because the pathways are in parallel.
  • NPMG and sulfadiazine combine in a synergistic manner.
  • gondii and prov ides a default pathway for the synthesis of ⁇ -aminole ulinic acid
  • the effects of gabaculine plus SHAM are not synergistic Cycloguanil which affects the plant-like DHFR-TS of 7 ' . gondii (McAuley et al, 1994) also is synergistic with NPMG and other inhibitors of enzymes in the shikimate pathway which provides an improved, novel method to treat this infection.
  • Use of synergistic combinations provide an improved strategy for the development of new medicines for the treatment of disease and eradication of the parasite.
  • Candidate inhibitors are administered to animals by daily intraperitoneal injection or by addition to the drinking water To inhibit EPSP synthase, //; vivo, NPMG is administered at a dose of lOOmg/kg/day a) Survival Five hundred tachyzoites of the RH strain are administered intraperitoneally to BALB/c mice. Cumulative mortality is followed in groups of mice given inhibitor compared to untreated controls b) Formation of Cysts- C3H/HeJ mice that have been infected perorally with the Me49 strain of T.
  • Cyst burden and pathology in the brains of inhibitor-treated and control mice are compared using methods described previously (Roberts, Cruickshank and Alexander, 1995, Brown et al., 1995, McLeod, Cohen, Estes, 1984, McLeod et al., 19SS) Cyst numbers present in a suspension of brain are enumerated, or cyst numbers in formalin fixed paraffin embedded sections are quantitated c) Persistence of Cysts C3H/HeJ mice are infected orally with 100 cysts of 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 receive antibody to gamma mterferon which leads to recrudescence of disease d) S nemy If marked synergistic effect is demonstrated /// viti o bv sho nm that the siibmhibitor. concentrations used touether exert an effect ureater than the additive effects of each used separately, for any combinations, their effect alone and together in vivo is compared. e). New Assays Which Determine the Effects of Antimicrobial Agents on 7 " .
  • gondii B l gene is amplified by PCR in the presence of a construct which produces a product slightly smaller than the wild type B 1 gene
  • the amount of construct can be quantitated to semiquantitate the amount of the competing wild type gene For example, presence of a greater amount of the wild type gene will result in lesser use of the competitor f)
  • Effect of Antimicrobial Agents on Apicomplexan Parasilcs In 1 ' ⁇ vo A demonstration of the effect of lnhibitors of plant-like metabolic pathwa in o ⁇ _, the Nv nei islic effect of NPMG and low dosage py rimethamine NPMG is an inhibitoi of iniection and piomotes su v al of mice infected w ith the v irulent RI I si tain of T.
  • 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. Percent survival is marked on the Y-axis and days post infection on the X-axis.
  • Tachyzoites are harvested in saline (0.9%) from the peritoneal cavity of euthanized mice and purified by filtration through a 3 ⁇ m filter. Bradyzoites are isolated as described herein in the Material and Methods. The tachyzoites are pelleted by centrifugation and the pellet is 5 fixed in 2.5% glutaraldehyde. Cysts and bradyzoites are purified from the brains of C57BL10/ScSn mice as described herein in the Materials and Methods and then fixed in 2.5% glutaraldehyde.
  • Immunoelectronmicroscopy localization is accomplished with Amersham Immunogold kit and cryosectioning using standard techniques in the electronmicroscopy facility at the University of Chicago or at Oxford University, Oxford, England. Extracellular organisms are studied as well as tachyzoites and bradyzoites at intervals after invasion.
  • Antibodies to the bradyzoite antigens (Weiss et al., 1992, and Bohne el al , 1993) and monoclonal and polyclonal antibodies to SAG1 (Kasper et al 19S3) as a marker for tachyzoite stage specific antigens are used for immunostaming of parasites to establish stage of the parasite Transgenic parasites with bradyzoite genes with reporter genes are also useful for such studies c) Inhibitors and Stage Switching
  • the bradyzoite stage relies on Var e rcspiration Inhibitors of conventional respiration favor tachyzoite to bradyzoite switching whereas inhibitors of alternative respiration inhibit tachyzoite and bradyzoite stages d)
  • 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 switching. For alternati e oxidase, immunostaining for bradyzoites and tachyzoite antigens is performed using gabaculine treated and control cultures.
  • bradyzoites utilize alternative oxidases exclusively, because gabaculine treatment of cultures would limit use of conventional oxidases and thereby select bradyzoites e) Western Blot Analysis, and ELISAs to determine stage specific expression of enzvmes
  • Brady zoites and tachyzoites also are compared dr e ⁇ ly for the relative amounts of alternative oxidase, using northern blot analyses, enzyme assays of parasites, isolation of mRNA and RT-PCR, using a competitor construct as an internal standard, and by Western blotting and ELISAs using antibodies to the enzymes (e g , alternative oxidase) UDP-glucose-starch glycosyl Iransferase. chorismate s nthase, isocitrate lyase, GS ⁇ also arc studied in a similar manner
  • DNA w ilh Homologous Plant-liUe Genes oi PoU itiallv l loniolouoiis Genes From Other I'.n avilcs The presence of the gsa genes, alternative oxidase genes, EPSP synthase genes 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 homologous gsa sequences in T. gondii. Probes are used on a membrane containing the genomic DNA of T.
  • T. gondii and soybean are used to probe other Apicomplexan DNA.
  • the gsa genes of Cryptosporidia, Etmeria, and Malaria are detected in the same manner as the T. gondii gsa.
  • DNA probes complementary to Trypanosome alternative oxidase are also used to probe other Apicomplexan DNA.
  • T. gondii alternative oxidase is identified by screening T. gondii cDNA expression libraries using the 7D3 monoclonal antibody or the tobacco alternative oxidase gene used as a probe and thus detecting the gene expressing the relevant protein This gene is used to detect the alternative oxidase genes of other Apicomplexan parasites by Southern analysis and screening other Apicomplexan cDNA libraries
  • 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 sequence Apicomplexan genes. Large numbers of T.
  • gondii genes from tachyzoite and bradyzoite cDNA libraries are being sequenced and deposited in Genbank Putative 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
  • Example 9 Identification of Genes Encoding Enzymes of the Plant-Like Biochemical Pathwavs in Apicomplexan Genes are isolated from a cDNA library by hybridization using specific probes to genes know n to encode enzymes in metabolic pathways of plants (see Example 9) Genes are cloned by complementation from a T. gondii cDNA expression library using a constitutes of E. coli mutants that lack these enzymes and thus depend on the addition of exogenous additives for their optimal growth Transformed bacteria arc used to isolate and sequence plasmid DNA and from those sequences, probes are generated to delcrmme w hether other Apicomplexans have genes homologous to those in T.
  • the titer of the amplified library is 1-2 X 10 l0 /ml.
  • Other cDNA libraries also are utilized.
  • the phagemids were excised with R408 or VCS-M13 helper phage and transduced into XL 1 -Blue Cells.
  • the plasmid DNA was purified using the Qiagen maxiprep system.
  • Other libraries e.g., early Me49 bradyzoite. in vivo Me49 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 presence of ihe sequences encoding enzymes required for specific algal or plant metabolic pathways.
  • Genomic DNA is extracted from Apicomplexan parasites by proteinase K digestion and phenol extraction DNA(5- 10 ⁇ g) is digested with restriction enzymes, clectrophorcscd through 1% Agarose and transferred to a nylon membrane 1 he ECL (Amersham) random prime system is used fo labeling of DNA probes, hybridization and chemiluminescence detection.
  • the Boehringer Mannheim Random Prime DNA labeling kit is used to label the DNA with j2 P with unincorporated nucleotides removed using G-50 Sephadex Spin columns.
  • Hybridization with the "P-labeled probe is carried out in [1M NaCl, 20 mM NaH 2 P04 pH 7.0, 1% SDS, 40% formamide, 10% dextran sulfate, 5 mg ml dry milk, 100 ⁇ g/ml salmon sperm DNA] at 37°C. Washes are optimized for maximum signal and minimum background. Probes are prepared from T. gondii cDNA clones obtained and characterized as described in Example 9. If lack of overall sequence conservation limits ability to detect homology, highly conserved regions are useful. For example, two highly conserved regions of the gsa gene are useful to generate oligonucleotide probes (Matters et al., 1995).
  • PCR An alternative approach for identifying genes encoding enzymes of the present inveiuion 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 DNA using primers generated from two highly conserved regions of GSAT.
  • the Neurospora crassa alternative oxidase gene has been isolated using degenerate primers designed from conserved regions in alternative oxidase sequences from plant species (Li et al, 1996) These primers are used to detect and clone the alternative oxidase gene from / gondii Candidate PCR products are cloned using the Invitrogen TA cloning kit 5) Sequencing: DNA from candidate cDNA clones is extracted using the Promega Wizard Miniprep System. Clones of interest are purified in large scale using the Maxiprep Protocol (Qiagen) and are sequenced by modified Sanger method with an automated sequencer (ABI Automated Sequencer) by the University of Chicago Cancer Research Center DNA Sequencing Facility.
  • T. gondii sequence data is available in Genbank. Sequences for plasmodia also are available as are some isolated sequences for the other Apicomplexan parasites. T. gondii sequences are searched for homologies to the known plant genes gsa, glutamyl-tRNA reductase, isocitrate lyase, malate synthase, alternative oxidase, EPSP synthase, and chorismate lyase using the BLASTN (DNA— DNA) and TBLASTN (Protein — » Conceptual Translation of DNA Sequence) programs. The conserved plant gene sequences for the shikimate pathway are those described by Kahn et al. (1977) and Maloy et al. 0980; 1982). conserveed plant gene sequences for comparison of homologies are outlined by Klee et al. ( 19S7). Similar libraries and sequence data for Plasmodia also are compared for homologies in the same manner.
  • the hemA gene encodes glutamate-tRNA reductase, an enzyme important in the C5-pathway for heme synthesis.
  • the liemB gene encodes ALA dehydratase, an enzyme common to both heme biosynthesis pathways that should be common to all organisms and is included as a positive control.
  • Mutant bacteria are made competent to take up DNA with CaClj treatment and are transformed with plasmids from the cDNA library. Briefly, chilled bacteria (O.D. 550nm ⁇ 0.4-0.5) are centrifuged to a pellet and resuspended in ice-cold 0.1M CaCb and incubated for 30 minutes on ice.
  • the cells are resuspended in 0.1M CaCb, 15% glycerol and frozen at -80°C in transformation-ready aliquots.
  • 0.2ml ice-thawed competent bacteria are incubated on ice for 30 minutes with approximately 50ng plasmid DNA.
  • Cells are placed at 43°C for 2.5 minutes and cooled on ice for 2 minutes.
  • 0.8ml Luria Broth cells are incubated at 37°C for 1 hour and 0.1 ml is plated onto M9 minimal media plates.
  • the M9 (Ausubel et al., 19S7) medium contains 0.2% glycerol as the carbon source, 1 M MgSO .
  • leucine, and thiamine No ⁇ selective medium contains 25 ⁇ g/ml ⁇ - aminolevulinic acid (lieni and hemA) or 4 ⁇ g/ml hemin (lie B).
  • bacteria can take up DNA by elcctroporalion Chilled bacteria arc prepared by a repetition of cciurifugation and resuspcnsion The cells arc w ashed in an equal volume of cold water, a '/: v olume of cold water, a I 5 .
  • Successful complementation of the E. coli mutants with a T. gondii gene is determined by plating the transformed bacteria onto minimal medium which lacks the supplement required for optimal growth of the E. coli mutant. Growth on the selective medium is compared to growth on nonselective medium, which contains 25 :g/m! ⁇ -aminolevulinic acid (hemL or hemA) or 4 ⁇ g/ml hemin (hemB). Clones that complement each E. coli mutant are tested for their ability to complement each of the other mutants. Clones of putative T. gondii gsa and glutamate-tRNA reductase should complement only emL and hemA mutants, respectively. Clones that suppress more than one he mutation are candidates for alternative oxidase gene clones. A cDN A clone containing the entire soybean gsa gene was able to transform the
  • E. coli hemL mutant from auxotrophic to prototrophic for ⁇ -aminolevulinic acid (ALA)
  • ALA ⁇ -aminolevulinic acid
  • the mutants used for complementation are as follows DV2 I A0 I laceA which lacks isocitrate lyase) and DV2 I A05 (aceli which lacks malate sv nt asel
  • the mutants for complementation are available and used as follows E. coli, AroA and yeast AR
  • 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 that lack functional mitochondria are used These 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 pathway is used at concentrations between 0 25 and 2 ⁇ g/ml in vitio, and 200 mg/kg/day orally or parenterally /// vivo alone or in conjunction with other inhibitory compounds
  • Other approaches include complementation of alternative oxidase- deficient /.
