EP1071959A2 - Procede d'identification de principes actifs chimiques et principes actifs destines a l'inhibition de la voie de biosynthese du 1-desoxy-d-xylulose-5-phosphate - Google Patents

Procede d'identification de principes actifs chimiques et principes actifs destines a l'inhibition de la voie de biosynthese du 1-desoxy-d-xylulose-5-phosphate

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Publication number
EP1071959A2
EP1071959A2 EP99920648A EP99920648A EP1071959A2 EP 1071959 A2 EP1071959 A2 EP 1071959A2 EP 99920648 A EP99920648 A EP 99920648A EP 99920648 A EP99920648 A EP 99920648A EP 1071959 A2 EP1071959 A2 EP 1071959A2
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EP
European Patent Office
Prior art keywords
phosphate
xylulose
deoxy
protein
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99920648A
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German (de)
English (en)
Inventor
Jomaa Hassan
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Bioagency AG
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Bioagency AG
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Filing date
Publication date
Priority claimed from DE19825585A external-priority patent/DE19825585A1/de
Priority claimed from DE19828097A external-priority patent/DE19828097A1/de
Priority claimed from DE19831639A external-priority patent/DE19831639C1/de
Priority claimed from DE19831637A external-priority patent/DE19831637A1/de
Application filed by Bioagency AG filed Critical Bioagency AG
Publication of EP1071959A2 publication Critical patent/EP1071959A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the invention relates to a method for identifying active substances which are suitable for the treatment of parasitic diseases caused by single or multi-cell parasites. / Fields of application of the invention are medicine and the pharmaceutical industry.
  • the invention further relates to proteins, as well as parts of proteins, furthermore DNA sequences which encode these proteins or parts of these proteins, de use of these DNA sequences, these proteins or their parts for the identification of substances with action against single or multicellular parasites , as well as the active ingredients identified in this way and their use for the manufacture of medicinal products.
  • parasites includes unicellular and multicellular parasites including helminths and anthropods. These cause infectious diseases in humans and animals.
  • unicellular parasites are protozoa.
  • Isoprenoids are molecules that are formed from individual isoprene units (isopentenyl diphosphate) and perform important functions in the cell. These include sterols, ubiquinones and other molecules that are important for the parasite household.
  • the previous approach was based on a model that was established m mushrooms and m suction cells. The subunit isopentenyl diphosphate is formed in mushrooms and suction cells based on the condensation of three molecules of acetyl-CoA to HMG-CoA.
  • HMG-CoA is then converted from the HMG-CoA reductase to mevalonate, which is then converted to isopentenyl diphosphate with mevalonate phosphate as an intermediate (see FIG. 7).
  • Inhibitors of HMG CoA reductase such as lovastatin, simvastatin and pravastatin have been used to inhibit the growth of the parasites. Malaria was able to achieve in vitro inhibition using very high doses of lovastatin and simvastatin, but the inhibition in vivo failed.
  • the inhibition of the DOXP metabolic pathway described above, in particular the enzymes DOXP synthase and DOXP reductoisomerase by the techniques known to the person skilled in the art, is suitable for the prevention and treatment of infections caused by single and multicellular parasites in humans and animal. Since this metabolic pathway is not available in humans, it is an excellent target for targeted chemotherapy of parasites.
  • the enzymes deoxyxylulose-5-phosphate synthase and de-soxyxylulose-5-phosphate reductoisomerase are particularly suitable as targets for chemotherapy.
  • the inhibition of the enzyme deoxyxylulose-5-phosphate reductoisomerase from malaria was particularly low in side effects and suitable, since humans have neither substrates and their precursors, nor the product of the enzyme nor the enzyme itself.
  • the present invention relates to methods for finding active substances which inhibit the DOXP metabolic pathway, and these active substances for the production of medicaments for the therapy and prophylaxis of infectious diseases caused by single or multi-cell parasites.
  • the object of the invention is to provide a new method for identifying active substances for the therapy of parasitic diseases in humans and animals. Another task is to develop a method for locating a drug that selectively kills the pathogen and has few side effects.
  • the present invention further relates to enzymes which are involved in this metabolic pathway and to parts of these enzymes. These enzymes are suitable proteins for carrying out the method according to the invention for identifying active substances.
  • the present invention further relates to DNA sequences which encode these enzymes or parts of these enzymes.
  • the present invention relates to a method and antibodies for identifying the enzymes or their parts and the production of the enzymes or their parts using recombinant technology.
