WO2005054457A1 - Verfahren zum identifizieren von fungizid wirksamen verbindungen basierend auf pyruvat-kinasen aus pilzen - Google Patents

Verfahren zum identifizieren von fungizid wirksamen verbindungen basierend auf pyruvat-kinasen aus pilzen Download PDF

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WO2005054457A1
WO2005054457A1 PCT/EP2004/013323 EP2004013323W WO2005054457A1 WO 2005054457 A1 WO2005054457 A1 WO 2005054457A1 EP 2004013323 W EP2004013323 W EP 2004013323W WO 2005054457 A1 WO2005054457 A1 WO 2005054457A1
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pyruvate kinase
activity
pyruvate
sequence
polypeptide
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English (en)
French (fr)
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Roland Ebbert
Edda Koopmann
Martin Adamczewski
Bernhard Grimmig
Karl-Heinz Kuck
Arnd Voerste
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Bayer Cropscience Aktiengesellschaft
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18Testing for antimicrobial activity of a material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37Assays involving biological materials from specific organisms or of a specific nature from fungi

Definitions

  • the invention relates to a method for identifying fungicides, the use of fungal pyruvate kinase to identify fungicides, and the use of inhibitors of pyruvate kinase as fungicides.
  • fungicides are often sought in essential biosynthetic pathways. Ideal fungicides continue to be those substances that inhibit gene products that are of crucial importance in the expression of the pathogenicity of a fungus.
  • the object of the present invention was therefore to identify a suitable new point of attack for potential fungicidal active substances and to make them accessible, and to provide a method which makes it possible to identify modulators of this point of attack which can then be used as fungicides.
  • Pyruvate kinase (EC No. 2.7.1.40), hereinafter also abbreviated to PK, is the last enzyme in glycolysis, the most important way of breaking down glucose in eukaryotic cells. Synonyms for pyruvate kinase are also the names phosphoenol pyruvate kinase, phosphoenol transphosphorylase or pyruvate 2-O-phosphotransferase.
  • the PK converts phosphoenolpyruvate and ADP into pyruvate and ATP.
  • the enzymatic activity of the PK can be represented schematically as follows:
  • Phosphoenolpyruvate + ADP + H + ⁇ > pyruvate + ATP.
  • the reaction can be divided into two halves (Muirhead et al., 1986). First, the phosphate of phosphoenol pyruvate is transferred directly to ADP with the formation of a pyruvate enolation (stabilized by a Mg 2+ ion). The pyruvate enolate is then protonated and isomerized to the pyruvate. This isomerization to the keto form is practically quantitative and is the driving force for the reaction.
  • PK is an important regulating point in the metabolism of a cell, since it has to be highly active for the glycolytic metabolism, but with active gluconeogenesis it has to be inactive in order to break down newly formed phosphoenolpyruvate in an idle cycle.
  • the kinetics and regulation of PK have been well investigated (summarized, for example, in Muirhead, 1987 and Boles, 1997).
  • the M 2 , L and R forms are regulated allosterically: fructose-l, 6-bisphosphate, phosphoenolpyruvate, ADP, K + - and NH 4 4 - ions have an activating effect, repressing citric acid and ATP.
  • the mi-enzyme is not allosterically regulated.
  • S. cerevisiae only has a PK isoenzyme that is similar in regulatory properties to the M 2 isoform from mammals.
  • Genes for pyruvate kinase have been cloned from various organisms, including the fungus Neurospora crassa (TrEMBL Accession No. EAA30602), Trichoderma reesei (Swissprot Accession No. P31865), Emericella nidulans (Swissprot Accession No. P22360), Aspergillus niger (Swissprot Accession No. Q122669), Aspergillus oryzae (TrEMBL Accession No. Q9HGY6), Kluyveromyces lactis (TrEMBL Accession No.
  • MG08063.1 access: http: // www- genome.wi.mit.edii / annotation fungi / magnaporthe / geneindex.html) or for Botrytis cinerea, Blumeria graminis, Mycosphaerella graminicola or Phytophthora infestans ( Sequences BfCon [1384], Bg [0304], mgall26f, PiCon [0026], access: http://cogeme.ex.ac.uk/search.html).
  • pyruvate kinase The importance of pyruvate kinase has already been explored in medical research. In contrast to most other tissues, tumor cells mainly contain the M 2 isozyme of PK (summarized in Mazurek and Eigenbrodt, 2003). Therefore, the PK is considered as possible Starting point for the fight against tumors viewed (WO 01/96535 A2, US 2001/0046997 AI, WO 02/095063 AI). Inhibitors of pyruvate kinase have also become known which can be used as pharmaceuticals for various diseases such as cancer or Alzheimer's (US 2001/0046997 AI). However, a possible role of pyruvate kinase as a target for fungicidal agents has not yet been investigated.
  • the object of the present invention was now to identify new points of attack by fungicides in fungi, in particular in phytopathogenic fungi, and to make a method accessible in which inhibitors of such a point of attack or polypeptide can be identified and their fungicidal properties can be tested.
  • the object was achieved by identifying the pyruvate kinase as a target for fungicidal active substances, which isolated nucleic acid coding for pyruvate kinase from a phytopathogenic fungus, Ustilago maydis, which was obtained from the encoded polypeptide and made available a method with the inhibitors of this enzyme can be determined.
  • the inhibitors identified with this method can be used against fungi in vivo.
  • FIG. 1 Schematic representation of the reaction catalyzed by the pyruvate kinase.
  • Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP.
  • PEP phosphoenolpyruvate
  • Figure 2 Sequence alignment to show the homology between pyruvate kinases from different fungi. Particularly homologous areas (consensus sequence) are shown with a black background.
  • Figure 3 Graphical representation of the kinetics of the NADPH decrease in a coupled activity test for the pyruvate kinase.
  • the reaction of the pyruvate kinase was coupled with the reaction of the lactate dehydrogenase.
  • Different concentrations of LDH were used in test batches of 200 ⁇ l per well. When using 1U LDH, the decrease in NADH concentration is particularly evident.
  • Figure 4 Graphical representation of the decrease in the activity of pyruvate kinase in the presence of an inhibitor. The course of the reaction is shown for batches with different concentrations of compound 1 and for the positive and negative control.