  • coli mutants to isolate and sequence the alternati e oxidase gene, immunostaming using antibodies for potentiallv homologous e ⁇ i7v ⁇ ncs, enzymatic ssay and the creation of mutant-knockouts for the alternative oxidase gene and studying stage specific antigens in such knockouts.
  • Inhibitor studies are carried out as described herein. SHAM concentrations are 0.25 to 2 mg/ml / ' // vitro and 200 mg/kg/day in vivo.
  • Immunostaining is performed on cultures of fibroblasts in Labtech slides infected with tachyzoites (RH strain) as described herein. Slides are stained 1, 2, 4, 6 and 8 days post infection with anti-BAG and antiSAGl .
  • RT-PCR is as performed using the protocol of Hill (Chaudhuri et al., 1996) with degenerate primers based on consensus sequences. The product is cloned, sequenced and homology with known alternative oxidases documents its presence.
  • Complementation is used to demonstrate function and is an alternative approach to isolate the gene. Proper function of the complementation system is demonstrated by- using complementation with a plant alternative oxidase gene Mutants suitable for use are hemL, hemA. liemB
  • the Var e oxidase gene, AOX. is cloned from a T. gondii cD A exp ⁇ e>sion library by complementation of the /:. coli hemL mutant HemL mutants of /..
  • the Escherichia coli 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° pfu are added to the XL I -Blue preparation
  • Approximately 6 X 10 s plaques are plated on agar onto 150 mm 2 petri dishes containing NZY medium, and grown at 42°C for 3 5 or 8 hours, depending upon which screening method is employed If antibodies are used for screening, IPTG-soaked nitrocellulose filters are placed on the plates after the short cubation period and the growth of the plaques is allowed to proceed for an equivalent period of time Filters are blocked in BLOTTO o ernight Screening is carried out under the same conditions which had been optimized during Western blotting with that primary antibody, and the appropriate secondary antibodv If DN ⁇ probes ai e used lor sciccnmg
  • 1993 1 is not absolutely required for growth when cv tochi ome o - idase can be activ e and mi ants are recov erable
  • 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.
  • Knockouts with a bradyzoite antigen reporter gene are produced and these 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. 8) Similar "knockouts" of EPSP synthase or chorismate synthase are produced.
  • Example I Production. Testing, and Use of Vaccines against Apicomplexa "Knock out" organisms (e.g., lacking GSAT, or alternative oxidase or EPSP- synthase or chorismate synthase or UDP-glucose starch glycosyl transferase) are produced as described herein.
  • 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 tested as described herein (see Materials and Methods) using unique genes and sequences ar.
  • Briellv. thev arc used to immunize C3H mice, and tissues of immunized and conlrol mice are subsequently examined for persistence of parasites. These immunized mice and controls are challenged perorally with 100 cysts of Me49 strain or intraperitoneally with 500 RH strain tachyzoites. Effect of immunizations on survival, and tissue parasite burden are determined (McLeod et al., 1988). Parasite burden refers to quantitation of numbers of parasites using PCR for the B 1 T.
  • gondii gene quantitating numbers of cysts in brain tissue, quantitating numbers of parasites by inoculating serial dilutions of tissues into uninfected mice when the RH strain of T. gondii is utilized and assessing survival of recipient mice as 1 parasite of the RH strain of 7 " . gondii is lethal.
  • Ability to prevent congenital transmission and to treat congenital infections is also a measure of vaccine efficacy.
  • 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 models.
  • Example 12 Nucleotide and Deduced Amino Acid Sequence of T. gondii Chorismate Svnthasc cDNA
  • T. gondii has the enzymes necessary to synthesize folates For this purpose.
  • T. gondii uses PABA. The biochemical events that lead to P BA production in / ' .
  • the shikimate pathway facilitates the conversion of shikimate to chorismate, a three step reaction catalyzed by three enzymes, shikimate kinase, 3-phospho-5-enolpyruvyl shikimate synthase (EPSP synthase) and chorismate synthase. Chorismate is then used as a substrate for the synthesis of PABA.
  • EPSP-synthase and chorismate synthase are encoded in the nucleus.
  • 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.
  • An inhibitor of the EPSP synthase, an essential enzyme in this pathway restricts the growth of 7 " . gondii. P. falciparum and C. parvum in vitro.
  • NPMG This inhibitor, synergizes with pyrimethamine and sulfadiazine to prevent T. gondii multiplication. NPMG also synergizes with pyrimethamine to protect mice against challenge with the virulent RH strain of T. gondii.
  • the sequence of a T. gondii gene that encodes a putative chorismate synthase, that has considerable homology with chorismate synthases from other organisms, provides information useful in developing novel antimicrobial agents.
  • a partial cD A sequence of approximately 250 bases was identified from the "Toxoplasma EST Project at Washington University.” This sequence, when translated. had approximately 30% homology with chorismate synthase from a number of organisms Bolh strands of the corresponding clone were sequenced and found to be 23 12 basc_. in length (FIG 9) Analysis revealed a large open reading frame of 160S base pairs which would encode a 536 amino acid protein. Homology was determined by the use of CLUSTAL X, a computer program that provides a new window base user interface to the CLUSTAL W multiple alignment program. (Thompson, 1994). The deduced amino acid sequence has considerable identity (44.5 to 51.4%) with chorismate synthases of diverse species (FIG. 10).
  • the putative T. gondii protein 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 chorismate synthases and is novel. The function of this region remains to be -* * determined.
  • the T. gondii chorismate synthase sequence was used in a search with the BLAST program. An EST from a Plasntodium falciparum cDNA library was located that has considerable homology with the T. gondii sequence. Chorismate synthase is also present in Mycobaclerium tuberculosis.
  • the nucleotide sequence of the cDNA which encodes a putative T. gondii 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 substantial homologies with chorismate synthases from diverse organisms including Solatium l ⁇ (.o ⁇ ers ⁇ cum (tomato), Synechocyslis species. Hemopliilus influenza. Saccharomyces cerevisiae, and Neurospora crassa (FIG. 10)
  • the Apicomplexan data base in Genbank was searched for homologies to the
  • Phagemid DNA was excised by simultaneously infecting XL 1 -Blue cells with the phage stock and VCS-M 13 helper phage. Purified phagemids.were used to infect XL 1 -blue cells. Infected XL 1 -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 previously described chorismate synthases from other organisms.
  • Sub-clones were made from restriction fragments isolated by agarose gel electrophoresis and purified using the Qiaex gel extraction kit Qiagen, Chatsworth CA. Double digestions of the plasmid. with Hinc II and Pst I resulted in 4 fragments of 500, 800, 300 and 4000 base pairs. The 800 bp fragment, corresponding- to the base pairs 800-1600 was ligated into the bluescript KS vector. The 1600-2400 base pair portion of the insert was obtained in a similar manner using Pst I and Xho I restriction enzymes and ligated into the bluescript KS vector. Ligatio ⁇ s were performed for 12 hours at 18 degrees centigrade on a PTC 100, programmable thermal cycler, MJ Research Inc Watertown. Massachusetts. Plasmids containing the restriction fragments were used to transform DH5 ⁇ competent cells. Plasmid DNA was purified using Qiagen maxi-prep kits
  • Pnme-j were designed based on the sequencing information obtained from restriction e zyme fragments
  • primers were designed from an ai ea app ⁇ 200-300 nucleotides 5 prime into the last obtained sequence
  • primers were designed lo be the complement and reverse of the same region.
  • Primers were designed to be 18-25 nucleotides in length and have a Tm of 55-60 degrees centigrade. G plus C content was 45-55 percent.
  • Primers were designed to have minimal self annealing and to have a low propensity for primer to primer annealing. Primers with the ability to form stable secondary structures were not designed.
  • a useful reporter protein for a chimeric construct is ⁇ glucoronidase of E. coli, expressed under the control of the 355 promoter of cauliflower mosaic virus The ⁇ glucoronidase alone is expressed, in parallel. The transit peptide chimeric construct is found in the plastid The control ⁇ glucoronidase is found in the cytoplasm
  • Another useful reporter system is green fluorescent protein
  • I 5 (gfp) Antibodies to the chorismate synthase protein are also used to detect the presence of the product of the gene (with the transit sequence) in the plastid and the product of a construct in which the transit sequence is not present in the cytoplasm only This is used to immunolocalize proteins in different life-cycle stages Further mutations a:"d deletions are made which identify the minimal transit sequence using the
  • This transit sequence also is useful because it provides a general extension of the concept of transit and targeting sequences in Apicomplexan parasites that enable targeting of other parasite.
  • the transit sequence.of Zea mays. and. T.. gondii. are. shown in Figure 11.
  • Example 14 Nucleotide and Deduced Amino Acid Sequences of f, falciparum Chorismate Svntliasc EST.
  • PFCS 1 AGC TAT TGG GTG GATC PFCS2 TCC ATG TCC TGG TCT AGG PFCS3 ATA A A ACA CAT TGA CTA TTC CTT C PFCS4 GGG GAT TTT TAT TTT CCA ATT CTT TG PFCS5 TTG , ⁇ CGT TGA ATG ATA AGA C PFCS6 TTT TAG ATC AGC AAT CAA ACC PFCS7 AAC TTT TTA TCT CCA TAC TTT G PFCS8 GAA GGA ATA GTC AAT GTG TTT TTA T PCFS9 GTA TTT TAC CAA GAT TAC CAC CC PFCS 10 CCC CCA ACA CTA TGT CG PFCS 1 1 CAG TGG GCA AAA TAA AGA PFCS 12 CCA GTG GGC AAA ATA A PFCS 13 GGA AGA GAA ACA GCC AC PFCS 14 TGC TGC TGG GGC GTG
  • Example 15 Southern Blotting Demonstrates Presence of Chorismate Svntliasc (and bv Inference nil of the Shikimate Pathway) in Apicomplexan Parasites Southern blotting using the T. gondii chorismate synthase gene as a " '2 P labeled probe demonstrated homology at moderate stringency (e.g 0 2 x SSC, 0. 1% SDS at 42°C) [more stringent conditions define greatest relatedness of genes] with Emieria bo vis and Cnpio. ⁇ oridiiiin parvum DNA
  • This 7 gondii cDNA also comprises a probe for screening cDNA libraries of all other Apicomplexa to identify their chorismate synthase genes
  • the same principles are applicable to all the othei enzymes in Table I
  • Example 16 Gene Expression.
  • Rccoiiihinant Protein Production of Antibody and Solving the T. gondii and P. falciparum Crystal Structures of chorismate svnthasc to establish their active site and secondary structure. These are done using standard techniques.
  • the gene construct is placed within a competent E. coli.
  • Recombinant enzyme is identified by homologous antibody reactivity and purified using affinity chromatography. Fusion proteins are useful for isolation of recombinant protein.
  • Protein is injected into rabbits and antibody specific to the protein is obtained and utilized to purify larger amounts of native protein for a crystal structure.
  • the crystal structure provides information about enzyme active site and facilitates rational drug design (Craig and Eakin, 1997). Recombinant proteins are used for high through put screens to identify new antimicrobial agents.
  • Example 17 Other Uses (e.g. in diagnostic reagents and vaccines) of the
  • T. gondii genes and proteins used as diagnostic reagents and as a vaccine to protect against congenital infection
  • Recombinant protein all or part of the enzyme
  • T-gondii chorismate svnthasc are used in ELISA (e g antibody to human IgG or IgM. or Ig ⁇ or Igl; attached to ELISA plate - sei m to be tested - antibody conjugated to enzyme - enzyme substrate)
  • ELISA e g antibody to human IgG or IgM. or Ig ⁇ or Igl; attached to ELISA plate - sei m to be tested - antibody conjugated to enzyme - enzyme substrate
  • the recombinant proteins, pooled human sera from known unmfected individuals (5 individual sera pooled) and infected individuals (5 individuals with acute infection sera pooled, 5 individuals with chronic infection sera pooled) are the controls This test is useful with serum or serum on filter paper
  • Another example of a diagnostic reagent are primers to amplify the target transit sequence or another portion of the chorismate synthase sequence unique to T.
  • gondii PCR with these primers is used with whole blood to detect presence of the parasite
  • Such assays have proven to be useful using the T. gondii B lgeneJKirisits. Mui, Mack, McLeod, 1996)
  • Another example of a diagnostic reagent is useful in outpatient settings such as an obstetrician's office or in underdeveloped areas of the world where malaria is prevalent
  • FABs of monoclonal antibodies which agglutinate human red cells when ligated
  • Kit, 19SS Antigen conjugated anti-red cell Fab also is used to detect antibody to the component
  • a positive test occurs when the enzyme or antibody is circulating in the patient blood and is defined by agglutination of red cells (in peripheral blood from the patient) mixed with the conjugated antibodies
  • Controls are the same as those specified for the ELISA
  • vaccines are protein, peptides. D ⁇ A encoding peptides or proteins These are administered alone or in conjunction with adjuvants Mich as 1SCOMS
  • mice 1 heie v accine preparations are tested first in mice then primates then in clinical it mis Lndpoints ai e induction of pi ieciiv c immune responses, protection measui ed as enhanced survival, reduced parasite burden, and absent or substantial reduction in incidence of congenital infection (McLeod et al., 1988).