  • the invention further relates to the use of these enzymes or their sections, or the use of the DNA sequences which encode these enzymes, or sections of these enzymes for the identification of substances with action against single or multicellular pathogens.
  • the invention further relates to active substances which are found with the aid of the enzymes according to the invention.
  • 1b shows the nucleotide sequence of the gene which encodes the 1-deoxy-D-xylulose-5-phosphate synthase from Plasmodium falciparum
  • FIG. 2a the nucleotide sequence of the gene encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Plasmodium falciparum and the corresponding amino acid sequence
  • FIG. 2b the nucleotide sequence of the gene, the 1-deoxy-D - Coded xylulose-5-phosphate synthase from Plasmodium falciparum and the corresponding amino acid sequence
  • Fig. 3a shows the amino acid sequence of the protein 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Plasmodium falciparum
  • 3b shows the amino acid sequence of the protein 1-deoxy-D-xylulose-5-phosphate synthase from the parasite Plasmodium falciparum
  • FIG. 4a shows a section of the nucleotide sequence according to FIG. 1b
  • FIG. 4b shows a section of the nucleotide sequence with the corresponding ammosaur sequence according to FIG. 2b
  • FIG. 4c shows a section of the amino acid sequence according to FIG. 3b
  • Fig. 5 In vivo data for the parasitemia values after 4 days of therapy with three doses of the substances in each case: formyl, which corresponds to 3- (N-formyl-N-hydroxylammo) propyl phosphonic acid monosodium salt, and Acetyl, which corresponds to 3- (N-acetyl-N-hydroxylammo) propylphosphonic acid monosodium salt,
  • Fig. ⁇ a the inhibition of the growth of P. falciparum after addition of 3- (N-formyl-N-hydroxylammo) propylphosphonic acid monosodium salt (open circles) and 3- (N-acetyl-N-hydroxylammo) propyl- phosphonic acid monosodium salt (closed circles) for the HB3 strain
  • 6b the inhibition of the growth of P. falciparum after addition of 3- (N-formyl-N-hydroxylamino) propylphosphonic acid monosodium salt (open circles) and 3- (N-acetyl-N-hydroxylamino) propyl- phosphonic acid monosodium salt (closed circles) for strain A2
  • FIG. 6c the inhibition of the growth of P.
  • the coding genes of the enzymes DOXP synthase and DOXP reductoisomerase were detected by means of genetic methods ( Figures la, lb, 2a, 2b). After enrichment by the polymerase chain reaction from the genome of P. falciparum, these genes were cloned in bacterial plasmids and their nucleotide sequence determined. The sequence data showed a high homology of these genes with the corresponding genes from algae, plants and bacteria. The very high homologies showed that the three genes encode the enzymes DOXP synthase and DOXP reductoisomerase from P. falciparum.
  • the enzymes were purified as recombinant proteins and used for activity studies in cell-free systems.
  • the activity of the DOXP synthase was measured by converting glyceraldehyde-3-phosphate and pyruvate to l-deoxy-D-xylulose-5-phosphate.
  • the activity of the DOXP reductoisomerase was determined by converting l-deoxy-D-xylulose-5-phosphate to 2-C-methyl D-erythr ⁇ tol-4-phosphate measured in the presence of NADPH.
  • the change in the NADPH concentration is measured via a parameter variation. This process is known to the person skilled in the art.
  • the enzymes can be defined via the DNA sequence coding them (FIGS. 1a, 1b, 2a, 2b) and the ammosaic sequence derived from them (FIGS. 3a and 3b).
  • the enzymes of the individual parasites can differ from parasite to parasite.
  • Such variations of the amino acids are usually ammosaurus exchanges.
  • deletions, insertions and additions of amino acids to the overall sequence can also result.
  • the enzymes according to the invention can be glycosylated or non-glycosylated, regardless of the size and type, depending on the cell and cell type in which they are expressed.
  • the enzymes or parts of these enzymes according to the invention are produced by expression of the DNA according to the invention in suitable expression systems, for example in bacteria, in particular in E. ccn, as a prokaryotic expression system or in a eukaryotic expression system, in particular COS cells or Dictyosteliu discoideum.
  • a) are the product of a prokaryotic or eukaryotic expression of an exogenous DNA
  • b) are encoded by a sequence in Figures la
  • lb, 2a and 2b c) are encoded by DNA sequences which correspond to those in Figures la, lb, 2a and DNA sequences shown in 2b or fragments of these DNA sequences (see, for example, FIGS. 4a and 4b) hybridize in the DNA region which encodes the mature protein
  • d) are encoded by DNA sequences which have no degeneration of the genetic code would hybridize to the sequences defined in b) to c) and encode a polypeptide with the same amino acid sequence.