  • SEQ ID NO: 1 nucleic acid sequence coding for the pyruvate kinase from Ustilago maydis.
  • identity is to be understood as the number of matching amino acids (identity) with other proteins, expressed in percent.
  • the identity is preferably determined by comparing a given sequence to other proteins with the aid of computer programs. If sequences which are compared with one another have different lengths, the identity is to be determined in such a way that the number of amino acids which the shorter sequence has in common with the longer sequence determines the percentage of identity.
  • identity can be determined by means of known computer programs which are available to the public, such as, for example, ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680).
  • ClustalW is made publicly available by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany.
  • ClustalW is also available from various websites, including the IGBMC (Institut de Genetique et de Biologie Moleisme et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and the EBI ( ftp://ftp.ebi.ac.uk pub / software /) and from all mirrored websites of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK). If the ClustalW computer program version 1.8 is used to determine the identity between e.g.
  • sequence database searches One way to find similar sequences is to perform sequence database searches. Here, one or more sequences are specified as a so-called query. This query sequence is then compared using statistical computer programs with sequences that are contained in the selected databases. Such database queries (“blast searches”) are known to the person skilled in the art and can be carried out at various providers.
  • proteins are to be understood which, when using at least one of the methods described above for identity determination, have an identity of at least 70%, preferably of at least 75%, particularly preferably of at least 80%, further preferably of at least 85 %, and in particular of at least 90%.
  • hybridize or “hybridization” as used herein describes the process in which a single-stranded nucleic acid molecule with a complementary strand undergoes base pairing.
  • DNA fragments from phytopathogenic fungi other than Ustilago maydis are isolated which code for pyruvate kinases which have the same or similar properties of one of the pyruvate kinase according to the invention.
  • Hybridization conditions are approximately calculated using the following formula:
  • Hybridization solution DIG Easy Hyb (Röche, ZZ) Hybridization temperature: 42 ° C to 70 ° C, preferably at 42-65 ° C (DNA-DNA) or 50 ° C (DNA-RNA).
  • particularly suitable stringent temperatures for the hybridization are between 50 and 65 ° C, a temperature of 65 ° C being a particularly suitable stringent temperature.
  • 1st washing step 2 x SSC, 0.1% SDS 2 5 min at room temperature;
  • 2nd washing step 1 x SSC, 0.1% SDS 2 x 15 min at 50 ° C; preferably 0.5 x SSC, 0.1% SDS 2 x 15 min at 65 ° C; particularly preferably 0.2 x SSC, 2 x 15 min at 68 ° C.
  • complete pyruvate kinase describes a pyruvate kinase encoded by a complete coding region of a transcription unit, comprising an ATG start codon and comprising all information-bearing exon regions of the gene present in the organism of origin, coding for a pyruvate kinase, and the signals necessary for a proper termination of the transcription.
  • enzyme or "biological activity of a pyruvate kinase” as used herein refers to the ability of a polypeptide to carry out the reaction described above, i.e. to catalyze the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP.
  • active fragment no longer describes complete nucleic acids coding for pyruvate kinase, but which still code for polypeptides with the biological activity of a pyruvate kinase and which can catalyze a reaction characteristic of the pyruvate kinase as described above. Such fragments are shorter than the complete nucleic acids encoding pyruvate kinase described above. Nucleic acids may have been removed both at the 3 'and / or 5' ends of the sequence, but parts of the sequence can also be deleted, i.e. have been removed that do not significantly affect the biological activity of the pyruvate kinase.
  • a lower or possibly also an increased activity, which, however, still allows the characterization or use of the resulting pyruvate kinase fragment, is understood to be sufficient in the sense of the expression used here.
  • the expression "active fragment” can also refer to the amino acid sequence of the pyruvate kinase and then applies analogously to the above statements for those polypeptides which, in comparison to the complete sequence defined above, no longer contain certain parts, the biological activity of the enzyme however not significantly impairing this is.
  • the fragments can have different lengths.
  • pyruvate kinase inhibition test or “inhibition test” as used herein refer to a method or a test which allows the inhibition of the enzymatic activity of a polypeptide with the activity of a pyruvate kinase by one or more chemical compounds ( "Candidate” or “test compound (s)”), whereby the chemical compound can be identified as an inhibitor of pyruvate kinase.
  • gene as used herein is the term for a portion of the genome of a cell that is responsible for the synthesis of a polypeptide chain.
  • fungicide or "fungicidal” as used herein refers to chemical compounds which are suitable for combating fungi which are pathogenic to humans, animals and plants, in particular fungi which are pathogenic to plants. Such phytopathogenic fungi are mentioned below, the list is not exhaustive: Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes, e.g.
  • Pythium species such as, for example, Pythium ultimum, Phytophthora species, such as, for example, Phytophthora infestans, Pseudoperonospora species, such as, for example, Pseudoperonospora humuli or Pseudoperonospora cubensis, Plasmopara species, such as, for example, Plasmopara viticola, Bremia species, such as, for example, Bremia lactucae, Peronospor Species such as Peronospora pisi or P.
  • brassicae Erysiphe species such as Erysiphe graminis, Sphaerotheca species such as Sphaerotheca fuliginea, Podosphaera species such as Podosphaera leucotricha, Venturia species such as Venturia inaequalis and Pyrenophora species , such as, for example, Pyrenophora teres or P.
  • Drechslera conidial form: Drechslera, Syn: Helminthosporium
  • Cochliobolus species such as, for example, Cochliobolus sativus
  • Uromyces species such as, for example, Uromyces appinia species
  • Pu such as, for example, Puccinia recondita
  • Sclerotinia species n such as Sclerotinia sclerotiorum
  • Tilletia species such as Tilletia caries
  • Ustilago species such as, for example, Ustilago nuda or Ustilago avenae
  • Pellicularia species such as, for example, Pellicularia sasakii
  • Pyricularia species such as, for example, Pyricularia oryzae
  • Fusarium species such as, for example, Fusarium culmorum, Botrytis species, Septoria
  • Fungicidal active ingredients which are found with the aid of the pyruvate kinases according to the invention from plant pathogenic fungi can also interact with pyruvate kinase from human pathogenic fungus species, the interaction with the different pyruvate kinases occurring in these fungi not always having to be equally strong.