  • Genomic clones are isolated from commercially available genomic libraries (AIDS repository) using the identified cDNA clones as probes in the screening process.
  • the genomic library as ⁇ phage, is isolated onto NZY agar plates using XL 1 -Blue E. coli as the host, resulting in plaques following a 37°C incubation.
  • the cDNA sequence is radiolabeled with 32 P and hybridized to nylon membranes to which DNA from the plaques has been covalentiy bound. Plasmids from candidates are excised and their restriction enzyme-digested inserts sequenced. Experimental details are as described in
  • Example 19 P. falciparum Chorismate Svnthase Genomic Sequence. This is done with a gene specific subgenomic library as described in
  • Example 18 (see example 41).
  • Other examples of enzymes and the genes which encode them and which are characterized as outlined above include: glutamyl-tRNA-synthetase; glutamyl-tRNA reductase; prephenate dehydrogenase aromatic acid aminotransferase (aromatic transaminase); cyclohexadienyl dehydrogenase tryptophan svnthase alpha subunit; tryptophan synthase beta subunit; tryptophan synthase beta subunit; indole-3 -glycerol phosphate synthase (anthranilateisomerase), (indoleglycerol phosphate synthase), anthranilate posphoribosyltransferase; anthranilate synthase component I; phosphobiosyl anthranilate isomerase anthranilate synthase component II; prephenate dehdryat
  • Example 20 T. gondii Chorismate Svnthasc. EPSP Svnthase. and Shikimate Kinase Activities were Demonstrated Assay for chorismate synthase, EPSP synthase and shikimate kinase in T. gondii were performed and demonstrated such activity.
  • Example 21 T. gondii Dehydroquinate Dehydratase Activity is Demonstrated
  • Example 22 GSAT activity is demonstrated in T. gondii taclivzoitc Ivsntcs
  • An er-.zymatic assay (Sangwan and O'Brian, 1993) demonstrates GSAT activity in /. gondii lysates.
  • the buffer contains 0. 1 M MOPS (3-f N- inorpholinoj rop.inesulfonic acid), pH 6 S. 0.3M glycerol.
  • Example 23 Isocitrate Ivase activity is demonstrated in T. gondii tachvzoite Ivsates
  • An enzymatic assay demonstrates isocitrate lyase activity in 7 " . gondii isolates prepared by disruption of the parasite membranes using french press or a lysis buffer. Demonstration that the lysis buffer does not alter enzyme activity is carried out by performing the assay with known substrate and enzyme in the lysis buffer and documenting presence of enzyme activity.
  • Example 24 Alternative oxidase activity is demonstrated in T. gondii preparations. 7 " .
  • gondii tachyzoites and bradyzoites are assayed for alternative oxidase activity and such activity is found to be present in greater amounts in bradyzoites
  • Example 25 Novel Substrate Competitors and Transition State Analogues of
  • inhibitors are competitiv substrates or transition state analogues and they aie utilized in the enzyme assay, in vitro with tachyzoite and bradyzoite preparations and w ith native enzyme, tissues culture assays and in in vivo models as described above These provide a model paradigm for designing inhibitors of any of the enzymes specified above. Briefly, inhibitors are produced as follows: Competitive substrates are produced by designing and synthesizing compounds similar to known compounds but modified very slightly. For example, inhibitors related to glyphosate are known. The structures of glyphosate, sulfosate and the precursor for EPSP have similarities (please see below).
  • Inhibitors are designed by modifying substrates in such a manner that the modification interferes with the enzyme active site. This can be performed using molecular modeling software. Similarly, halogenated substrates for other enzymes have functioned effectively as nontoxic inhibitors. The principles are applicable to the design of inhibitors for any of the unique enzymes with well characterized substrates and active sites.
  • the structure of the target is modeled using the three- dimensional coordinates for amino acids in a related enzyme
  • An example of this is that the crystal structure of GSAT from a plant has been sol ed and its active site is known
  • Drug resistant mutants are produced in vitro following mutation with nitrosoguanidine and culture with the inhibitor.
  • the surviving organisms have acquired resistance to the inhibitor.
  • This process is carried out either with the Apicomplexan parasite or with bacteria or yeast complemented with the gene encoding the enzyme or part of the gene (e.g., without the transit sequence).
  • PCR amplifies the relevant cDNA and this cDNA encoding the resistant enzyme is cloned and sequenced. The sequence is compared'with that of the enzyme that is not resistant. With the information about the inhibitor target and three-dimensional structure, the point mutations which cause resistance are analyzed with computer graphic display.
  • This information provides the mechanism for altered binding of the drug, and the inhibitory compound is then modified to produce second generation medicines designed to treat resistant pathogens prior to their development in nature.
  • An example of the use of toxic analogues to kill parasites used by others provides a means whereby there is production of analogues toxic to parasites.
  • the purine analogue prodrugs, 6 sulfanyipurinol. 6 thioguanine, 6 thioxanthine and allopurinol interact with hypoxanthine phosphoribosyltransferase which is responsible for salvage of purines used to produce .AMP and GMP.
  • Such toxic analogues are effective against the plant-like enzymes i .
  • Multisubstrate analogues are useful because they markedly enhance the binding affinity to the enzyme Similarly, if enzymes in a cascade are linked in such a manner that the substrate for one reaction provides the substrate for the next reaction, multisubstrate analogues are more useful
  • Co-crystal zation of inhibitors with target enzymes of host and pathogen enable three-dimensional analysis of molecular constructs and atomic interactions between inhibitors and enzymes and redesign of lnhibitors (leads) to enhance their affinity for the pathogen enzyme Iterative crystallography, lead redesign and inhibitor testing /// vili o and in vn o enable design and development of potent selective inhibitors of the target of the pathogen enzyme Recombinant methods for screening large numbers of analogues for those that bind selectiv elv to the enzy mes of specific parasites prov ide justification for inclusion of the analogues which bind best in the design of transition-state or multisubstrate analogues
  • Inhibitors that effect components of these pathways are halogenated substrates or analogues which are effective competitors.
  • Inhibitor of Isoleucinc/valinc biosynthctic pat .. ay are noncompetitive inhibitors as is shown by the lack of relatedness of the inhibitors (e.g., imidazolinones, sulfonylureas) to ihe target enzymes
  • Example 26 Modifications of Inhibitory Compounds to Improve Oral Absorption Tissue Distribution (especially to brain and eve ' ). Tissue distribution is characterized using radiolabeled inhibitor administered to mice with its disposition to tissues measured by quantitation of radtolabel in tissues Compounds are modified to improve oral absorption and tissue distribution by standard methods
  • Example 27 Efficacy of Antimicrobial Compounds Alone. Together and In Conjoii't Infections in Murine Models Inhibitors of plant-like pathways are effective against the Apicomplexan infection alone, together with the bacterial and/or fungal infections and also treat the bacterial and fungal infections alone
  • Tissue Parasite Burden Is determined by quantitating brain and. eye cyst numbers.
  • Inflammatory Response This is noted in histopathologic preparations. Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM and atovaquone, NPMG and pyrimethamine, NPMG and SHAM.
  • Example 29 Determining whether there is Carcinogcnicitv and Teratogenicitv: Standard assays to evaluate carcinogenicity and teratogenicity include administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity (i.e. development of malignancies).
  • Observation includes general physical examination, autopsy and histopathologic studies which detect any teratogenic or carcinogenic effects of medicines.
  • Example 30 Constructs to Measure Parasitemia: Portions of genes are deleted and the shorter gene is used as an internal standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui,
  • Example 31 Vaccine Constructs and Proteins and their Administration: These are prepared, as described. They include DNA constructs (Ulmer, Donnelly and Liu, 1996) with the appropriate gene or portions of the gene alone or together, with adjuvants. Representative adjuvants include ISCOMS, nonionicsurfactant, vesicles, cytokine genes in the constructs and other commonly used adjuvants Native and recombinant proteins also are used in studies of vaccines
  • Suitable examples of plant-like enzymes which make parasites less able to switch from or persist in a specific life cycle stage include: alternative oxidase, enzymes critical for amylopectin synthesis such as starch synthases, UDP glucose-glucosyl starch transferase and branching (Q) enzymes Exa ple 33.
  • alternative oxidase enzymes critical for amylopectin synthesis such as starch synthases
  • UDP glucose-glucosyl starch transferase UDP glucose-glucosyl starch transferase and branching (Q) enzymes Exa ple 33.
  • Preparation of Diagnostic Test Reagents and Diagnostic Tests are as described (Boyer and McLeod, 1996). Sensitivity and specificity are established as is standard in the field.
  • Tests and reagents include ELISAs in which antibodies to the proteins or peptides and recombinant proteins of this in ention such as chorismate synthase ⁇ Awe) are used and PCR methodology in which primers to amplify DNA which encodes the enzymes, or parts of this DNA, are used
  • a test useful in an outpatient setting is based on conjugation of a monoclonal a iibodv to human red blood cells with antibody to plant-like peptides or proteins based on an assav described by Kemp et al (Kemp et al , 198S)
  • the red cells are cross linked ia the monoclonal antibody moietv, resulting in agglutination of the red blood cells in the blood sample if the antigen or antibody to the parasite component is present in the blood sample ELISA and PCR can be utihzed with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates outpatient and
  • Antisense ohgonucleotides directed against the nucleic acids which encode the enzymes of the essential parasite metabolic process described herein are effective medicines to treat these infections
  • Antisense oligonucleotides also are directed against transit sequences in the genes
  • Antisense ohgonucleotides are short synthetic stretches of DNA and RNA designed to block the action of the specific genes described abo e for example chorismate svnthase of T gondii or P fa/cipai um, by binding to their RNA transcript They turn oft the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRN A and no translation into protein
  • antisense do not contain cytosine nucleotides
  • Intracellular Antibodies to Target Essential Enzymes Proteins and Organdies Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes of this invention or portions of them (e.g , anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, a's described under vaccines.
  • Example 37 Development of New Antimicrobial Compounds Based on Lead Compounds:
  • the herbicide inhibitors comprise lead compounds and are modified as is standard Examples are where side chain modifications or substitutions of groups are made to make more active inhibitors (Table 1)
  • Their mode of action and structure as well as the enzyme and substrate structures are useful in designing related compounds which better abrogate the function of the enzymes Examples of such substrate or active site targeting are listed in Table I
  • Endpoints for vaccine effect and efficacy are development of antibody and cell- mediated immunity to T. gondii (effect) and most importantly, prevention of T. gondii congenital infections.
  • Assay for efficacy is via a serologic screening program to detect newborn congenital toxoplasmosis (described in Boyer and McLeod, 1996) with usual testing to document whether seropositive infants are infected (described in Boyer and McLeod, 1996) Example 40.
  • Vaccines are live attenuated, genetic constructs or recombinant protein
  • Intra-muscular, sub-cutaneous and oral are the preferred routes, although intravenous intraperitoneal and intradcrmal routes may also be used Scottish blackface or/and swaledale ewes foui to years old are tested for luG antibodies to I oxoplusnta gondii using an ELI SA assay
  • Only scro-negative animals are used for the study Three groups of 10-15 ewes are used for each experiment.
  • Groups 1 are vaccinated, while group 2 and 3 are not Three months later all ewes are synchronized for estrous and mated. At 90 days gestation the ewes in groups 1 and 2 are given 2000 sporulated oocyst of T. gondii. The outcome of pregnancy is monitored in all groups Aborted lambs or those dying soon after birth are examined histologically and by PCR for the B 1 gene or sub- inoculation into mice or tissue culture, for the presence of T. gondii. All placentas are examined histologically and as above for parasites.
  • Lambs are weighed at birth, Pre- colostral serum is taken from each lamb Congenital transmission is assessed by performing ELISA assays on the serum for IgG or IgM. Protection is measured as a decrease in congenital transmission, a decrease in the incidence or severity of congenital disease, or a decrease in abortion
  • E.xamp_e l T. yff.tttfrChorismate Svnthase Genomic Se uenrp Used to Produce "Knockouts" (Attenuated Vaccine
  • genomic sequence of chorismate synthase is in FIG. 13. As with other genomic sequences herein, it provides an example of a gene which is "knocked out" to produce an attenuated vaccine and also can be utilized as described in other parts of this document.
  • a chorismate synthase knock out parasite was produced as follows: The genomic T. gondii chorismate synthase sequence consists of 9 exons. To prepare the knockout construct, this sequence was digested with EcoN I to remove a 1.8 kb fragment that included exons 2, 3, and 4. The EcoNl digested ends were blunt ended followed by dephosphorylation. A 1.9 kb piece bearing HXGPRT flanked by the 5' promoter region and 3' untranslated region of dhfr (called dhfr HXGPRT) was isolated by digestion of a construct, obtained from J. Boothroyd, and Xbal and hoi.