  • Enzymes are preferred which are encoded by the nucleotides from FIGS. 1a, 1b, 2a and 2b or by DNA sequences which, owing to the degeneration of the genetic code, would encode a polypeptide with the same amino acid sequence.
  • the two enzymes according to the invention (sequence in FIGS. 3a and 3b) can be regarded as new prototypes of specific proteins of single-cell and multi-cell parasites, in particular of the single-cell parasites.
  • An object of this invention are nucleic acid sequences which encode the enzymes and are selected from the group
  • the invention further relates to enzymes from any parasites which condense essentially pyruvate and glyceraldehyde-3-phosphate to give l-deoxy-D-xylulose-5-phosphate (DOXP synthase) and l-deoxy-D-xylulose. Convert 5-phosphate to 2-C-methyl-D-erythritol-4-phosphate (DOXP reductoisomerase).
  • enzymes which are analogous to the enzymes from malaria parasites, can be obtained by screening a cDNA library or genomic library of the corresponding parasite using a hybridization sample which contains sequences encoding malaria parasite methods, or by comparing the sequences the DNA and protein sequence for enzymes from malaria parasites with other parasite enzymes. Enzymes according to the invention can be obtained in large quantities in a reproducible manner with the aid of the nucleic acids.
  • the nucleic acid is integrated into suitable expression vectors by methods known to the person skilled in the art.
  • Such an expression vector preferably contains a regulatable / inducible promoter.
  • Suitable host cells are prokaryotic cells, such as, for example, E. coli, and eukaryotic cells, in particular yeasts (for example Saccharomyces cervisiae, Schizosaccharomyces pombe, Pichia pastoris), insect cells (for example cell lines from Drosophila melanogaster such as S2 cells, Spodoptera) frugiperda, Trichoplusia ni), vertebrate cell lines, especially teratocarcinoma cell lines such as CHO or COS cells, and plant cell lines.
  • prokaryotic cells such as, for example, E. coli
  • eukaryotic cells in particular yeasts (for example Saccharomyces cervisiae, Schizosaccharomyces pombe, Pichia pastoris), insect cells (for example cell lines from Drosophila melanogaster such as S2 cells, Spodoptera) frugiperda, Trichoplusia ni), vertebrate cell lines, especially tera
  • the enzymes according to the invention can also be expressed in transgenic plants and animals (e.g. mice, sheep, goats, pigs, guinea pigs).
  • the expression system is advantageously designed by techniques known to those skilled in the art so that the enzymes produced are excreted in the milk of the animals or can be obtained from easily obtained plant parts (fruits, leaves, flowers, shoots and roots).
  • Systems which are derived from papilloma viruses for example SV40), retroviruses, Sindbis viruses, cytomegaloviruses and vaccinia viruses are particularly suitable as expression vectors for vertebrate cells.
  • papilloma viruses for example SV40
  • retroviruses for example SV40
  • Sindbis viruses for example SV40
  • cytomegaloviruses for example SV40
  • vaccinia viruses are particularly suitable as expression vectors for vertebrate cells.
  • insect cells the baculovirus system, for plant cells systems based on the Ti plasmid from Agrobacterium tumefaciens and the bombardment of the cells with nucleic acid-coated particles.
  • the expression of the enzymes according to the invention in slime molds such as Dictyostelium discoideum, Polysphondylium pallidum and Physarum polycephalum is of particular importance since their cells can be cultured inexpensively in large quantities on simple media.
  • the use of Dictyostelium discoideum has the further advantage that this organism uses similar codons for the respective amino acids as Plasmodium falciparum and thereby a particularly effective production of the enzymes according to the invention is achieved.
  • inducible promoters e.g. due to lack of food
  • expression vectors for Dictyostelium discoideum are known. This can further increase the yield of recombinant enzyme.
  • Particularly suitable for the expression of the enzymes according to the invention are those host cells and organisms which have no intrinsic enzymes and which condense pyruvate and glyceraldehyde-3-phosphate to give 1-deoxy-D-xyluiose-5-phosphate (DOXP synthase) and 1 - Convert deoxy-D-xylulose-5-phosphate to 2-C-methyl-D-erythritol-4-phosphate (DOXP reductoisomerase).