  • the present invention therefore also relates to the use of inhibitors of pyruvate kinase for the preparation of agents for the treatment of diseases caused by fungi which are pathogenic to humans.
  • Dermatophytes such as Trichophyton spec, Microsporum spec, Epidermophytonfloccosum or Keratomyces ajelloi, which cause foot mycoses (tinea pedis), for example,
  • Yeasts such as Candida albicans, which causes thrush esophagitis and dermatitis, Candida glabrata, Candida krusei or Cryptococcus neoformans, which e.g. can cause pulmonary cryptococcosis and also torulose,
  • Mold such as Aspergillus fumigatus, A. flavus, A. niger, e.g. cause bronchopulmonary aspergillosis or fungal sepsis, Mucor spec, Absidia spec, or Rhizopus spec, e.g. Cause zygomycoses (intravascular mycoses), Rhinosporidium seeberi, which e.g. chronic granulomatous pharyngitis and tracheitis, Madurella myzetomatis, which e.g. causes subcutaneous mycetomas, Histoplasma capsulatum, which e.g.
  • Coccidioides immitis which e.g. pulmonary coccidioidomycosis and sepsis
  • Paracoccidioides brasiliensis which e.g. Brazilian blastomycosis
  • Blastomyces dermatitidis which e.g. Gilchrist's disease and North American blastomycosis
  • Loboa loboi which e.g. Keloid blastomycosis and Lobo's disease
  • Sporothrix schenckii e.g. Sporotrichosis (granulomatous skin mycosis).
  • Fungicidal active ingredients which are found with the aid of a pyruvate kinase obtained from a specific fungus, here from Ustilago maydis, can therefore also interact with pyruvate kinase from other numerous fungus species, especially also with phytopathogenic fungi, the interaction with the different pyruvate kinases occurring in these fungi does not always have to be equally strong. Among other things, this explains the observed selectivity of the substances active on this enzyme.
  • homologous promoter refers to a promoter which controls the expression of the gene in question in the original organism.
  • heterologous promoter refers to a promoter which has different properties than the promoter which controls the expression of the gene in question in the original organism.
  • competitive refers to the property of the compounds to compete with other compounds that may be identified in order to compete for binding to the pyruvate kinase and to displace or be displaced by the enzyme.
  • inhibitor or "specific inhibitor” as used herein denotes a substance that directly inhibits an enzymatic activity of the pyruvate kinase.
  • Such a Inhibitor is preferably "specific", ie the inhibition of PK requires a lower concentration of the inhibitor than for another, independent effect.
  • the concentration is preferably two times lower, particularly preferably five times lower and very particularly preferably at least ten times or 20 times lower than the concentration of a compound which is required to produce an unspecific effect.
  • modulator represents a generic term for inhibitors and activators.
  • Modulators can be small organic chemical molecules, peptides or antibodies which bind to the polypeptides according to the invention or which influence their activity.
  • modulators can be small organic chemical molecules, peptides or antibodies which bind to a molecule which in turn binds to the polypeptides according to the invention and thereby influences their biological activity.
  • Modulators can represent natural substrates and ligands or structural or functional mimetics thereof.
  • modulator as used herein is preferably, however, those molecules which do not represent the natural substrates or ligands.
  • the complete sequence of a pyruvate kinase from the plant-pathogenic fungus Ustilago maydis is made available, which enables further research of pyruvate kinases, in particular from plant-pathogenic fungi, and thus the development of a new target protein for the identification of new fungicidal active substances.
  • pyruvate kinase in fungi can be a target protein (a so-called "target") of fungicidally active substances.
  • target a target protein of fungicidally active substances.
  • pyruvate kinase is an enzyme that is particularly important for fungi and is therefore particularly suitable for use as a target protein in the search for further and improved fungicidally active compounds.
  • Inhibitors of pyruvate kinase with a fungicidal activity have not previously been described. So far, there is also no indication that the pyruvate kinase in phytopathogenic or human or animal pathogenic fungi can be influenced, for example inhibited, by active substances and whether it is possible in vivo to combat fungi, in particular phytopathogenic fungi, with an active substance which modulates the pyruvate kinase is. Pyruvate kinase has not yet been described as a target protein for fungicides. There are no known active ingredients which have a fungicidal action and whose site of action is pyruvate kinase.
  • the pyruvate kinase can be a target or “target” for fungicidal active compounds in phytopathogenic fungi, so that the inhibition of the pyruvate kinase could lead to damage or death of the fungus.
  • pyruvate kinase can be used to identify modulators or inhibitors of its enzymatic activity in test procedures, which is possible with various theoretically interesting targets, e.g. are already known as essential for an organism.
  • inhibitors of pyruvate kinase which could be identified in such processes, can be used as fungicides.
  • a method was developed which is suitable for determining the activity of the pyruvate kinase and the inhibition of this activity in a so-called inhibition test, in this way inhibitors of the enzyme e.g. identified in HTS and UHTS procedures.
  • the compounds identified with the aid of this method according to the invention were tested in in vivo tests on fungi.
  • pyruvtakmase can also be inhibited in vivo by active substances and that a fungal organism treated with these active substances can be damaged and killed by treatment with these active substances.
  • the inhibitors of a fungal pyruvate kinase can therefore be used as fungicides, in particular in crop protection or as antifungals in pharmaceutical indications.
  • the inhibition of pyruvate kinase with a substance identified in a method according to the invention leads to the death or inhibition of growth of the treated fungi in synthetic media or on the plant.
  • Pyruvate kinases can be obtained from various phytopathogenic fungi or from human or animal pathogenic fungi, e.g. as shown in the present invention from the plant pathogenic fungus U. maydis.
  • the gene can e.g. recombinantly expressed in Escherichia coli and an enzyme preparation made from E. coli cells.
  • Pyruvate kinases from phytopathogenic fungi are preferably used to identify fungicides which can be used in crop protection. If the goal is to identify fungicides or antimycotics that are to be used in pharmaceutical indications, the use of pyruvate kinases from human or animal pathogenic fungi is recommended.
  • the associated ORF was amplified by means of PCR using selected primers using methods known to the person skilled in the art.