  • the 1.9 kb fragment was cloned into the chorismate synthase construct so that dhfr HXGPRT replaced chorismate synthase exons 2, 3 and 4.
  • This construct was used for knockout of the wild type chorismate synthase gene.
  • the sequence of the construct was verified by PCR. Following transfection into T. gondii (deficient in HXGPRT) and selection in medium containing 25 ⁇ g/ml mycophenolic acid and 50 ⁇ g/ml Xanthi ⁇ e, successful transfection was confirmed by PCR of the chorismate synthase/dhfr HXGPRT junction and sequence the product.
  • Aromatic compound deficient medium with 10% AlbuMax® as a serum substitute was prepared. Final concentrations of aromatic compounds in the supplemented medium are 0.1M phenylalanine, tyrosine, tryptophan, PABA, 2,3 dihydroxybenzoate and p- hydoxybenzoate. DNA was extracted from those replicate cultures of parasite clones that grew only in the presence of aromatic compound
  • RECTIFIED SHEET (RULE 91) supplementation.
  • PCR primers were designed to confirm presence of t he knockout construct and demonstrated that homologous recombination occurred resulting in replacement of exons 2-4 with the dhfr HXGPRT sequence.
  • the knockout parasite was passaged in aromatic compound supplemented medium. Whether this selection clearly demonstrates inability of the knockout parasite to grow in aromatic compound deficient medium, but ability to grow in aromatic compound sufficient medium using a uracil assay.
  • aro deficient strains of bacteria have been used as vaccines precisely because they are nonpersistent.
  • the resulting construct was transfected intc the chorismate synthase knockout parasite described above. Proof that the construct produces mRNA for chorismate synthase is with northern and western blotting. The lack of ability of the knockout and the ability of the complemented parasite to grow in folate and other aromatic compound deficient medium indicates a functional construct. This knockout organism is suitable for use as an attenuated vaccine strain.
  • Example 42 T. eondii Chorismate Svnthase cDNA Sequence in a
  • DNA Vaccine Vector Elicits Antibodies.
  • Antibody production is detected on Western blot and in other immunoassay systems. This is an example of
  • RECTIFIED SHEET (RULE 91) a recombinant vaccine and a system to produce antibody reagents useful in diagnostic tests without the need to produce recombinant protein.
  • Protein Construct is Made and Used in Parasite Survival Assavs to Test Antimicrobial Agents.
  • a T. gondii chorismate synthase-green fluorescent protein DNA construct elicits a fusion (reporter) protein detectable with conventional immunofluorescence microscopy and deconvolution microscopy (FIG. 15) and other techniques known in the art to detect fluorescence.
  • This construct accelerates the growth rate of the parasite and is useful for measuring effects of antimicrobial agents on the parasite by detecting relative amounts of the green fluorescent reporter protein. This is useful for testing antimicrobial agents.
  • Example 44 Chorismate Svnthase and Life Cvcle. Chorismate synthase is differentially located and expressed in different life cycle stages indicating that it can be an antimicrobial agent target in, and reagent to detect, specific stages of the parasite.
  • Immunostaining This is performed as is standard in the art with tachyzoites, converting organisms, intestinal life cycle stages using specimens produced in vivo and in vitro.
  • chorismate synthase was concentrated in a small area contiguous to the nucleus in the area of the plastid (FIG. 16A).
  • T. gondii chorismate synthase In other life cycle stages it was distributed diffusely throughout the cytoplasm (FIG. 16B, C). It was most abundant in bradyzoites and macrogametes. A C-terminal green fluorescent protein reporter alters its localization in tachyzoites (FIG. 15). Unique stage-associated expression and subcellular localization of T. gondii chorismate synthase is identified in tachyzoites, bradyzoites and in the stages of the parasite in the cat intestine including macrogametes, microgametes but not schizonts. Stage-associated expression of T. gondii chorismate synthase (FIG.
  • 16A-C is an example of the expression and differential subcellular localization of this protein. This stage-associated expression demonstrates
  • Chorismate Svnthase Chorismate synthase was expressed in E. coli using a pGEX expression system (Pharmacia). Briefly, PCR was used to amplify the coding region and to introduce
  • the pUC18 plasmid containing the insert was digested with EcoRl and BamHI and following purification by electrophoresis, the insert was eluted from an agarose gel and then cloned into pGEX-2T. DNA sequencing confirmed that the nucleotide sequence was in frame and that no PCR errors had been introduced. Following transformation the protein was expressed in BL21. To optimize expression and to test protein for enzymatic activity, expression is increased using BL21 Codon Plus
  • RECTIFIED SHEET (RULE 91) allo s the production of recombinant proteins which have either C- terminal or N-terminal protein C tags.
  • the protein C tag is used to purify protein using an antibody that binds the protein C tag only in the presence of Ca "2 . Calcium chelation then provides a gentle means of elutin ⁇ the purified protein from the antibody.
  • the Pichia Expression System (Invitrogen) is also used. This system offers advantages of bacterial systems such as high-level expression and ability to use large scale cultures. In addition, it offers certain advantages of eukaryotic expression systems that facilitate protein processing, folding and post-translational modifications.
  • the system makes use of the powerful alcohol oxidase promoter (AOX1) to aid high expression levels. Tranformants are selected by Zeocin resistance and inframe C-terminal His tag allows purification by metal-chelating resins and detection through an anti-; ⁇ vc antibody. This produces additional recombinant chorismate synthase protein, in order to produce polyclonal antisera to chorismate synthase. Antisera is employed to determine subcellular localization of T.
  • AOX1 alcohol oxidase promoter
  • gondii chorismate synthase gondii chorismate synthase.
  • Recombinant protein also is used for later crystallography studies and for high throughput screens. Production of anti-chorismate svnthase antibody
  • a commercial source for immunization of rabbits is also suitable. Preimmune sera and sera containing polyclonal antibody, is obtained 7 days after the second immunization.
  • anti-peptide antibodies to specific regions of the protein also is produced in rabbits by a commercial laboratory (Alpha Diagnostic, San Antonio, TX).
  • RECTIFIED SHEET (RULE 91) they recognize native folded protein, and of the anti-peptide antibodies is that when they recognize native protein, peptide epitopes are defined.
  • Development of enzvme assav for high throughput assays To measure chorismate synthesis, a phosphate release assay is performed using a malachite green dye and the product is detected spectrophotometrically with a plate reader. This is adapted for large scale screening for high throughput screens. This assay is performed anaerobically (i.e., in a nitrogen environment) using polyethylene bags. Substrate EPSP will be synthesized as described previously.
  • Example 46 Antibody to Recombinant Chorismate Svnthase is
  • Example 47 Isocitrate Lvase. T. gondii isocitrate lyase activity was demonstrated and has the same uses as chorismate synthase activity, and other enzymes, e.g., it is useful for high throughput screens of T. gondii, Isocitrate lyase enzyme activity (FIG. 17C, D) and its inhibition by 3 nitropropionic acid (3NPA) (FIG. 17D) was identified.
  • Use of a knockout microorganism complemented with the parasite ICL gene is another example of a method useful for high throughput screens to idnetify an inhibitor of ICL. sequences Antisense gene sequences to interfere with parasite growth or survival. This is a representative example of inhibition of this enzyme in this pathway.
  • This enzvme is potentially useful in development of antimicrobial agents, diagnostic reagents or vaccines.
  • Example 4 ⁇ The T. gondii Isocitrate Lvase Binding Pocket anri Active Site Form a Basis for Rational Antimicrohint Agent Development .
  • FIG. 20, box were identified and have absolute homologies with all other isocitrate lyases and not with other partially homologous enzymes such as CPEP mutase.
  • a yeast with a mutation in a base encoding a lysine (K) only in this area produced an inactive isocitrate lyase. This observation is useful for development of antimicrobial agents as described for other sequences herein.
  • Example 49 T. gondii Isocitrate Lvase Genomic Sequence is Useful for Vaccine Development
  • a genomic ICL sequence is in FIG. 21 and is useful for vaccine development as described for thoer genomic sequences.
  • T. gondii isocitrate lyase stage associated protein is present in bradyzoites and is useful as described herein for producing diagnostic reagents, identifying anti-microbial agents and for vaccines.
  • T. gondii, isocitrate lyase stage-associated protein is present in bradyzoites (FIG. 22) and there is stage associated mRNA expression and protein (FIG. 23). This observation is useful in the same manner as other examples of mRNA and protein described herein in for diagnostic reagents, antimicrobial agent and vaccines.
  • Example 51 Additional Inhibitors of Apicomplexan Isocitrate Lvase are Based on Compounds that Inhibit Isocitrate Lvases of Other Organisms. Additional inhibitors of apicomplexan isocitrate lyases are identified and designed. They are used as lead compounds for designing new inhibitors as described herein and this is useful for development of
  • RECTIFIED SHEET (RULE 91) diagnostic reagents, antimicrobial agents and vaccines as described for other enzymes herein.
  • Example 52 Genetic, E ⁇ zvmatic and Functional Evidence and Active Inhibitors of Apicomplexan Acetyl coA Carboxylases Such as Clodinafop Provide a Basis for Development of
  • FIG. 24 presents enzymatic, genetic and functional evidence of a wheat-like T. gondii acetyl coA carboxylases. Partial gene sequences were identified for T. gondii, Plasmodia and Cryptosporidia acetyl coA carboxylases. Inhibitors of T. gondii acetyl coA carboxylase inhibited parasite survival in vitro. This is useful for diagnostic reagents, antimicrobial agents and vaccines as described for other sequences herein.
  • Example 53 Synergism of Antimicrobial Agents that Inhibit Apicomplexan Lipid Synthesis.
  • Aryloxyphenoxy-propionate Herbicides Targeting Acetyl-CoA Carboxylase.
  • plastid-like organeUes in apicomplexan parasites provide new targets for antimicrobial agents.
  • Aryloxyphenoxypropionates known inhibitors of the plastid Acetyl-Co Carboxylase (ACC) of grasses, inhibit Toxoplasma gondii ACC by 50% at a concentration of 20 ⁇ M Clodinafop, the most effective of the herbicides
  • RECTIFIED SHEET (RULE 91) tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 ⁇ M and is not toxic to the host cell even at much higher concentrations.
  • Infected fibroblasts treated with Clodinafop for two days show a substantial reduction in the number of T. gondii cells at 10 ⁇ M and almost complete removal of parasites at 100 ⁇ M. Longer treatments are even more effective. Fragments of genes encoding biotin carboxylase domain of multi-domain ACCs were cloned. One ACC from T.
  • ACC1 clusters with the putative Cyclotella cryptica chloroplast ACC and Plasmodium ACC
  • ACC2 clusters with Cryptosporidium ACC, probably the cytoplasmic form.
  • genes encoding enzymes for fatty acid synthesis, including various subunits of ACC except one, are present in the nuclear genome and their protein products are imported and function in plastids.
  • ACC catalyzing the first committed step of de novo fatty acid biosynthesis, is a known selective target of aryloxyphenoxypropionate ("fops”) and cyclohexanedione (“dims”) herbicides in sensitive species.
  • the molecular mechanism of inhibition/resistance of the enzyme is not know but there is a strong correlation between the enzyme structure and its origin.
  • the high molecular weight multi-domain ACC that is localized in plastids of grasses is extremely sensitive to these herbicides.
  • All of the multi-subunit chloroplast enzymes of dicot plants and bacteria as well as other multi-domain cytosolic ACCs, such as those from man, chicken, rat and yeast, are resistant.
  • ACC activity is conveniently measured in vitro by the incorporation of the carboxyl group from bicarbonate into an acid-stable form using crude protein extracts after Sephadex G50 filtration.
  • Anti-parasite activity and toxicity for four fops and one representative dim were determined. Pyrimethamine and sulfadiazine, antimicrobial agents which are known inhibitors of folate synthesis, were included as positive control. The combination of candidates inhibited uracil incorporation by T. gondii by more than 95% without toxicity for fibroblasts. Consistent with the data for ACC activity in vitro, the inhibitory activity of the fops and the dim on T. gondii growth in fibroblasts was in the same concentration range. Clodinafop was even more active in this assay than in the enzyme assay, giving 70% inhibition at 10 ⁇ M. With regard to toxicity, fops are mildly toxic at the highest concentration, 400 ⁇ M.
  • Clodinafop was assessed by light microscopy. Micrographs showed infected fibroblasts treated with Clodinafop at 10 and 100 ⁇ M compared with control infected cells without herbicide and uninfected cells. There is substantial reduction of the number of Toxoplasma tachyzoites at 10 ⁇ M and almost complete removal at 100 ⁇ M. The effectiveness of Clodinafop at 10 ⁇ M is greatly enhanced by a 4-day treatment with one change of medium and inhibitor after 2 days. In this experiment, cultures were incubated for 2 more days without the inhibitor. No parasite cells were found in infected fibroblasts treated in this way.