  • DOE synthase 1-deoxy-D-xyluiose-5-phosphate
  • DOXP reductoisomerase 2-C-methyl-D-erythritol-4-phosphate
  • the enzymes according to the invention are advantageously expressed in eukaryotic cells when post-translational modifications and a native folding of the polypeptide chain are to be achieved.
  • introns are eliminated by splicing the DNA and the enzymes are produced in the polypeptide sequence characteristic of the parasites. Sequences coding introns can also be removed from the DNA sequences to be expanded by recombinant DNA technology or inserted experimentally.
  • the protein can be isolated from the host cell or the cell supernatant of the host cell by methods known to those skilled in the art. It may also be necessary to reactivate the enzymes in vitro.
  • the enzymes according to the invention or partial sequences of the enzymes can be expressed as fusion proteins with different pepti ⁇ chains. Oligo-histidm sequences and sequences derived from glutathione-S-transferase, thioredoxm or calomodm-binding peptides are particularly suitable for this purpose. Fusions with thioredoxm-derived sequences are particularly suitable for prokaryotic expression, since this increases the solubility of the recombinant enzymes.
  • the enzymes according to the invention or partial sequences of the enzymes can be expressed as fusion proteins with peptide chains known to the person skilled in the art that the recombinant enzymes are transported in the extracellular environment or in certain compartments of the host cells. the. This enables both the purification and the investigation of the biological activity of the enzymes to be facilitated.
  • the enzymes according to the invention can be obtained under standardized conditions by techniques known to the person skilled in the art by in vitro translation. Suitable systems are rabbit reticulocyte and wheat germ extracts. In vitro transcribed mRNA can also be translated into Xenopus oocytes.
  • Oligo- and polypeptides can be produced by chemical synthesis, the sequences of which are derived from the peptide sequence of the enzymes according to the invention. With a suitable choice of the sequences, such peptides have properties which are characteristic of the complete enzymes according to the invention. Such peptides can be produced in large quantities and are particularly suitable for Studies on the kinetics of enzyme activity, the regulation of enzyme activity, the three-dimensional structure of the enzymes, the inhibition of enzyme activity by different chemicals and pharmaceuticals and the binding geometry and binding affinity of different ligands.
  • a DNA with the nucleotides from the sequences shown in FIGS. 1a, 1b, 2a and 2b or a fragment according to FIGS. 4a and 4b is preferably used.
  • the invention further relates to processes for obtaining the enzymes which are involved in the DOXP pathway, in particular the enzymes DOXP synthase and DOXP reductoisomerase by isolation from the parasites.
  • the enzymes are isolated from parasite extracts using chromatographic, electrophoretic and other methods known to the person skilled in the art.
  • the enzymes are determined by measuring the respective enzymatic activity or reactivity with appropriate antibodies.
  • the detection of transformed, transfected or transduced host cells which produce the enzymes recombinantly, and the purification of the protein are preferably carried out using antibodies which bind to these enzymes.
  • Such antibodies can be obtained with the aid of the enzymes according to the invention or parts of the enzymes as an antigen or immunogen in a simple manner by known processes.
  • homologous or cross-reacting proteins of other parasites can be detected, for example, by Western blotting analyzes.
  • Another object of this invention are methods for determining the enzymatic activity of the DOXP enzymes, in particular the enzymes DOXP synthase and DOXP reductoisomerase. This can be determined according to the known instructions (Sprenger et al. PNAS, 94 (1997) 12857-62 and Kuzuyama et al. Tetrahedron Letters 39 (1998) 4509-12).
  • Such derivatives can, for example, be modified in single or more amino acids by substitution, deletion or addition.
  • the derivatization can take place, for example, via site-directed mutagenesis (site-specific mutagenesis).
  • site-directed mutagenesis site-specific mutagenesis
  • the detection methods described above can be used in suitable test kits for screening for antiparasitic the physical effects of substances can be used.
  • These include methods which are known to the person skilled in the art and are suitable for screening natural substances from flora and fauna, from plants, algae, bacteria or animals, and their derivatives, chemical libraries, including libraries which are used by techniques known to the person skilled in the art, including combinatorial chemistry (Pindur et al. Pharmacy in our time 26 (1997) 24-30; Broach et al. Nature 384 (1997) 14-6; Lack et al. Chimia 50 (1996) 445-7; Czarnik and Ellmann Accounts of chemical research 29 (1996); Chemical and engineerings News 74 (1996) 28-73; Lorin et al. Chemical reviews 96 (1996) 555-600; Weber et al. News from Chemistry, Technology and Laboratory 42 (1994 ) 698-702).