  • the polypeptide was then transformed and expressed in E. coli AD494 (DE3) Comp. Cells from Novagen (Example 1).
  • the Ustilago maydis gene coding for pyruvate kinase has a size of 1587
  • the present invention thus also provides a complete genomic sequence of a phytopathogenic fungus coding for a pyruvate kinase, and describes its use or the use of the polypeptide encoded thereby for the identification of inhibitors of the enzyme.
  • the present invention therefore also relates to the nucleic acid from the Ustilago maydis fungus coding for a polypeptide with the enzymatic function of a pyruvate kinase according to SEQ ID NO: 1.
  • Pyruvate kinases share homologous regions (see Figure 2). Typical of pyruvate kinases is a conserved region that includes a lysine residue that is likely to act as an acid / base catalyst in the conversion of pyruvate to phosphonenolypruvate. The conserved region also includes a glutamic acid residue that is involved in the binding of the magnesium ion.
  • sequence motif which is typical for pyruvate kinases, can be reproduced in the form of a prostite motif (Hofmann et al., 1999). It can be represented as follows:
  • residue K stands for lysine in the active center and the residue E for the glutamine involved in the binding of the magnesium ion.
  • PROSITE enables polypeptides to be assigned a function and thus to recognize pyruvate kinases as such.
  • the "one-letter code” is used to display the Prosite motif.
  • the symbol “x” stands for a position where every amino acid is accepted.
  • a variable position at which various specific amino acids are accepted is shown in square brackets "[...]", whereby the possible amino acids at this position are listed.
  • Amino acids that are not accepted at a certain position are enclosed in curly brackets " ⁇ ... ⁇ ”.
  • a dash “-” separates the individual elements or positions of the motif. If a certain position, for example "x”, repeats several times in succession, this can be represented by specifying the number of repetitions in a subsequent bracket, for example "x (3)”, which stands for "xxx”.
  • a Prosite motif ultimately represents the components of a consensus sequence, as well as the distances between the amino acids involved, and is therefore typical for a certain enzyme class.
  • further polypeptides from phytopathogenic fungi can be identified or assigned on the basis of the nucleic acids according to the invention which belong to the same class as the polypeptide according to the invention and can therefore also be used in the manner according to the invention.
  • this motif is present, as is the case with Neurospora crassa, Trichoderma reesei, Emericella nidulans, Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe, Agaricusthisophiae infestus, S. c Figure 2).
  • Prosite motif or the specific consensus sequence are typical of the polypeptides according to the invention, which can be structurally defined on the basis of these consensus sequences and are therefore also clearly identifiable.
  • the present invention therefore also relates to polypeptides from phytopathogenic fungi with the biological activity of a pyruvate kinase, which have the aforementioned prosite motif [LIVAC] -x- [LlvTsI] (2) - [SAPCV] -K- [LIV] -E-
  • Those polypeptides which comprise the prosite motif [IV] -x- [IV] -I- [SPACV] -K- [ ⁇ V] -E- [NS] -X- [EQ] -G- [LIVM] are preferred .
  • pyruvate kinases from other phytopathogenic fungi can also be identified and used to achieve the above object, ie they can also be used to identify inhibitors of a pyruvate kinase, which in turn can be used as Fungicides can be used in crop protection.
  • pyruvate kinases from other phytopathogenic fungi can also be identified and used to achieve the above object, ie they can also be used to identify inhibitors of a pyruvate kinase, which in turn can be used as Fungicides can be used in crop protection.
  • another fungus which is not pathogenic to the plant, or its pyruvate kinase or the sequence coding therefor, in order to identify fungicidal inhibitors of pyruvate kinase.
  • nucleic acids coding for pyruvate kinases from other (phytopathogenic) fungi, for example by PCR.
  • nucleic acids and their use in methods for identifying fungicidal active substances are considered to be encompassed by the present invention.
  • nucleic acid sequence according to the invention With the aid of the nucleic acid sequence according to the invention and sequences obtained from other phytopathogenic fungi according to the methods described above, further nucleic acid sequences coding for a pyruvate kinase from other fungi can be identified.
  • the present invention therefore also relates to the use of nucleic acids from phytopathogenic fungi which code for a polypeptide with the enzymatic activity of a pyruvate kinase, in particular polypeptides which comprise the motif described above, for identifying inhibitors of fungal pyruvate kinases.
  • the present invention also relates to the nucleic acid coding for the Ustilago maydis pyruvate kinase with the SEQ ID NO: 1 and the nucleic acids coding for the polypeptides according to SEQ ID NO: 2 or active fragments thereof.
  • the nucleic acids according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • Preferred embodiments are fragments of genomic DNA, and in particular cDNAs.
  • the nucleic acids which can be used in the manner according to the invention comprise a sequence of phytopathogenic fungi coding for a polypeptide with the enzymatic activity of a pyruvate kinase selected from
  • polypeptides with the activity of a pyruvate kinase can also be obtained from other fungi, preferably from phytopathogenic fungi, which then e.g. can be used in a method according to the invention.
  • the pyruvate kinase from Ustilago maydis is preferably used.
  • the present invention furthermore relates to DNA constructs which comprise a nucleic acid according to the invention and a homologous or heterologous promoter.
  • heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression.
  • heterologous promoters are the 35S promoter of cauliflower mosaic virus for plant cells, the alcohol dehydrogenase promoter for yeast cells, the T3, T7 or SP6 promoters for prokaryotic cells or cell-free systems.
  • Promoters which are also suitable for the expression of the UMP / CMP kinases in gram-negative bacterial strains are, for example, the cos, tac, trp, tet, lpp, lac, laclq, T5, gal, trc , ara, 1-PR or 1-PL promoter.
  • the promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, AOX1 and GAP can also be used for expression in yeast strains.
  • yeast strains for example, the polyhedrin promoter and the p10 promoter (Luckow, V.A. and Summers, M.D. (1988) Bio / Techn. 6, 47-55) can be used.
  • Fungal expression systems such as e.g. the Pichia pastoris system can be used, the transcription here being driven by the methanol-inducible AOX promoter.