  • esters which are converted to free acids by plant esterases.
  • the true inhibitor of ACC is the free acid.
  • Clodinafop (Topik) have no effort on T gondii ACC activity in crude extracts and were relatively inactive in the uracil incorporation assay except for Topik that was as active as the free acid, suggesting significant level of hydrolysis of this ester. In general, in this assay fop esters are not more effective than free acids.
  • parvum were determined from PCR-cloned gene fragments.
  • a phylogenetic analysis was performed based on amino acid sequence comparisons of the two candidate ACCs from T. gondii with those of other BC domains.
  • Three apicomplexan sequences (T. gondii, P. knowlesii, and P. falciparum) cluster together with Cyclotella cryptica ACC, an enzyme thought to be in the diatom chloroplast. This isozyme, called ACC1 in T. gondii, is likely the plastid form. This assignment awaits cloning and sequencing of the 5'-terminal portion of the cDNA, where a sequence encoding a signal/transit peptide ought to be found.
  • the other ACC clusters with the ACC of C. parvum. These two are probably cytosolic forms.
  • the partial genomic sequences revealed differences in intron number and location before ACC1 and ACC2 of T. gondii, and the three ACC genes from the other apicomplexa.
  • One of the other PCR products encoded a BC domain similar to that of pyruvate carboxylases.
  • Deduced amino acid sequences encoded by the remaining two PCR products were similar to the BC domains of rat ACC and prokaryotic-type biotin-dependent carboyxlases, respectively. These fragments were assumed to encode the host mouse ACC and a carboxylase from a bacterial comaminat.
  • Protein gels blotted with streptavidin revealed pyruvate caroxylases (130 kDa) in addition to ACC (240 kDa), but no bacterial-type biotin carboxyl carrier protein (20 kDa) or biotinylated subunit of propionyl-CoA carboxylase (70 kDa).
  • the target for sensitivity is likely in to a region encompassing the ⁇ domain of carboxytransferase, based on experiments using yeast gene replacement strains, in which chimeric genes encoding wheat ACCs replace the yeast ACC1 gene. Such strains are herbicide-sensitive if they contain a gene encoding sensitive ACC. Availability of the genes encoding T. gondii ACCs may clarify which of the isozymes is targeted to the plastid and whether one or both of them are sensitive to fops (the majority of the activity in the protein extracts was inhibited). Inhibition of T.
  • isocitrate lyase and malate synthase key enzymes unique to the glyoxylate cycle. Enzymes of the glyoxylate cycle were detected in protein extracts of T. gondii. Polyclonal antibodies to cotton malate syntase and isocitrase lyase were used to detect heterologous apicomplexan proteins by western blot analysis. A protein band of approximately 64 kD was detected using antibodies to cotton isocitrate lyase and malate synthase in lysates of T. gondii tachyzoites. Isocitrate lyase was also sought, and found in western blots of T.
  • gondii bradyzoites Antibody to cotton isocitrate lyase also was used for immunohistochemistry to study bradyzoites within cysts in brain tissue. Isocitrate lyase was identified in bradyzoites. Whether there was stage related expression of isocitrate lyase in T. gondii was studied by using smaller number of parasites in semiquantitative western blots.
  • isocitrate lyase is regulated at a number of different steps.
  • E. coli there is an ace operon comprised of ace B, A, and K encoding malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase phosphatase, respectively.
  • ace operon is under the transcriptional control of two genes, the iclR gene and fadR.
  • the fadR is also involved in the regulation of fatty acid degradation. It has been suggested that these genes encode repressor proteins, which act independently or in concert, to repress the ace operon.
  • functionally related isoenzymes with distinct roles in the metabolic pathways needed for growth under different minimal conditions also have been described.
  • different iso forms of the isocitrate lyase enzyme related to the age of the organism have been identified.
  • isocitrate lyase plays a time-limited role with decline in isocitrate lyase activity in the senescent endosperms.
  • NPA Nitropropionic acid
  • This sequence when translated had an open reading frame (ORF) of 857 base pairs, had over 30% homology with isocitrate lyases from varied organisms (range: 29 - 53% identities; 43 - 67% positives).
  • ORF open reading frame
  • a T. gondii RH strain genomic Lambda DASH II library (Stratagene) was then screened using TgESTzz53c08.rl as a probe, and a genomic clone was obtained and sequenced (GenBank accession number to be assigned). The binding pocket and catalytic site that are absolutely conserved among other isocitrate lyases was identified in the T. gondii gene.
  • the deduced amino acid sequence also showed partial homology with putative carboxyphosphonoenolpyruvate phosphonomutase from E. coli and Salmonella species. Two regions of isocitrate lyase have been implicated as part of the active site. The motif KKCGHM(L) is conserved in all isocitrate lyases, and it is proposed that the cysteine is a critical active site residue. The absolute identity of the T.
  • mice 12-15 mice per assay were infected intraperitoneally with T. gondii tachyzoites (Rh strain, 2xl0 7 per mouse) 2 days prior to assay. Tachyzoites were extracted with a peritoneal lavage using 5 ml of sterile saline per mouse.
  • the PTg strain of 7. gondii was cultured as tachyzoites or tachyzoites induced to become bradyzoites, as described 8 .
  • T. gondii tachyzoites or bradyzoites were obtained at indicated time points from host cells by scraping the monolayer, passing the infected cells through a syringe with a 25g needle twice to disrupt them, and then organisms were counted and centrifuged at 2000 rpm for 10 minutes at 4°C to pellet the parasites. The supernatant was discarded and the pellet was suspended in SDS PAGE loading buffer (with 2 mercaptoethanol) at a concentration of l l O 5 parasites per ⁇ l and boiled for 10 minutes. Unless otherwise,
  • RNA messenger RNA, isolated on oligo dT solid phase matrix columns and reverse transcribed using a random priming method, was used for semi-quantitative PCR analysis of tachyzoite surface antigen (SAG) l, bradyzoite cytosolic antigen (BAG) 1-5, and isocitrate lyase (ICL), relative to beta tubulin (TUB).
  • SAG tachyzoite surface antigen
  • BAG bradyzoite cytosolic antigen
  • ICL isocitrate lyase
  • the primer sets were as follows: SAG1 (5'-CGC?
  • the BAG 1-5 primers flank an intron serving as a control for genomic DNA contamination, yielding a PCR product of 784 bp.
  • cDNA from T. gondii tachyzoites of the RH strain and induced bradyzoites from the Me49 strain were used as templates.
  • elution buffer 100 M KCl. 20% glycerol, 7 mM 2-mer
  • a discontinuous method described by Ko and McFadden 17 was employed with minor modifications to measure the ability of isocitrate lyase to convert isocitrate to succinate and glyoxylate.
  • This method utilizes the colorimetric reaction between the phenylhydrozone of glyoxylate and ferricyanide.
  • Reaction mixtures (92 mM MOPS, . 5 mM MgCI,, 1 mM DTT, 1% phenylhydrazine, 4.4 mM isocitrate, in 0.5 ml with fractionated parasite lysate) were incubated in a 37 "C water bath for a determined amount of time. After incubation, enzymatic reactions were stopped with concentrated HCl, mixed with 25% (w/v) potassium ferricyanide, and then measured in a spectrophotometer at 520 nm. Culture of parasites in vitro with inhibitors
  • the purified phage DNA was digested with Notl; xhol or EcoRI enzymes, run on a l agarose gel, transferred onto a nylon membrane probed with the biotinylated probe (above). A -4kb band which was identified with the probe and was cloned into pBluescript KS * and sequenced. DNA sequencing and sequence analysis
  • DNA sequencing was performed using an automated DNA sequencer. This sequence was compared to peptide sequence databases at The National Center for Biotechnology Information (NCBI) using the program TBlastX or BlastP (for derived open reading frames). Gene construction using the sequence obtained was also performed utilizing the Baylor College of Medicine program. Primers for sequencing were made at Integrated DNA Technology. Sequence analysis was carried out by software programs MacNector, ClustalX and MACH Box.
  • HFF Human foreskin fibroblasts
  • IMDM Iscoves' Modified Dulbecoes Medium
  • Confluent cells are removed by trypsinization and washed in IMDM They are used in a growth phase for toxicity assays or when 100% confluent for parasite inhibition assays b) Tachyzoites: Tachyzoites of the RH and pTg strains of T.
  • Bradyzoites are used at concentrations of 2x10 ' ', 2xl0 4 and 2xl0 5 per ml in parasite growth inhibition assays pH shock is also used to retain organisms fn bradyzoite stage when such pH does not interfere with inhibitor activity d)
  • Inhibitors Inhibitor compounds are tested over a range of concentrations for toxicity against mammalian cells by assessing their ability to prevent cell growth as measured by tritiated thymidme uptake and inspection of the monolayer using microscopic evaluation A range of concentrations that are non-toxic in this assay are tested for their ability to prevent the growth of 7 " .
  • Oilier inhibitors of this pathwav include 4 amm - ⁇ -hew noic acid and 4-aminofluoropentano ⁇ c acid which provide additional corroborative evidence that this pathway is present ii) Glyoxylate Cycle
  • the inhibitor of isocitrate lyase is 3 nitropropionic acid (ranging from 0 005 to 5 mg/ml m vitro) iii) Alternative Oxidase T.
  • gondii bradyzoites use unique alternative oxidases.
  • Alternative oxidase is necessary and sufficient for bradyzoite survival.
  • Methods to characterize plant alternative oxidases are described (Hill, 1976, Kumar and Soil, 1992, Lambers, 1994; Li et al, 1996, Mclntosh, 1994)
  • cell lines that lack functional mitochondria are used. These cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent of their effects on the host cell mitochondria
  • Two cell lines, a human fibroblast cell line ( 143B/206) lacking mitochondrial DNA. and the parental strain ( 143 B) which poses functional mitochondria are used These cell lines have been demonstrated to support the growth of 7 " .
  • product rescue assay is performed with PABA g) Assays for synergy in vitro. This is an assay in which ⁇ 50% inhibitory concentrations of two antimicrobial agents are added alone and together to determine whether there is an additive, synergistic or inhibitory interaction All other aspects of this assay are as described herein.
  • the target for PCR amplification is the 35 fold repetitive B 1 gene of T gondii and the amplification was performed using primers previously 5 reported
  • a competitive internal standard is generated using a linker primer and the original B 1 primers
  • Competitive PCR was performed by spiking individual reactions (containing equal amounts of genomic DNA) with a dilution of the internal standard Since this internal control contains the same primer template sequences, it competes 0 with the B 1 gene of 71 gondii for primer binding and amplification
  • the sensitivity of the PCR reaction in each sample can be monitored Following competitive PCR, the PCR products are distinguished by size and the amount of products generated by the target and internal standard can be compared on a gel
  • the amount of competitor DNA yielding equal amounts of products gives the initial amount of target gene 5 6.
  • Tachyzoites and bradyzoites (McLeod et al. 1984, 1988; Denton et al., 1996a, b), or their mitochondria and plastids are isolated as previously described. Equivalent numbers of tachyzoites and bradyzoites are separately solubilized in 2x sample buffer and boiled for 5 minutes Samples are electrophoresed through a 10 percent SDS- polyacry ⁇ mide gel. Proteins are transferred to a nitrocellulose membrane at 4°C, 32V with 25mM Tris and 192mM glycine, 20% v/v methanol, pH 8.3. Blots are blocked in PBS (pH 7.2) containing 5% powered milk and 0.
  • Antibodies to various enzymes e.g. , soybean GSAT, barley GSAT, synechococcus GSAT, plant and/or trypanosome alternative oxidase, cotton isocitrate lyase, cotton malate synthase, soybean malate synthase, petunia EPSP synthase were used to determine whether homologous enzymes are present in 71 gondii tachyzoites, bradyzoites mitochondrial and plastid enriched preparations
  • Antibodies used include monoclonal antibodies to I panosoina bi nceii and Voo Doo Lily (Chaudhuri el al 1996) altei nativ oxidase and polyclonal antibody to hi uceii alternati e oxidase
  • the hybridizations with antibodies to plant and related protozoan alternative oxidases demonstrated the relatedness of 71 gondii metabolic pathways to those of plants and other non-Apicomplexan proto
  • Genomic Sequence Genomic clones are identified and sequenced in the same manner as described above for cDNA except a genomic library is used Analysis of unique promoter regions also provide novel targets
  • J bi adv zoite are meme ilicd as desu ibed ci ein Tiie pai asites aie Iv sed in 50mM HEPES (pH7 4) containing 20% glycerol, 0 25% Triton X- 100 and proteinase inhibitors (5n ⁇ M PMSF, 5FM E64, 1 FM pepstatin, 0 2mM 1 , 10-phenanthroiine)
  • This method has proven successful for measurement of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD- and NADH-linked isocitrate dehydrogenases and succinic dehydrogenase
  • GSAT activity is measured by the method of Jahn et al., (1991), which uses GSA as substrate. GSA is synthesized according to methods of Gough et al. (1989) Heat-inactivated (60°C, 10') lysates are employed as non-enzymatic controls. ALA is quantified following chromatographic separation (Weinstein and
  • ALA Synthase To determine whether parasites contain ALA synthase, an activity also present in mammalian host cell mitochondria, cell fractions from purified parasites are assayed. (Weinstein and Beale, 19S5) ALA produced from added glycine and succinyl CoA is quantified as for GSAT.