  • the present invention also relates to the use of proteins or sections of these proteins, including proteins or sections of proteins with or without enzymatic activity in techniques known to those skilled in the art for determining structures of the protein, in particular the characterization of the binding sites which are suitable for development of agents with an inhibitory effect on the enzymatic activity.
  • Active ingredients that are found with the help of the proteins according to the invention are of great interest for medicine and veterinary medicine.
  • the active substances which are found with the aid of the proteins according to the invention are suitable, with favorable warm-blood toxicity, for combating pathogenic parasites which are useful in humans and in animal husbandry and animal breeding in utility, breeding, zoo, laboratory, experimental and Hobby animals occur. They are effective against all or individual stages of development of the pests and against resistant and normally sensitive parasites. By combating the parasites, diseases, deaths and reduced performance (for example in the production of meat, milk, wool, hides, eggs, etc.) are to be reduced, so that the use of the active compounds enables more economical and simple animal husbandry
  • the results from the enzyme assays could also be confirmed in malaria culture (see examples) and in animal experiments (see examples).
  • the inhibitors determined by means of these enzyme assays were able to inhibit the growth of malaria parasites in vitro and in vivo.
  • Treatment of the animals for 8 days showed healing of the animals.
  • the acetyl form was three times more effective than the formyl form. This result is very surprising, since much higher (up to 100x) concentrations of 3- (N-acetyl-N-hydroxyammo) propylphosphonate are required to inhibit bacterial growth.
  • the method according to the invention for identifying tion of active substances and the active substances according to the invention are suitable for the therapeutic and prophylactic treatment of infections in humans and animals which are caused by parasites, fungi or viruses.
  • the compounds are used as prophylaxis against and for the treatment of infections caused by pathogens of malaria and sleepiness as well as Chagas disease, toxoplasmosis, amoebic dysentery, Leishmaniosis, Tricho ⁇ toniasis, pneumocystosis, balantidiosis, cryptosporidiosis, Sarcocystosis, Acanthoma, Naeglerose, Coccidiosis, Giardiosis and Lambliosis.
  • the methods and active substances according to the invention are particularly suitable for the treatment of malaria, sleeping sickness and leishmaniasis.
  • the active compounds according to the invention are also suitable for inhibiting the metabolic pathway of bacteria and of plants.
  • substances which are identified according to the invention as inhibitors of the DOXP pathway are also suitable for use as herbicides and for use in the treatment of bacterial infections in humans and animals.
  • Livestock and breeding animals suitable for treatment include mammals such as cattle, horses, sheep, pigs, goats, camels, water buffalos, donkeys, rabbits, salt and freshwater fish, such as. B. trout, carp and eels.
  • Suitable laboratory and experimental animals include mice, rats, guinea pigs, golden hamsters, dogs, cats and pigs.
  • Suitable pets include dogs and cats.
  • the application can be prophylactic as well as therapeutic.
  • the application of the active ingredients is carried out directly or in the form of suitable preparations known to those skilled in the art, such as enterally, parenterally, dermally or nasally.
  • the active compounds according to the invention can be used in combination with all antiinfectives known to the person skilled in the art. These include substances that have antibacterial, antiparasitic, antiviral or fungicidal effects. These include anti-infectives, which are listed in the Red List and in the specialist literature (general and special pharmacology and toxicology by Forth et al. Bl-Wissenschaftsverlag, Mannheim 1998; antibiotic therapy by Simon and Stille, Schattauer-Verlag, Stuttgart 1993).
  • the invention further relates to the combination of inhibitors of the DOXP pathway with agents that inhibit the fat pathway, including inhibitors of synthesis or lipid uptake, especially inhibitors of the mevalonate pathway.
  • the inhibitors of the enzymes HMG-COA synthase and inhibitors of HMG-CoA reductase may be mentioned here in particular.
  • HMG-CoA reductase inhibitors include, in particular, lovastatin and derivatives, mevastatin and derivatives, compactin and derivatives, simvastatin and derivatives, pravastatin and derivatives, atorvastatin and derivatives, fluvastatin and derivatives and cerivastatin and derivatives.
  • the gene encoding the P. falci parum DOX reductoisomerase was cloned by PCR amplification of the corresponding sequences of genomic DNA as a template.
  • genomic DNA the P. falciparum strain HB3 was cultivated according to the candle pot method (Tranger and Jensen (1976), Science 193, 673-675).