  • the present invention furthermore relates to vectors which contain a nucleic acid according to the invention, a regulatory region according to the invention or a DNA construct according to the invention. All phages, plasmids, phagmids, phasmids, cosmids, YACs, BACs, artificial chromosomes or particles which are suitable for particle bombardment can be used as vectors.
  • Preferred vectors are, for example, the commercially available fusion and expression vectors pGEX (Pharmacia Biotech Ine), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein, or Protein A, the pTrc vectors (Amann et al., (1988), Gene 69: 301-315), the "pKK233-2n (CLONTECH, Palo Alto, California) and the" pET “and” pBADH vectors -series from Stratagene, La Jolla.
  • vectors for use in yeast are pYep Secl (Baldari et al. (1987), Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982), Cell 30: 933-943), pJRY88 (Schultz et al. (1987), Gene 54: 113-123), and pYES derivatives, pGAPZ derivatives, pPICZ derivatives, the vectors of the Pichia Expression Kit '(Tnvitrogen Corporation, San Diego, CA), the p4XXprom.
  • Vector series (Mumberg et al., 1995) and pSPORT vectors (Fa. Life Technologies) for bacterial cells, or gateway vectors (Fa. Life Technologies) for various expression systems in bacterial cells, plants, P. pastoris, S. cerevisiae or insect cells.
  • the present invention also relates to host cells which contain a nucleic acid according to the invention, a DNA construct according to the invention or a vector according to the invention.
  • host cell refers to cells which do not naturally contain the nucleic acids according to the invention.
  • Suitable host cells are both prokaryotic cells, preferably E. coli, and eukaryotic cells, such as cells from Saccharomyces cerevisiae, Pichia pastoris, insects, plants, frog oocytes and cell lines from mammals.
  • the present invention furthermore relates to polypeptides with the biological activity of a pyruvate kinase which are encoded by the nucleic acids according to the invention.
  • polypeptides which can be used in accordance with the invention preferably comprise an amino acid sequence selected from phytopathogenic fungi
  • polypeptides refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, as well as longer amino acid chains commonly referred to as proteins. It encompasses amino acid chains that can be modified either by natural processes, such as post-translational processing, or by chemical processes that are state of the art. Such modifications can occur at various locations and multiple times in a polypeptide, such as, for example, on the peptide backbone, on the amino acid side chain, on the amino and or on the carboxy terminus.
  • acetylations include, for example, acetylations, acylations, ADP ribosylations, amidations, covalent linkages with flavins, heme components, nucleotides or nucleotide derivatives, lipids or lipid derivatives or phosphatidylinositol, cyclizations, disulfide bridging, demethylations, cystine formation, formylations gamma carboxylations, glycosylations, hydroxylations, iodinations, methylations, myristoylations, oxidations, proteolytic processing, phosphorylations, selenoylations and tRNA-specific additions of amino acids.
  • polypeptides according to the invention can be in the form of "mature” proteins or as parts of larger proteins, e.g. as fusion proteins. Furthermore, they can have secreting or "leader” sequences, pro sequences, sequences which enable simple purification, such as multiple histidine residues, or additional stabilizing amino acids.
  • the proteins according to the invention can also be present as they are naturally present in their organism of origin, from which they can be obtained directly, for example. Active fragments of a pyruvate kinase can also be used in the method according to the invention, as long as they enable the determination of the enzymatic activity of the polypeptide or its inhibition by a candidate compound.
  • polypeptides used in the methods according to the invention can have deletions or amino acid substitutions in comparison with the corresponding regions of naturally occurring pyruvate kinases, as long as they still show at least the biological activity of a complete pyruvate kinase.
  • Conservative substitutions are preferred. Such conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:
  • Aromatic residues Phe, Tyr and Trp.
  • a possible purification method of pyruvate kinase is based on preparative electrophoresis, FPLC, HPLC (for example using gel filtration, reverse phase or slightly hydrophobic columns), gel filtration, differential precipitation, ion exchange chromatography or affinity chromatography (see Example 2).
  • a rapid method for isolating pyruvate kinase synthesized by host cells begins with the expression of a fusion protein, whereby the fusion partner can be easily affinity-purified.
  • the fusion partner can be, for example, an MBP tag or a His tag (cf. Example 2).
  • the fusion partner can be separated by partial proteolytic cleavage, for example on linkers between the fusion partner and the polypeptide according to the invention to be purified.
  • the linker can be designed to include target amino acids, such as arginine and lysine residues, that define sites for trypsin cleavage. Standard cloning techniques using oligonucleotides can be used to create such linkers.
  • purification processes are based on preparative electrophoresis, FPLC, HPLC (e.g. using gel filtration, reverse phase or slightly hydrophobic columns), gel filtration, differential precipitation, ion exchange chromatography and affinity chromatography.
  • composition containing the polypeptides according to the invention is preferably enriched at least 10-fold and particularly preferably at least 100-fold with respect to the protein content compared to a preparation from the host cells.
  • polypeptides according to the invention can also be affinity-purified without a fusion partner using antibodies which bind to the polypeptides.
  • polypeptides with pyruvate mase activity e.g. the pyruvate kinase from U. maydis is characterized by
  • the cells thus obtained containing the polypeptide according to the invention or the purified polypeptide thus obtained are suitable for use in methods for identifying pyruvate kinase modulators or inhibitors.
  • the present invention also relates to the use of polypeptides from fungi, preferably from phytopathogenic fungi, which have at least one biological activity of a pyruvate kinase in methods for identifying fungicides, it being possible for the pyruvate kinase inhibitors to be used as fungicides.
  • the pyruvate kinase from Ustilago maydis is particularly preferably used.
  • Fungicidal active ingredients which are found with the help of a pyruvate kinase from a particular fungal species, can also interact with pyruvate kinase from other fungal species, although the interaction with the different pyruvate kinase occurring in these fungi does not always have to be equally strong. This explains, among other things, the selectivity of active substances.
  • the use of the active ingredients found with a specific pyruvate kinase as a fungicide in other fungi can also be attributed to the fact that pyruvate kinases from different fungus species are very close and show pronounced homology in larger areas. Figure 2 clearly shows that such homology exists over considerable sequence sections between all examined fungi and thus the effect of e.g. substances found with the help of pyruvate kinase from U. maydis is not necessarily limited to U maydis.