  • 5-Enolpyruvylsh ⁇ kimate 3-phosphate synthase is assayed in forward and reverse directions as described previously (Mousdale and Coggins 1984) 5 Shikimate NADP oxidoreductase (shikimate dehydrogenase), shikimate kinase,
  • 3-Dehvdroqu ⁇ nase are assayed Assay mixtures contained in a total volume of 1 ml 100 mM potassium phosphate (pH 7 0) and 0 8 mM ammonium 3-dehydroqu ⁇ nate 3-Dehydroqu ⁇ nate synthase is assayed by coupling for forward reaction to the 3-dehydroqu ⁇ nase reaction, assay mixtures ' contained in a total volume of I ml 10 mM potassium phosphate (pH 7 0), 50 ⁇ M ⁇ ⁇ D 0 1 mM CoCN 0 5 nkat partiallv -punlled Lsc ei idua t ali DHQase and (to initiate assay) 0 4 mM DAHP
  • the DAHP is prepared from E. coli strain AB2847A and DHQase from E. coli strain ATCC 14948
  • Assay of DAHP synthase is by a modification of the method of Sprinson et al Assay mixtures contained in a total volume of 0.5 ml. 50 mM 1 ,3-bis [tris(hvdroxymethyl)-methylamino] propane-HCI (pH 7.4), 1 mM erythrose 4- phosphate, 2 mM phosphoenolpyruvate and 1 mM CoCl 2 .
  • reaction is initiated by the addition of a 50 to 100 ⁇ l sample containing DAHP synthase and terminated after 10 min at 37°C by 100 ⁇ l 25% (w/v) trichloroacetic acid The mixture was chilled for 1 h and centrifuged to remove precipitated protein A 200 ⁇ l aliquot of the supernatant was mixed with 100 ⁇ l 0.2 M Nal0 4 in 9 M
  • Another representative assay is an assay for chorismate lyase which is as desc ⁇ bed by Nichols and Green 1992 Chorismate lyase assays are earned out in a v olume of 0 5 ml containing 50 mM
  • chorismate lyase activity is defined as the amount of enzyme required to produce 1 nmol of 4-hydroxybenzoate in 30 min at 37°C.
  • Assays for 4-aminobenzoate and 4-amino-4-deoxychorismate are performed as described previously.”
  • Enzyme Assays The 5-enolpyruvyIshikimate-3-phosphate (EPSP) synthase assay entailed monitoring the generation of EPSP using HPLC. Reaction components were separated using a Hypersil H3APS2 HPLC column (Hichrom Limited, Reading. UK) and a NaH2P04 elution gradient (50-400 mM).
  • UV spectra (200-320 nm) of the column eluate were collected to identify eluants.
  • Shikimate-3-phosphate and 5-enolpyruvylshikimate-3-phosphate synthesized enzymatically and purified to at least 95% purity as described (12), eluted after 3.9 and 6.S min, respectively; phosphoenolpyruvate did not interfere with the EPSP detection and eluted after 5.3 min.
  • the peaks at 21 5 nm were integrated; the EPSP produced was quantified using a standard curve of authentic EPSP.
  • Parasite extracts were produced at 4 ; C by suspension of pure tachyzoites in extraction buffer (50 mM
  • the resulting supernatant was diluted 6-fold with extraction buffer and loaded onto a ResourceQ column (1 ml, Pharmacia) equilibrated with extraction buffer.
  • the bound protein was eluted in a single step using extraction buffer containing 500 mM KCl.
  • the eluted material was used for enzyme assay.
  • the assay mix contained 1 mM phosphoenolpyruvate, 1 mM SP and 50 mM HEPES, pH 7.5.
  • gene knockout organisms are generated by the method of Roos et al., 1996. Specifically, the strategy for creating mutants is with homologous recombination and to generate a targeted gene knock-out a sequential positive/negative selection procedure is used (Roos et al., 1996). In this procedure positive and negative selectable markers are both introduced adjacent to, but not within the cloned and suitably mutated locus.
  • This construct is transfected as a circular plasmid Positive selection is applied to yield a single-site homologous recombinant that is distinguished from non-homologous recombinants by molecular screening In the resulting 'pseudodiploid.' mutant and wild-type alleles flank selectable marker and other vector sequences
  • parasites are removed from positive selection, which permits recombination betw een the duplicated loci This event appears to occur at a frequency of 2 x 10 ' per cell generation
  • These recombinants are isolated with negative selection Next, they are screened to distinguish those that have recombined in a manner that deletes the mutant locus and yields a wild-type revertant from those that deleted the wild-type gene to leave a perfect allelic replacement.
  • An example is a knockout created for the chorismate synthase gene It also can be made more general to include knockout of other genes for attenuated vaccines such as EPSP synthase and alternative oxidase
  • the parasite with the gene of interest to be knocked out is grown ("manufactured") in vitro in presence of product, but when used /// ⁇ ⁇ o (he needed pi oduct is not pr esent
  • the pai asite functions as an attenuated v accine s des_.nbed below under vaccines ⁇ specific example follows Specilicaliv the strategy of product inhibition discussed above is also useful for growing gene knockout parasites (which lack a key gene for their survival) in vitro by providing the essential product and thus bypassing the need for the gene during /// wtr ⁇ propagation of the parasite
  • Such gene knockouts cultivated /// vitro in this manner are useful attenuated organisms that are used as attenuated vaccines
  • the chorismate synthase cDNA clones are used as hybridization probes for recovering genomic clones from a 71 gondii genomic cosmid library. Coding regions are mapped onto the genomic clones using the cDNA clones as a guide Appropriate sections are sequenced to verify the gene location. Ultimately, full genomic sequences are obtained. Enough of the genomic clones are sequenced to develop a strategy for generating a putative null allele.
  • chorismate synthase -null and chorismate synthase-neutral plasmids are transfected into HXGPRT-negative strains of 7 gondii (strains RH(EP) ⁇ XGPRT [a ME49 derivative] Numerous independent clones are selected for survival on mv cophenolic acid to select for insertion of the pla>mtd These strains are scr eened bv Souther n analysis designed to detect the pi csence oi both the nor ma! and modified copies of the chorismate synthase gene and for tandem location of the two copies (with the vector HXGPRT gene between).
  • HXGPRT clones with verified pseudodiploid structure of the- chorismate : synthase alleles are selected for loss of HXGPRT using 6-thioxanthine (the "run" part of the protocol). Numerous clones are selected. If the loss of HXGPRT is based upon random homologous exchange between the two chorismate synthase pseudodiploid alleles, theoretically half of the events should lead to excision of the modified chorismate synthase allele along with the HXGPRT, leaving the original wild type allele in the chromosome.
  • the other half should excise the wiJ type allele, leaving the modified allele in the chromosome.
  • the medium is supplemented with chorismate at the concentration determined to best rescue cells from inhibitor toxicity.
  • the purpose of the supplementation is to enhance the chances of recovering chorismate synthase-null strains.
  • the genomic structure of the selected clones is examined by Southern analysis to confirm loss of the vector HXGPRT and of one copy of the chorismate synthase and to identify the remaining allele of chorismate synlhase
  • the ratio of mutant to w ild type is tabulated
  • the chorismate synthase-neutral allele is intended as a positive control to confirm that either allele (wild type or mutant) can be lost in this procedure. If chorismate synthase- neutral strains can be recovered but chorismate synthase-null strains cannot, the conclusion is that the chorismate synthase gene is essential for growth.
  • chorismate synthase-null strains they are subjected to further phenotypic analysis, first, using immunoblotting of electrophoretically separated cell extracts to confirm absence of chorismate synthase protein, then, determining if these strains sho hypersensitivity to inhibitors of the alternative oxidase or to any of the other potential inhibitors. Sensitivity to chorismate. synthase inhibitors is analyzed to. determine the relative specificity of inhibition. If chorismate synthase is the sole target of the inhibitors, then the null mutants should be insensitive to further inhibition. Sensitivity analysis is conducted /// vitro as described herein.
  • strains show alterations in expression of the alternative oxidase or in any stage-specific antigens is of interest. These analyses are conducted by immunoblotting of electrophoretically separated cell extracts. /// vivo analysis using a mouse model is conducted to determine if these strains are infective and what stages of parasites can be detected following infection.
  • stage switch Genetically altered 71 gondii organisms are used to infect C3H/HeJ mice by the intraperitoneal route Mortality is monitored and brains examined for cysts at 30 days post infection Knockouts with bradyzoite reporter genes are useful to determine whether these enzymes influence stage switch Stage switch also is characterized by quantitating relative amounts of parasite mRNA present in each stage of parasite using Northern blotting, isolation of mRNA and RT-PCR using a competitive inhibitor, and enzyme assay.
  • flanking sequences Functions as either a positive or negative selection marker (using 6-thio.xanthine or mycophenolic acid, respectively) in suitable host strains.
  • RH Wild-type host strain RH (highly pathogenic in mice).
  • Antibody to soybean, barley and synechococcus GSAT are polyclonal antibodies with preimmune sera the control for the barley and synechococcus antibodies.
  • T. gondii contains a glyoxylate cycle that allows growth using lipids as a carbon source, thus the lipid mobilization pathway of T. gondii is similar to the pathway of plants (Tolbert, 19S0)
  • a similar approach using polyclonal antibodies to isocitrate lyase and to malate synthase and preimmune control sera are used
  • Antibodies to acetohydroxy acid synthase are used.
  • Antibodies to starch synthesis, branching (Q) enzymes and UDP glucose starch glycosyl transferase are used.
  • the E. coli AroC mutant which lacks chorismate synthase was obtained from the E. coli genetic stock center.
  • AroC bacteria is made competent to fake up DNA by transformation with CaCI 2 treatment.
  • the cells are electroporated to take up DNA.
  • the presence of the plasmid is demonstrated in this system by growth on media which contains ampicillin, as the plasmid contains an ampicillin resistance gene. Complementation is confirmed by demonstrating growth on media lacking the product catalyzed by (i.e., chorismate).
  • this transformation complementation is used with the T. gondii cDNA librar, system or a construct which contains some or all of the chorismate synthase gene to transform the AroC mutant. Functional enzyme is then demonstrated.
  • mice As an alternative approach if complementation studies are unsuccessful and the monoclonal antibodies to a plant protein are not cross reactive, purified plant protein is used to immunize mice to raise polyclonal antibodies to each enzyme Where necessary, antibodies to the pertinent enzymes are generated in mice, ND4 outbred mice are immunized with 20 ⁇ g of enzyme emulsified in Titermax complete adjuvant injected intramuscularly into their gluteal muscle Two weeks later mice are immunized with a further 20 ⁇ g of enzyme emulsified in Titermax. After a further 2 weeks mice receive a further boost of enzyme alone in PBS by the intraperitoneal route. Mice are bled and the serum tested for specificity by the standard Western blotting technique. K. Immunofluorescence
  • Antibodies used to identify enzymes in the Apicomplexan metabolic pathways disclosed here are used for immunofluorescence studies. Examples are demonstration of alternative oxidase in 71 gondii by immunofluorescence assay (IFA). T. gondii alternative oxidase is immunolocalized to mitochondria. L. ELISAs
  • ELISAs are used for documenting the presence and quantitating the amounts of alternative oxidase
  • a useful reporter pi otem for a chimeric construct is ⁇ glucoronidase. expressed in / • .. coli undo: comi ol of ihe 355 promoter of cauliflower mosaic vu us
  • the glucoronidase alone without the transit sequence is expressed in parallel
  • the transit peptide construct is found in the plastid
  • the control glucoronidase is found in the cytoplasm
  • Antibodies to the chorismate synthase protein are also used to detect the presence of the product of the gene (with the transit sequence) in the plastid and the product of a construct (in which the transit sequence is not present) in the cytoplasm only Further mutations and deletions are made which identify the minimal transit sequence using the same techniques as described above for the entire peptide
  • Antisense, ⁇ bozyme or intracellular antibodies directed against the transit sequence nucleic acid or translated protein are useful as medicines
  • the amino acid ofnucleic acid which encodes the transit sequences are the bases for development of
  • Tissue distribution is characterized using radiolabeled inhibitor administered to mice with its disposition to tissues measured Compounds are modified to improve oral absorption and tissue distribution O.