  • RPMI 1640 As a culture medium, RPMI 1640 (with HEPES and L-glutamine, Gibco) was supplemented with 10% human serum, 0.3 ⁇ g / ml gentamycin and 0.1 mM hypoxanthine, and a haematocrit of 5% was set with human erythrocytes.
  • the free parasites were washed twice by centrifugation (10 min, 10,000 rpm, 4 ° C.) with a solution of 1% BSA in carrier buffer.
  • the DNA preparation from the free parasites obtained was carried out according to standard protocols.
  • the parasites were first digested with Proteinase K. The mixture was then extracted four times with phenol / chloroform, the DNA solution was dialyzed overnight against TE and then precipitated with isopropanol.
  • the following primers were used for the PCR amplification:
  • the batch for the complete extension of all products was cubed 10 mm at 72 ° C.
  • the PCR product from 4 such batches was combined and purified on a 0.7% agarose gel.
  • the DNA was eluted from the agarose block using the "Kit for DNA extraction" (Millipore, Cat. No. S667).
  • the eluted DNA was precipitated with ethanol and 10 ⁇ l of H 2 O.
  • the PCR product was then processed according to the instructions from the manufacturer with the TA-clonmg kit (Invitrogen), using 20 mg msert-DNA for a ligation approach.
  • desired recombinant plasmid were identified by analytical plasmid preparation and EcoRI digestion of the plasmids.
  • the cloned PCR products were then sequenced using standard forward and reverse primers; the sequences were completed with the primer walking technique.
  • a PCR product which was present in the corresponding orientation in the pCR2.1 vector, was cloned into the expression vector pBK-CMV (Stratagene). The cloning took place via the interfaces of the restriction enzymes Not I and BamH I, which occur in the polylinker of both vectors.
  • the expression vector with the PCR product as an insert was prepared by anion exchange chromatography (Qiagen) on a preparative scale. All methods used for cloning are described in detail in J. Sambrook, E.F. Fritsch, T. Maniatis (1989), Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Habor Laboratory Press, Cold Spring Habor, USA.
  • the COS-7 cells were cultured in DMEM medium with 10% FCS under standard conditions. 30 ml of culture medium were calculated per cell culture bottle. Cells at approx. 50% confluence were used for the transfection, which had been freshly split the day before.
  • DOTAP Boehringer was used as the transfection reagent. 40 ul DNA solution (0.5 ug / ml) were mixed with 110 ul 20 mM HEPES (pH 7.4). In addition, 100 ul DOTAP were mixed with 230 ul 20 mM HEPES (pH 7.4) in a polystyrene reaction vessel. Then the DNA solution was pipetted into the DOTAP solution and incubated for 15 min at room temperature.
  • the mixture was then mixed with 20 ml of culture medium and the medium of the COS-7 cells was replaced by this mixture. The following day the cells were transferred to new cell culture areas with fresh medium. After a further 48 hours of incubation, the transfected COS-7 cells were harvested. For this purpose, the cells were scraped off and washed three times by centrifugation in assay buffer (100 mM TrisHCl (pH 7.5), 1 mM MnCl 2 ). The cells were resuspended in a minimal volume of assay buffer and disrupted by freezing three times (in liquid nitrogen) and thawing. Cell fragments were centrifuged in a 1.5 ml reaction vessel (13,000 rp, 10 min, 4 ° C.) and the supernatant was used directly for measuring the enzyme activity or for purifying the enzyme.
  • assay buffer 100 mM TrisHCl (pH 7.5), 1 mM MnCl 2 .
  • the recombinant P. fal ciparum DOXP reductoisomerase expressed in COS-7 cells was purified to a high degree of homogeneity.
  • the purification was carried out via an affinity chromatography and a gel permeation chromatography step.
  • affinity chromatography column antibodies against the P. falciparum DOXP reductoisomerase were first prepared.
  • sections were selected from the amino acid sequence derived from the DNA sequence for which a particularly high antigenic effect could be predicted.
  • Corresponding peptides were synthesized and used for the immunization of rabbits.
  • the quality of the antisera obtained was determined both by their reactivity with the synthetic ones Peptides, as well as confirmed by Western blot analyzes. Extracts from P. falciparum and recombinant COS cells were used for the Western blot analyzes (BM Western Blottmg Kit, Boehrmger).
  • the antiserum was dialyzed against PBS to remove low molecular weight nurses.
  • the antibodies were then bound to Protein A-Sepharose and covalently coupled to DMP by cross-Img (IgG Oentation Kit, Pierce).