  • the present invention therefore also relates to a method for identifying fungicides by testing potential inhibitors or modulators of the enzymatic activity of pyruvate kinase (“candidate compound” or “test compound”) in a pyruvate kinase inhibition test.
  • Methods which are suitable for identifying modulators, in particular inhibitors or antagonists, of the polypeptides according to the invention are generally based on the determination of the
  • test systems that aim to test compounds and natural extracts are preferably geared towards high throughput numbers in order to maximize the number of substances tested in a given period of time.
  • Test systems that are based on cell-free work require purified or semi-purified protein. They are suitable for a first test, which primarily aims to detect a possible influence of a substance on the target protein. Once such a first test has been carried out and one or more compounds, extracts etc. have been found, the effect of such compounds can be examined in a more targeted manner in the laboratory.
  • the inhibition or activation of the polypeptide according to the invention can be checked again in vitro in order to then test the effectiveness of the compound on the target organism, here one or more phytopathogenic fungi.
  • the compound can then optionally be used as a starting point for the further search and development of fungicidal compounds based on the original structure, but e.g. are optimized in terms of effectiveness, toxicity or selectivity.
  • modulators e.g. incubating a synthetic reaction mix (e.g. products of in vitro transcription) or a cellular component such as a membrane, a compartment or any other preparation which contains the polypeptides according to the invention together with an optionally labeled substrate or ligand of the polypeptides in the presence and absence of a candidate molecule become.
  • the ability of the candidate molecule to inhibit the enzymatic activity of the polypeptides of the invention will e.g. recognizable by a reduced binding of the optionally labeled ligand or by a reduced implementation of the optionally labeled substrate.
  • Molecules that inhibit the biological activity of the polypeptides according to the invention are good antagonists or inhibitors.
  • reporter systems include, but are not limited to, colorimetrically or fluorimetrically detectable substrates that are converted to a product, or a reporter gene that is responsive to changes in the activity or expression of the polypeptides of the invention, or other known binding assays.
  • Another example of a method with which modulators of the polypeptides according to the invention can be found is a displacement test in which, under suitable conditions, the polypeptides according to the invention and a potential modulator with a molecule which is known to bind to the polypeptides according to the invention, such as a natural one Bring substrate or ligands or a substrate or ligand mimetic.
  • the polypeptides according to the invention themselves can be labeled, for example fluorimetrically or colorimetrically, so that the number of polypeptides which are bound to a ligand or which have undergone a reaction can be determined exactly.
  • the binding can also be followed by means of the optionally labeled substrate, ligand or substrate analog. In this way, the effectiveness of an antagonist can be measured.
  • Test systems check both inhibitory or suppressive effects of the substances as well as stimulatory effects.
  • the effectiveness of a substance can be checked using concentration-dependent test series. Control approaches without test substances or without enzyme can be used to evaluate the effects.
  • Another possibility for identifying inhibitors of the polypeptide according to the invention is based on so-called in vivo or cell-based methods, which are based in principle on the following steps: (1) Production of transgenic organisms which are capable of transformation with a nucleic acid according to the invention to express a UMP / CMP kinase, (2) applying a test compound to the organism from step (1) and for control to an analog, non-transformed organism, (3) determining the growth or survivability of the transgenic and a non- transformed organism after the application of the test compound from step (2), and (4) selection of test compounds which, in comparison with the growth of the transgenic organism, result in reduced growth, reduced viability and / or reduced pathogenicity of the non-transgenic organism.
  • An analog, non-transformed organism is to be understood as an organism that was used as the starting organism in step (1).
  • the transformation can be carried out using a vector according to the invention or the nucleic acid according to the invention itself.
  • Fungi are particularly suitable as organisms which are transformed with the nucleic acid or vector according to the invention, particularly preferably the phytopathogenic fungi mentioned at the beginning.
  • the host cells containing nucleic acids coding for a pyruvate kinase according to the invention which are available on the basis of the present invention also enable the development of test systems based on cells for the identification of substances which modulate the activity of the polypeptides according to the invention.
  • the modulators to be identified are preferably small organic chemical compounds.
  • a method for identifying a compound which modulates the activity of a pyruvate kinase from fungi and which, if appropriate in a suitable formulation, can be used as a fungicide in crop protection is preferably that
  • a polypeptide according to the invention or a host cell containing this polypeptide is brought into contact with a chemical compound or with a mixture of chemical compounds under conditions which allow the interaction of a chemical compound with the polypeptide,
  • the compound which specifically inhibits the activity of the polypeptide according to the invention is particularly preferably determined.
  • activity refers to the biological activity of the polypeptide according to the invention.
  • the fact is used that the pyruvate formed in the reaction of the pyruvate kinase is converted to lactate by the lactate dehydrogenase (LDH) using NADH. This also creates NAD0.
  • LDH lactate dehydrogenase
  • the cosubstrate NADH consumed in the reaction of the LDH has an extinction maximum at 340 nm.
  • the reaction is allowed to run and the decrease in absorbance is monitored as a function of time (kinetic measurement).
  • the enzymatic activity of the pyruvate kinase can be followed by monitoring the decrease in NADH by absorbance measurement at 340 nm.
  • a lower or inhibited activity of the pyruvate kinase is recognized by the fact that the NADH concentration (determined by photospectrometry) decreases more slowly or not at all.
  • the measurement can also be carried out in formats more common for HTS or UHTS assays, e.g. in microtiter plates in which e.g. a total volume of 5 to 50 .mu.l per batch or per well is presented and the individual components are present in one of the final concentrations given above.
  • the compound to be tested, potentially inhibiting or activating the activity of the enzyme is e.g. in a suitable concentration in test buffer containing ATP, phosphoenol pyruvate, and NADH and the auxiliary enzyme lactate dehydrogenase.
  • the polypeptide according to the invention is added in the above-mentioned test buffer and the reaction is started with it.
  • the approach is then e.g. incubated for up to 30 minutes at a suitable temperature and measured the decrease in the NADH concentration.
  • a further measurement is carried out in a corresponding approach, but without adding a candidate molecule and without adding a polypeptide according to the invention (negative control).