  • Infections are established and influence of an inhibitor or combination of inhibitoi s on outcomes arc as outlined below Infections Intections with l ⁇ xoplas a gondii Pneumocy stis cat mil. M ⁇ cobacle ⁇ nun luhei Lidosis ⁇ l ⁇ ⁇ ohu ie ⁇ nun cn ium intracellular and C ⁇ ⁇ piospo ⁇ idiiim parv um ar e estabhshed alone and together using an immunosuppressed rodent model Endpoints in these infections are
  • Tissue Parasite Burden This is determined by quantitating brain and eye cyst numbers Inflammatory Response This is noted in histopathologic preparations Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM and atovaquone, NPMG and pyrimethamine, NPMG and SHAM P. Testing of Antimicrobial Compounds
  • the testing in murine models includes standard Thompson tests Testing of antimicrobial agents for efficacy and safety in primate models for malaria is performed Dosages are selected based on safety information available from data bases of information concerning herbicides and the literature Measurements of serum and tissue levels of antimicrobial compounds are performed using assays which detect inhibitor concentrations and concentrations of their metabol.tes Representative assays are high performance liquid chromatographv and assaying i.>>ues for percentage of l adiolabeled compounds admmistei cd using liquid scintilla; , ⁇ and other assav s also are used R. Carcinogenicity and Tcratogcnicitv
  • Standard assays to evaluate carcinogenicity include administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity. Observation includes general physical examination, autopsy and histopathologic studies which detect any teratogenic or carcinogenic effects of medicines. S. Constructs to Measure Parasitemia
  • Tests and reagents include DNA constructs (Tine et al., 1996) with the appropriate gene or portions of the gene alone or together. with adjuvants.
  • Representative adjuvants include ISCOMS, nonionicsurfactant vesicles, cytokine genes in the constructs and other commonly used adjuvants.
  • Native and recombinant proteins also are used in studies of vaccines Protection is measured using immunologic //; vitro assays, and by assessing survival and reduction of parasitemia and tissue parasite burden and prevention of congenital infection (McLeod ei al., 19SS) U. Preparation of Diagnostic Test Reagents and Diagnostic Tests:
  • test useful in an outpatient setting is based on conjugation of a monoclonal antibody to human red blood cells with antibody to peptides or proteins.
  • the red cells are cross linked if the antibody to the parasite component interacts with the parasite component and agglutinates the red cells in the blood sample ELISA and PCR can be utilized with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates outpatient and field use.
  • Antisense oligonucleotides are short synthetic stretches of DNA and RNA designed to block the action of the specific genes described above, for example, chorismate sv nthase of T. gondii or P. falciparum, by binding to their RNA transcript They turn off the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRNA and no translation into protein Antisense reagents have been found to be active against neoplasms, inflammatory disease of the bo el (Crohn's Disease) and HI V m early trials Antisense oligonucleotides directed against the nucleic acid> w hich encode the essential parasite metabolic process described hei ein ai e efleeiiv e medicines to tr eat these infections Antisense o gonucleotides also ai e dn ecicd , ⁇ g...
  • Antisense w ill not contain cytosine nucleotides followed by guanines as this generates extreme immune responses (Roush, 1997).
  • Antisense oligonucleotides with sequence for thymidine kinase also is used for regulatable gene therapy.
  • Ribozvmes and Other Toxic Compounds Ribozymes are RNA enzymes (Mack, McLeod. 1996) and they and toxic compounds such as ricins (Mahal et al, 1997) are conjugated to antisense oligonucleotides (see V, DNA), or intracellular antibodies (see X, for proteins), and these constructs destroy the enzyme.
  • X. Intracellular Antibodies Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes or portions of them (e.g., anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, as described under vaccines.
  • the herbicide inhibitors comprise lead compounds and are modified as is standard For example, side chain modifications or substitutions of groups are made to make more active inhibitors Their mode of action and structure as well as the enzyme and substrate structures are useful in designing related compounds which better abrogate the function of the enzymes. Examples of such substrate or active site targeting are described above
  • Nativ e or recombinant protein is used in enzymatic assays and //; vitro assays described abov e are used to test activity of the designed newly synthesized compounds Subsequently, they will be tested in animals Z. Trials to Demonstrate EfTicacv for H uman Disease
  • phosphate synthase (3-phospl ⁇ osl ⁇ ikimatc-l- carboxyvinyltransferase): An enzyme which functions in chorismate synthesis
  • 3-NPA An inhibitor of isocitrate lyase in the glyoxylate pathway and also of succinate dehydrogenase.
  • 3-oxtaprenyI-4-hydroxybenzoatc carboxylyase An enzyme which functions in ubiquinone synthesis.
  • 4-hydroxybenzoate octaprenyltransferase An enzyme which functions in ubiquinone synthesis.
  • 8-OH-qui ⁇ oline An inhibitor of the alternative oxidase.
  • Abscissic Acid Metabolism in Plants A 15-carbon sequiterpenoid synthesized partly in plastids by the mevalonic acid pathway Abscissic acid protects plants against stress and is.a marker of the plant's maturation and activation of transcription, and causes dormancy Inhibits protein synthesis and leads to specific activation and deactivation of genes
  • Acctohydro ⁇ acid synthase Enzyme w hich catalyzes production of acetohydroxy acids (the branched chain amino acids valine. leucine and isoleucine in plants)
  • Alternative ovidase An enzyme important in the alternative pathway of respiration
  • Altered gene includes knockouts.
  • Amide The R portion of the amino group has an amino group connected to a carbonyl carbon. Glutamine and asparagine are amides. Important for nitrogen transport and storage.
  • Amylopcctin A branched starch of plants Also found in T gondii bradyzoites.
  • Amyloplast Storage granule for starch in plants. Derived from chloroplasts. Amylosc: An unbranched starch of plants
  • Anabolism Formation of large molecules such as starch, cellulose, proteins, fats and nucleic acids from small molecules. Requires input of energy.
  • Anthranilate phosporibolsyltransfcrase An enzyme which functions in tryptophan synthesis
  • Anthranilate svnthasc component I An enzyme which functions in tryptophan synthesis
  • Anthranilate synthase component II An enzyme which functions in tryptophan synthesis
  • Antimicrobial agent A chemical, for e xample a protein or antisense nucleic acid which effectiv ely inhibits or kills a pathogenic microbe
  • a pathogenic microbe There are examples (Schwab ei al . 1994 Strath e/ al . 1993 , Beckers _ ⁇ . al 1 995 Blais et al.. 1993 , Fichcra et al . 1995, Pfefferkorn &. Borotz, 1994, Pfefferkorn et al. , 1992, Pukivittaykamee et a/. , 1994)
  • Apicomplcx The common feature of Apicomplexan parasites including a conoid and rhoptry organeUes and micronemes at the apical end of the parasite.
  • Apicomplexan parasite A microorganism that belongs to the Apicomplexan group of parasites.
  • Apicomplexan parasites include Toxoplasma gondii, Plasmodium, Cryptosporidia and Emieria Aromatic acid aminotransfcrase (aromatic transaminase): An enzyme which functions in tyrosine synthesis.
  • glutamate and glutamine synthesis Involve glutamine synthase and glutamate synthetase and are plastid associated in plants Glutamine synthase in plants is inhibited by the herbicide glufosinate (2 am ⁇ no-4-fhydroxymethyiphosphinyl) butanoic acid Glutamine synthase also is present in animals
  • ATP-phosphofructokinasc (ATP-PFK) May exert control ov er glycolytic pathway because a step when hexoses phosphate cannot also be used to form sucrose or starch Nearly all animals lack PPi-PFK with plant-like substrate specificity (i c PPi, not ATP)
  • i c PPi, not ATP plant-like substrate specificity
  • Bradyzoite The slowly replicating life cycle stage of the Apicomplexan parasite Toxoplasma gondii This stage is responsible for latent and recrudescent infection due to this parasite
  • the morphologic features which characterize this parasite stage are electron dense rhopt ⁇ es and amylopectin granules Bradyzoites contain a plastid organeUe as do other life cycle stages of this parasite
  • This parasite stage also has specific antigens w hich other life cycle stages do not have, including bradyzoite surface antigen 4 and bradv zoite antigen 5 (lactate dehydrogenase), w hich is an intracellular and cyst matrix antigen Bradyzoites exist together in a structure called a cyst which has a cyst wall and matrix Cysts contain a few to thousands of bradyzoites
  • the cyst containing bradyzoites is a major means of transmission of the organism Toxoplasma gondii hen it
  • Branching or Q enzyme Forms branches in amylopectins between C6 of the main chain and C I of the branch chain. 5 Catabolism: Degradation or breakdown of large molecules to small molecules, often releasing energy.
  • Calmodulin is a calcium binding protein (Robson et al, 1993)
  • Chloroplast A DNA-containing multilamellar organeUe of plants and algae associated with metabolic pathways important for photosynthesis and other energy production. Chloroplasts utilize proteins encoded in their ow n DNA and also proteins encoded by nuclear DNA
  • Chorismate The product of the action of the enzyme EPSP synthase on shikimate 15
  • Cliorisniate lvase An enzyme responsible for the conversion of chorismate to
  • Cliorisniate s nthase An enzyme responsible for the conversion of 3-phospho 5- — '-' enolpyruvy! shikimate to chorismate
  • Chorismate The product of the action of the enzvme EPSP sv nthase on shikimate
  • Competitive inhibitors Structures sufficiently similar to the substrate that they compete for the active site of the enzyme. Addition of more natural substrate overcomes effect of the inhibitor.
  • Components includes nucleic acids, proteins, peptides, enzymes, peptide targeting sequences, transit peptides, carbohydrates, starch, lipids, hormones, for example those listed in Table 1 and other constituents of metabolic pathways or products derived from these components.
  • Cryptosporidiosis The disease due to the Apicomplexan parasite Cryptosporidium parvum. It causes self-limited diarrhea or no symptoms in immunologically normal individuals. In individuals who have immunocompromising illnesses, such as the acquired immune deficiency syndrome, Cryptosporidiosis causes life-threatening, persistent, copious, watery diarrhea.
  • Cryptosporidium parvum Cryptosporidium parvum is an Apicomplexan parasite which causes cryptosporidiosis
  • Cyanidc-inscnsilivc, non-liciuc "alternative" oxidase is a metabolic activity that is found in most eukaryotic plants and algae and is absent from multicellular animals
  • the alternative oxidase is a single polypeptide enzvme that lacks heme and can serve as the lei minal election acceptor t ⁇ support respiratory growth of /-.
  • Cyclohexadienyl dehydrogenase An enzyme which functions in tyrosine synthesis
  • Cytochrome oxidase An enzyme utilized in the conventional pathway of energy generation
  • Dehydroquinate dehydratase An enzyme which functions in chorismate synthesis
  • Dcoxyribonuclcascs Enzymes which are hydrolases which hydrolyze DNA (phosphate esters)
  • Eimeria maxima and Eimeria tenella cause eimeriosis in chickens
  • Eimeria A group of Apicomplexan parasites which cause gastrointestinal disease in agnculturallv important animals including poultry and cattle These economically important parasites mclude l.imei ia tenella /. " maxima and L hovis Endosymbiont: An organism which is taken up by another organism and then lives within it
  • Enzyme A protein which catalyzes (makes more rapid) the conversion of a substrate into a product
  • Enzymes are catalysts which speed reaction rates generally by factors between 10 s and I O 20 They may require ion or protein cofactors Control is by products and environmental changes There are more than 5000 enzymes in living systems. Enzymes are named with common or trivial names, and the sufFix-ase which characterizes the substrate acted upon (e g , cytochrome oxidase removes an electron from a cytochrome) Sequential series of steps in a metabolic pathway Enzymes that govern the steps in a metabolic pathway are sometimes arranged so that a kind of assembly-line production process occurs
  • EPSP synthase An enzyme important in the conversion of shikimate to chorismate EST: Expressed sequence tag, a short, single pass cDNA sequence generated from randomly selected library clones Eukaryote: Microorganism or phylogenetically higher organism, the cells of which have a nucleus with a limiting membrane
  • Fragment refers to a sequence of nucleic acids or aminoacids, where a fragment is sufficient to function as a component of or product derived from an Apicomplexan as defined herein
  • Gabaculine An inhibitor of the enzyme GSAT in the heme synthesis pathway Gene: Nucleotide sequence which encodes an amino acid sequence or another nucleotide sequence
  • GSAT in the heme synthesis pathway Gene
  • Plant hormones which promote plant growth, overcome dormancy, stimulate GI to S transition and shorten S phase of cell cycle, increase hydrolysis of starch and sucrose into glucose and fructose They are derivatives of ent-gibberellane skeleton synthesized from a 2acetyl CoA to mevalonic acid to isopenternyl pyrophosphate to 4 isopentenyl pyrophosphate to geranylgeranyl pyrophosphate to copalylpyrophosphate to kaurene to kaurenol to keaurenal to kaurenoic acid to GA
  • Glyoxylate pathway The pathway important for lipid degradation which takes acetyl CoA and converts it to CoA-SH through the conversion of isocitrate to C4 acids including succinate. This pathway utilizes isocitrate lyase and also converts glyoxylate to malate, a reaction catalyzed by the enzyme malate synthase.