  • the protein extract was obtained as described in Example 1 from 55 cell culture areas with transfected COS-7 cells and loaded onto the column equilibrated with assay buffer. After washing excessively with assay buffer, the column was eluted with elution buffer (100 mM GlycmHCl (pH 2.8), 0.4% CHAPS). The eluate was immediately neutralized with 1 M TrisHCl (pH 7.5). The main fractions were identified by west blot analysis.
  • biotmylated antibodies were used for the detection in order to avoid interference by antibodies eluted from the column in small amounts.
  • the main fractions were pooled, dialyzed against assay buffer and concentrated by ultrafiltration (30 kDa, Amicon).
  • the further purification was carried out by gel permeation chromatography (Superdex 200, Pharmacia) with assay buffer as the start and elution buffer.
  • the main fractions were identified, pooled and concentrated as described above, 20% Glygerm was added and frozen at -70 ° C.
  • the purified P. falciparum DOXP reductoisomerase was shown as a uniform band at 54 kDa by SDS-PAGE (12% acrylamide) under reducing conditions and silver staining (gel code Color Silver Stam Kit, Pierce).
  • the DOXP reductoisomerase activity of the purified enzyme was confirmed in an in vitro test system.
  • 100 ⁇ l assay buffer with 0.3 mM NADPH, 0.3 mM DOXP and 10 ⁇ g recombinant enzyme was used.
  • the reaction was started by adding DOXP to the complete batch.
  • the oxidation of NADPH was monitored photometrically at 340 nm m micro quartz cuvettes at 37 ° C. This experimental system was used to demonstrate the inhibition of P. falciparum recombinant DOXP reductoisomerase by various substances.
  • the various derivatives were tested according to the modified Peters' test.
  • the substances were applied in a quarter of the half-lethal dose (LD50).
  • LD50 half-lethal dose
  • ten mice were infected with Plasmodium vmcken, the causative agent of mouse malaria. After confirmation of the infection by blood test, the treatment was carried out in four mice. Six mice that were not treated served as controls.
  • the treated group was free from live parasites after only one day.
  • the control mice had to be killed on day 5 after infection with a parasitemia of> 80%.
  • mice were still free of parasites 8 weeks after the end of treatment. Further experiments showed an efficacy of 50 mg / kg / d 3- (N-formyl-N-hydroxylamino) propyl-phosphonic acid monosodium salt in mice with a parasitemia of 80%. These mice were also free of live parasites after 1 day. The further results for 3- (N-formyl-N-hydroxylamino) propylphosphonic acid monosodium salt and 3- (N-acetyl-N-hydroxylamino) propylphosphonic acid monosodium salt are shown in FIG.
  • mice Male mice (BALB / c strain) weighing 20 to 25 g.
  • mice One day before infection, four mice were treated intraperitoneally with 50 mg / kg of 3- (N-formyl-N-hydroxylamino) propylphosphonic acid monosodium salt.
  • the mice were then infected with Plasmodium vinckeii.
  • Mice not pretreated with the substance served as a control. No infection was detected in the treated mice, whereas the control mice were killed after 5 days with a parasitemia above 80%. The treated mice were also free of parasites 8 weeks after infection.
  • Example 6 Example 6
  • the malaria parasites are first cultivated for a complete 48-hour cycle in the presence of inhibitors. In the subsequent 24 hours, the survival rate was measured by [ J H] hypoxanthine cultivation. A dilution series of 3-iN-formyl-N-hydroxylammo) propylphosphonic acid monosodium salt in a 10-fold concentration of 20 ⁇ l aliquots is placed on a microtiter plate. Then 180 ⁇ l of parasite suspensions are added to each well in culture medium. Asynchronous cultures with approx. 0.4% parasitemia and 2% hematotope are used. The microtiter plates are then incubated for 48 h.
  • Plasmodium falciparum HB3 (Honduras) is resistant to pyrimethamine.
  • Plasmodium falciparum Dd2 (Indochina) is resistant to cloroquin, quinine, py ⁇ methamine, cycloguanil and sulfadoxine.
  • Plasmodium falciparum A2 (Gamoia) is resistant to chloroquine and cycloguanil. No cross-resistance with anti-malaria drugs was found.

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Abstract

L'invention concerne un procédé permettant de trouver des principes actifs chimiques adaptés au traitement d'infections dues à des parasites monocellulaires ou pluricellulaires. Ce procédé consiste à mettre en contact des protéines qui participent à la voie de métabolisme du 1-désoxy-D-xylulose-5-phosphate, ou de leurs dérivés d'action identique avec les principes actifs dont on veut examiner l'action vis-à-vis des parasites. Ce procédé consiste ensuite à sélectionner les principes actifs qui inhibent les protéines ou leurs dérivés. L'invention concerne également les principes actifs trouvés et destinés à la production de médicaments efficaces contre les infections parasitoses.