  • a further measurement is again carried out in the absence of a candidate molecule, but in the presence of the polypeptide according to the invention (positive control). Negative and positive control thus give the comparison values for the batches in the presence of a candidate molecule.
  • inhibitors of pyruvate kinase could be identified with the method according to the invention.
  • Table I shows examples of compounds which could be identified as inhibitors of pyruvate kinase using a method according to the invention. This shows that it is possible to successfully identify inhibitors of pyruvate kinase using a method according to the invention.
  • the compounds 1 and 2 are known cyclopentabenzofuran derivatives which have been described in connection with the therapy of NF- ⁇ B-dependent diseases (WO 00/08007 A2). Synthetic routes for the preparation of compounds 3 and 4 are described in Example 8.
  • the pyruvate kinase inhibitors found do not measurably inhibit the lactate dehydrogenase used as auxiliary enzyme.
  • inhibitors of a pyruvate mase according to the invention identified with the aid of a method according to the invention are suitable for damaging or killing fungi in a suitable formulation.
  • a solution of the active substance to be tested can be pipetted into the cavities of microtiter plates, for example. After the solvent has evaporated, medium is added to each cavity. A suitable concentration of spores or mycelium of the fungus to be tested is added to the medium beforehand. The resulting concentrations of the active ingredient are, for example, 0.1, 1, 10 and 100 ppm.
  • the plates are then incubated on a shaker at a temperature of 22 ° C. until sufficient growth can be determined in the untreated control.
  • the evaluation is carried out photometrically at a wavelength of 620 ⁇ m.
  • the dose of active ingredient can be determined from the measurement data of the different concentrations, which leads to a 50% inhibition of fungal growth compared to the untreated control (ED 50 ).
  • the present invention therefore also relates to the use of pyruvate kinase modulators from fungi, preferably from phytopathogenic fungi, as fungicides.
  • the present invention also relates to fungicides which have been identified using a method according to the invention.
  • Table ⁇ shows the results of such a test as ED 50 values for compounds found in a process according to the invention (cf. Table I) as an example.
  • the compounds have a fungicidal action on various fungi.
  • Table II shows the results of such a test as ED 50 values for compounds found in a process according to the invention (cf. Table I) as an example.
  • the compounds have a fungicidal action on various fungi.
  • the active ingredient to be tested is mixed with a solvent and emulsifier and the concentrate is diluted with water to the desired concentration.
  • the protective activity of the compound young tomato plants are sprayed with the active compound preparation and inoculated 1 day after treatment with a spore suspension of Phytophthora infestans. The plants are then placed in a climatic cell and the experiment is evaluated after a few days (example 7).
  • Table HI shows the results of such a test in the form of an efficiency (WG, percentage effect) for a given compound found in a process according to the invention (cf. Table I) (cf. Example 7).
  • 0% means an efficiency which corresponds to that of the control, while an efficiency of 100% means that no infection of the plant by the fungus is observed.
  • inhibitors of fungal pyruvate kinase have a fungicidal action and can be used as fungicides.
  • the present invention therefore also relates to the use of modulators of pyruvate kinase from fungi, preferably from phytopathogenic fungi, as fungicides or to processes for Control of unwanted fungal growth based on bringing an inhibitor of fungal pyruvate kinase into contact with the unwanted fungus and or its habitat.
  • the present invention also relates to fungicides which have been identified using a method according to the invention.
  • the identified active ingredients can be converted into the customary formulations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, very fine encapsulations in polymeric substances and in coating compositions for seeds , as well as ULV cold and warm fog formulations.
  • formulations are prepared in a known manner, for example by mixing the active ingredients with extenders, that is to say liquid solvents, pressurized liquefied gases and / or solid carriers, if appropriate using surface-active agents, that is to say emulsifiers and or dispersants and / or foam-generating agents. If water is used as an extender, organic solvents can, for example, also be used as auxiliary solvents.
  • extenders that is to say liquid solvents, pressurized liquefied gases and / or solid carriers, if appropriate using surface-active agents, that is to say emulsifiers and or dispersants and / or foam-generating agents.
  • surface-active agents that is to say emulsifiers and or dispersants and / or foam-generating agents.
  • organic solvents can, for example, also be used as auxiliary solvents.
  • aromatics such as xylene, toluene or alkylnaphthalenes
  • chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloromethylene or methylene chloride
  • aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions
  • alcohols such as butanol or Glycol and its ethers and esters
  • ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone
  • strongly polar solvents such as dimethylformamide and dimethyl sulfoxide, and water.
  • Liquefied gaseous extenders or carriers mean liquids which are gaseous at normal temperature and under normal pressure, for example aerosol propellants, such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.
  • aerosol propellants such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.
  • solid carriers for example, natural rock powders such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth and synthetic rock powders such as highly disperse silica, aluminum oxide and silicates.
  • Possible solid carriers for granules are: e.g.
  • Suitable emulsifiers and / or foaming agents are: for example nonionic and anionic Emulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and protein hydrolyzates.
  • Possible dispersing agents are, for example, lignin sulfite waste liquor and methyl cellulose.
  • Adhesives such as carboxymethyl cellulose, natural and synthetic powdery, granular or latex-shaped polymers can be used in the formulations, such as gum arabic, polyvinyl alcohol, polyvinyl acetate, and also natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids.
  • Other additives can be mineral and vegetable oils.
  • Dyes such as inorganic pigments, e.g. Iron oxide, titanium oxide, Fe ⁇ ocyanblau and organic dyes such as alizarin, azo and metal phthalocyanine and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc can be used.
  • the formulations generally contain between 0.1 and 95 percent by weight of active compound, preferably between 0.5 and 90%.
  • the active compounds according to the invention can also be used in a mixture with known fungicides, bactericides, acaricides, nematicides or insecticides, in order, for example, to broaden the spectrum of activity or to prevent the development of resistance.
  • fungicides bactericides
  • acaricides nematicides or insecticides
  • synergistic effects are obtained, i.e. the effectiveness of the mixture is greater than the effectiveness of the individual components.
  • the application rates can be varied within wide ranges depending on the application.
  • Plants are understood here to mean all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants).
  • Crop plants can be plants which can be obtained by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods, including the transgenic plants and including the plant cultivars which can or cannot be protected by plant breeders' rights.