  • the glyoxysome or Glyoxylate pathway which is cytoplasmic in certain algae involves isocitrate lyase and malate synthase to metabolize lipids and provide C4 acids.
  • a metabolic distinction between autotrophic eukaryotes and heterotrophs is the presence of a glyoxylate cycle
  • This cycle employs two enzymes, isocitrate lyase and malate synthase, to bypass the two decarboxvlation steps of the TCA cycle and enables the utilization of carbon stored in fatty acids for growth.
  • the enzymes of the glyoxylate cycle are compartmentalized within a unique single-membrane-bound organeUe, the glyoxysome In certain algae, the cycle is entirely cytoplasmic In plants, these enzvmes are most abundant during germination and senescence In animals, the glyoxylate cycle enzymes have been described as being present onlv during starvation
  • Glyoxysome An organeUe which in some instances contains enzymes important in the glyoxylate cvcle
  • GSAT Glutamate-I semialdehyde aminotransferase is the enzyme important in heme synthesis for the conversion of glutamate semialdehyde to ALA ( ⁇ -aminolevuhnic acid)
  • Heme synthesis pathway A metabolic pathway important for generation of heme, porphyrins and other iron sulfated proteins used in mitochondria in the conventional pathway of energy generation This pathway occurs in plant chloroplasts and uses the nuclear encoded enzyme GSAT
  • a metabolic distinction between plants and animals occurs in the heme biosynthesis pathway
  • photosynthetic organisms including plants, algae and cyanobacte ⁇ a, E coli
  • Imidazolinones Inhibitor of acetohydroxy acid synthase (an enzyme involved in the synthesis of branched chain amino acids, a pathway not in or rarely present in animals, Indolc-3-glyceroI phosphate synthase (antliranilatcisomerasc), (indolcglyccrol phosphate synthase): An enzyme which functions in tryptophan synthesis.
  • Inhibitor A compound which abrogates the effect of another compound.
  • Isocitrate lyase An enzyme which functions in glyoxylate cycle.
  • Isomcrascs Enzymes which rearrange atoms of a molecule to form a structural isomer.
  • Isoprenoid Metabolism in Plants Terpenes are isoprenoids that lack oxygen and are pure hydrocarbons; 5 carbon units with some of the general properties of lipids.
  • Latency The dormant form of the parasitic infection.
  • One example is with
  • Toxoplasma gondii in which the infection is not active and the parasite is primarily within cysts in the bradyzoite phase of the life cycle.
  • Another example is the hypnozoite phase of ' Plasntodium falciparum.
  • Ligases or Synthetascs Enzymes which join two molecules coupled with hydrolysis of ATP or other nucleoside triphosphate.
  • Lipascs Enzymes which are hydrolases which hydrolyze fats (esters) Lipid and terpene synthesis associated with plant plastids. Also see fatty acid synthesis and terpenes.
  • Lysnscs Enzymes which form double bonds by elimination of a chemical group.
  • Malaria Disease due to pathogenic Plasmodia. Examples are Plasntodium falciparum, Plasmodium virax, Plasmodium ovale, Plasmodium malaria, in humans and Plasmodium knowlesii in monkeys
  • Malate synthase An enzyme which functions in glyoxylate cycle
  • Metabolic pathways Both anabolism and catabolism consist of metabolic pathways in which an initial Compound A is conv erted to another B, then B is converted to C, C to D and so on until a final product is formed
  • glucose is the initial compound
  • CO CO
  • H 0
  • metabolic pathways There are approximately 50 distinct reactions in respiration but other metabolic pathways have fevvci reactions
  • metabolic pathways and “biochemical pathways” are used interchangeably.
  • Metabolism Chemical reactions that make life possible. Thousands of such reactions occur constantly in each cell.
  • Microbes Organisms which are visible only with use of a microscope Some cause disease (are pathogenic).
  • Microbicidal An agent (e.g., an antibiotic or antimicrobial compound) which kills microbes.
  • Mitochondria An organeUe responsible for the generation of energy.
  • Multilamellar An adjective which refers to the multiple membranes within an organeUe.
  • Noncompctitivc inhibitors Combine with enzymes at sites other than active site
  • NPMG An inhibitor of EPSP synthase in the shikimate pathway
  • Nucleic Acid Deoxyribonucleic acid and ribonucleic acid molecules are constructed of a sugar phosphate backbone and nitrogen bases, important in the encoding, transcription and synthesis of proteins
  • Oocyst A life cycle stage of a parasite, e.g . Toxoplasma gondii that contains sporozoites 7 gondii sporozoites and oocvsts form only in the cat intestine This foim of the par asite is able to persist in nature in warm, moist soil for up to a year and is highh infectious Spoiulation occui s sev eral d ⁇ s after excretion of oocvsts by members of the cat family (e.g., domestic cats or wild cats such as lions or tigers)
  • members of the cat family e.g., domestic cats or wild cats such as lions or tigers
  • OrganeUe A structure within a cell. Examples are plastids, mitochondria, rhoptries, dense granules and micronemes
  • Oxidorcductases Oxidorcductases (oxidases, reductases, dehydrogenases): Remove and add electrons or electrons and hydrogen. Oxidases transfer electrons or hydrogen to 0 2 only.
  • PABA Paraminobenzoic acid
  • Parasite .An organism which lives in or on a host for a period of time during at least one life-cycle stage.
  • Phagcmid Plasmid packaged within a filamentous phage particle.
  • Phosphoribosyl anthranilate isomerase An enzyme which functions in tryptophan synthesis
  • Plant-like Present in algae and higher plants, but not or only rarely, or in unusual circumstances in animals
  • Plasmodium falciparum One species of Plasmodium which causes substantial human disease
  • Plasmodium knowlesii A species of Plasmodium which causes malaria in monkeys
  • Plastid A uliilamellar organeUe of plants, algae and Apicomplexan parasites which contains its ow n DNA separate from nuclear DNA Plastids have been described in studies of Apicomplexan parasites which used electron micrographs (Siddall, 1992, Williamson e. ⁇ //., 1994, Wilson et ai, 1991 , Wilson et al., 1994, Wilson et al , 1996,
  • Polymerascs Enzymes which link subunits (monomers) into a polymer such as RNA or DNA PPi phosphofructokinase Type I : An enzyme present in plants that functions in glycolysis and in a number of organisms regulates glycolysis In plants and protozoans
  • PPi not ATP (as in animals) is utilized to synthesize Fru-1-6P 2 from Fru 6P. Activity is not stimulated in protozoa by Fru-2-6-P 2 (Peng & Mansour, 1992, Denton et al.,
  • Prephenate dehydrogenase An enzyme which functions in tyrosine synthesis
  • Prosthetic group Smaller organic nonprotein portion of an enzyme essential for catalytic activ ity Flavin is an example
  • Proteinascs Enzymes which are hydrolases which hydrolyze proteins (peptide bonds)
  • PS II Important alternative means for producing energy within chloroplasts and apparently also described as being present in Apicomplexans
  • Pyrimethamine An inhibitor of the conversion of folate to folinic acid and thus an inhibitor of nucleic acids production effective against Toxoplasma gondii
  • Recrudescence Reactivation of the parasite Toxoplasma gondii from its latent phase
  • Respiration Major catabo c process that releases energy in all cells It involves breakdown of sugars to C0 2 and H 2 0
  • Ribonuclcases Enzymes which are hydrolases which hydrolyze RNA (phosphate esters) Salicylic Acid Metabolism in Plants: Salicylic acid is a plant hormone which promotes activity of cyanide resistant respiration SHAM: An inhibitor of the alternative oxidase
  • Shikimate dehydrogenase An enzyme which functions in chorismate synthesis
  • Shikimate kinase (shikimate 3-phosphotransferase) An enzyme which functions in chorismate synthesis
  • Shikimate pathway A pathway that involves the conversion of shikimate to chorismate and subsequently the production of folate, aromatic amino acids, and ubiquinone This pathway contains enzymes which lead to production of folic acid, ubiquinone, and aromatic amino acids Folate, ubiquinone, and aromatic amino acids are products derived from this pathway in plants There is sequential use of products of these pathways as reactants in subsequent enzymatically catalyzed reactions For example, ubiquinone is an essential coenzyme for both conventional and alternative respiration There are examples in plants, bacteria and fungi (Bornemann et al , 1995 Marzabadi et al , 1996, Ozenberger el al .
  • Stage specific A characteristic of the parasite which is expressed or present only in a single life cycle stage or in some but not all life cycle stages.
  • Starch Degradation in Plants 3 enzymes: ⁇ amylase (attack 1, 4 bonds of amylopectin (to maltose) and amylase (to dextrin). Many activated by Ca++. Located in chloroplasts. ⁇ amylase hydrolyzes starch to maltose; starch phosphorylase degrades starch beginning at nonreducing end. (Starch + H2P04 *•* glucose + - Phosphate) Only partially degrades amy lopectin debranching enzymes hydroxy 1.6 branch linkage in amylopectin.
  • Starch synthesis is dependent on starch synthase ' and branching Q enzymes Mutations in genes encoding these enzymes lead to diminished production of starch In addition, amylopectin synthesis predominates in plant mutants without UDP-glucose-starch glycosyl transferase whereas wild type plants with this enzyme make predominantly amylose and a smaller amount of amylopecti In the mutant UDP-glucose-starch glycosyl transferase appears to be transcriptionally regulated Amino acid motifs that target proteins to plant plastid organeUes have been identified in UDP-glucose starch glycosyl transferase, as have other motifs that determine transit into plastids and mitochondria and these have been used to target the transported proteins in plants.
  • Branching or Q enzymes form branches in amylopectins between C6 of the main chain and CI of the branch chain. There are examples in plants (Abel et al., 1996; Van der Leif et al., 1991 ; Van der Steege et ⁇ /., 1992).
  • Starch synthase catalyzes reaction. ADPG + small amylose (n-glucose units) ⁇ larger amylose n+l glucose units + ADP and is activated by K+. Thus, sugars not starch accumulate in plants deficient in K+.
  • Starch Major storage carbohydrate of plants, used for energy regeneration
  • the two types are amylose and amylopectin
  • Amylopectin is highly branched with the branches occurring between C-6 of a glucose in the main chain and C- 1 of the first glucose in the branch chain (- 1 ,6 bonds)
  • Amyloses are smaller and have fewer branches
  • Amylopectin becomes purple or blue when stained with iodine- potassium-iodine solution.
  • Amylopectin exhibits a purple red color.
  • Control of starch formation is by K- and a light activated sucrose phosphate synthase enzyme, invertase enzymes and the allosteric effect of fructose 2.
  • ADPG phiphosphate adenosine diphosphoglucose
  • Substrate The protein on which an enzyme acts that leads to the generation of a product.
  • an additive effect is when the effect of the compounds used together is simply the sum of the effects of each inhibitory compound used alone
  • Tachyzoite The rapidly replicating form of the parasite Toxoplasma gondii
  • Thcileria An Apicomplexan parasite infecting cattle.
  • Toxoplasma gondii A 3-5 micron, obligate, intracellular, protozoan parasite which is an Apicomplexan.
  • Toxoplasmosis Disease due to Toxoplasma gondii.
  • Transit (translocation) peptide sequence Amino acid sequence which results in transit into or out of an organeUe.
  • Triazinc An inhibitor of PS II complex.
  • Tryptophan svnthasc alpha subunit An enzyme which functions in tryptophan synthesis.
  • Tryptophan synthase beta subunit An enzy me which functions in tryptophan synthesis T ⁇ pc 1 PPi phosiihofi'uctokiiiasc is another enzyme present in plants and there is different substrate utilization In phosphofructokmases of animals UDP glucose starch glycosyl transferase: An enzyme involved in production of amylose in plants. The absence of this enzyme leads to starch formation as amylopectin rather than amylose.
  • McLeod R D Mack and C Brown (1991) Exper Parasitol 72 109-121 McLeod R, D Mack, R Foss, K Boyer, S Withers, S Levin and J Hubbel ( 1992) Antimicrob Ag Chemother 36 1040-104S
  • Rhoads David M., Mclntosh, Lee (1992) The Plant Cell 4: 1 131-1 132.
  • Wilson R J ⁇ 1 G • drier M J . Fcag:-- J FI W illiamson D I I ( l °9 l ) PawisiloI Todav 7 134- 130 Wilson, R.J.M., Williamson, D.H., and Preiser, P. ( 1994) Infectious Agents and Disease 3.29-37.

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