EP99920648A 1998-04-14 1999-04-13 Procede d'identification de principes actifs chimiques et principes actifs destines a l'inhibition de la voie de biosynthese du 1-desoxy-d-xylulose-5-phosphate Withdrawn EP1071959A2 (fr)

Applications Claiming Priority (15)

Application Number Priority Date Filing Date Title
DE19816196 1998-04-14
DE19816196 1998-04-14
DE19825585 1998-06-09
DE19825585A DE19825585A1 (de) 1998-04-14 1998-06-09 Verwendung von Aminohydrocarbylphosphonsäurederivaten zur therapeutischen und prophylaktischen Behandlung von Infektionen, die durch Parasiten, Pilze oder Viren hervorgerufen werden
DE19828097 1998-06-24
DE19828097A DE19828097A1 (de) 1998-06-24 1998-06-24 Kombinationspräparat von Aminohydrocarbylphosphonsäurederivaten und Hemmern des Fettstoffwechsels
DE19831637 1998-07-15
DE19831638 1998-07-15
DE19831639A DE19831639C1 (de) 1998-07-15 1998-07-15 Phosphororganische Verbindungen und ihre Verwendung
DE19831639 1998-07-15
DE19831638 1998-07-15
DE19831637A DE19831637A1 (de) 1998-07-15 1998-07-15 Verwendung von phosphororganischen Verbindungen zur therapeutischen und prophylaktischen Behandlung von Infektionen, die durch Parasiten hervorgerufen werden
DE19843279 1998-09-22
DE19843279 1998-09-22
PCT/EP1999/002463 WO1999052938A2 (fr) 1998-04-14 1999-04-13 Identification de principes actifs chimiques destines a l'inhibition de la voie de biosynthese du 1-desoxy-d-xylulose-5-phosphate dans des parasites

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CA (1) CA2328157A1 (fr)
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Publication number Priority date Publication date Assignee Title
CA2339519A1 (fr) * 1998-08-05 2000-02-17 Sungene Gmbh & Co.Kgaa Sequence adn codant pour une 1-deoxy-d-xylulose-5-phosphate synthase et sa surproduction dans les plantes
AP2001002104A0 (en) * 1998-09-22 2001-03-31 Jomaa Pharmaka Gmbh Genes of the 1-desoxy-d-xylulose biosynthetic pathway.
JP2002532101A (ja) * 1998-12-14 2002-10-02 バッハー，アーデルバート テルペノイド類生合成阻害剤のためのスクリーニング
GB9901902D0 (en) * 1999-01-28 1999-03-17 New Royal Holloway & Bedford Manipulating isoprenoid expression
MXPA01011894A (es) * 1999-05-21 2002-06-21 Jomaa Pharmaka Gmbh Uso de genes de las trayectorias biosinteticas de fosfato de desoxi-d-xilulosa para alterar la concentracion de isoprenoides.
ATE373715T1 (de) 1999-08-04 2007-10-15 Adelbert Bacher Isoprenoid biosynthese
DE10021688A1 (de) * 2000-05-05 2001-11-15 Hassan Jomaa Gene des 1-Desoxy-D-xylulose-Biosynthesewegs
DE10027821A1 (de) 2000-06-05 2001-12-06 Adelbert Bacher Der Mevalonat-unabhängige Isoprenoidbiosyntheseweg
DE10201458A1 (de) 2001-04-11 2002-10-17 Adelbert Bacher Intermediate und Enzyme des Mevalonat-unabhängigen Isoprenoidbiosyntheseweg
CN102860999B (zh) * 2012-10-12 2014-04-09 西北大学 1-脱氧-d-木酮糖5-磷酸还原异构化酶抑制剂及其制备方法

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DE19752700A1 (de) * 1997-11-28 1999-06-02 Hoechst Schering Agrevo Gmbh 1-Desoxy-D-xylulose-5-phosphat Synthase, Verfahren zur Identifizierung von Effektoren der 1-Desoxy-D-xylulose-5-phosphat Synthase und Effektoren der 1-Desoxy-D-xylulose-5-phosphat Synthase

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IL138721A0 (en) 2001-10-31
OA11500A (en) 2004-05-14
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AU4481699A (en) 1999-11-01
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