  • Plant parts are to be understood to mean all above-ground and underground parts and organs of plants, such as shoots, leaves, flowers and roots, examples being leaves, needles, stems, stems, flowers, fruiting bodies, fruits and seeds as well as roots, tubers and rhizomes.
  • the plant parts also include crops and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offshoots and seeds.
  • the treatment of the plants and parts of plants with the active compounds according to the invention is carried out directly or by acting on their environment, habitat or location according to the customary treatment methods, for example by immersion, spraying, evaporation, atomizing, scattering, spreading and, in the case of propagation material, in particular seeds single or multi-layer wrapping.
  • the gene with gene-specific oligonucleotides (forward primer: gcg ctg gcc cat atg atc tac gct cct att gcc aag aca c; reverse primer: g cca gca gct agc ggc cgc aac ctg agt gcc ctc gag ctt ) amplified by PCR. After digestion with the restriction enzymes Ndel and Notl, the PCR product is cloned into the similarly cut expression vector pET-21b (+) from concertovagen. The resulting plasmid pEB017 thus codes for a pyruvate kinase with a C-terminal His 6 tag
  • the plasmid pEB017 is transformed into E. coli AD494 (DE3).
  • An overnight culture (Ü ⁇ K) is set up by inoculating 25 ml LB-ampicillin medium (c [ampicillin]: 100 ⁇ g / ml) in a 100 ml Erlenmeyer flask with some cell material (individual colonies) from a freshly grown plate ( ⁇ 1 week) these are incubated at 37 ° C. and 200 rpm in a shaker cabinet overnight.
  • the cultures are distributed to 10 50 ml Falcon tubes and ⁇ 20 min. centrifuged at 4000 rpm and 4 ° C. After the first centrifugation, the supernatant is discarded and the rest of the cultures are redistributed to the tubes, so that a 100 ml pellet results per Falcon tube. After centrifugation, the pellets can be shock-frozen in liquid nitrogen and frozen at -85 ° C.
  • a cell pellet of 0.5 g is mixed with 2.8 ml lysis buffer, 50 mM Tris-Cl, pH 8.0, 300 mM NaCl, 10% glycerol, 6 mM DTT, the 1 mg / ml lysozyme and 30 ⁇ l Sigma Protease- Inhibitor P8849 be added.
  • the resuspended pellets are then 20 min. shaken at 30 ° C and ⁇ 160 rpm (digestion of the cell wall by lysozyme). Subsequently, the cell suspensions are divided into 4 15 ml Falcon tubes and disrupted with the ultrasonic wand (SONIFIER) for approx.
  • SONIFIER ultrasonic wand
  • Frits are inserted into empty PDIO columns, checked for leaks or permeability with water or lysis buffer and hung in the drip rack.
  • the Ni-NTA enzyme mixture is carefully applied to the frits (in equal parts) and the flow rate is collected.
  • the mixture is then washed with imidazole concentrations of 5 and 10 mM in lysis buffer (see above) or eluted with imidazole concentrations of 100 mM.
  • a protein determination according to BioRad and an activity test of the PK-containing elution fractions are then carried out, and all samples are checked by SDS-PAGE. After checking the activity, fractions with sufficient activity are pooled and desalted or concentrated using the Millipore Ultrafree-15 centrifugation units, pore size 30 kDa.
  • the elution fractions (100 mM imidazole) are pooled and stored in storage buffer (50 mM MES, pH 6.2, 10% glycerol, 100 mM KC1, 15 mM MgSO 4 , 6 mM phosphoenol pyruvate, 6 mM DTT) Make up buffer to a total of 70 ml.
  • the enzyme solution thus filled up is distributed over 6 Millipore Ultrafreel5 filters / 30kDa and 30 min. Centrifuged at 2000 xg and 20 ° C.
  • Test batch 200 ⁇ l 0.2; 1; 5 and 10 U LDH (corresponding to final concentrations of
  • the microtiter plate is incubated at room temperature.
  • the decrease in optical density at 340 nm is measured over 15-30 minutes.
  • the inhibition test for finding modulators is carried out in 384-well microtiter plates.
  • the volume of the batches is 50 ⁇ l.
  • the enzyme preparation described in Example 2 is thawed and in dilution buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgSO 4 ; 30% (v / v) glycerol; 0.6 mM phosphoenol pyruvate; 6 mM DTT ; 20 ⁇ g / ml BSA) depending on the activity of the enzyme preparation diluted to 0.5 - 2 ⁇ g / ml protein concentration.
  • This enzyme solution is stored at room temperature.
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgSO 4 .
  • concentration of the substances is measured so that the final concentration is between 2 ⁇ M and 10 ⁇ M.
  • 10 ⁇ l of the enzyme solution are added and the reaction by adding 35 ⁇ l of substrate mix (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4 ; 0.36 mM ADP; 0.25 mM phosphoenolpyruvate; 0.36 mM NADH; 1.43 mM fructose-1, 6-bisphosphate and 7.1 U LDH / ml; stored on ice).
  • substrate mix 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4 ; 0.36 mM ADP; 0.25 mM phosphoenolpyruvate; 0.36 mM NADH; 1.43 mM fructose-1, 6-bisphosphate and 7.1 U LDH / ml; stored on ice.
  • the microtiter plate is incubated at room temperature. The decrease in optical density at 340 nm is measured over 15-30 minutes.
  • a measurement is used as a negative control in which no substance to be tested, but 5 ⁇ l of 0.5% DMSO in test buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4 ) is introduced and no enzyme solution, but purer Dilution buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4 ; 30% (v / v) glycerin; 0.6 mM phosphoenolpyruvate; 6 mM DTT; 20 ⁇ g / ml BSA) without Pyruvate kinase is added. Otherwise, this approach is treated like the approaches with substances to be tested.
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • purer Dilution buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4 ;
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • This approach is also treated like the approaches with substances to be tested.
  • the pyruvate kinase is omitted from the first solution and phosphoenol pyruvate is replaced by pyruvate.
  • the pyruvate is only added in the first solution, since in the presence of pyruvate in the substrate mix, the reaction already took place in the substrate mix.
  • a corresponding solution without pyruvate kinase with 1.5 mM pyruvate is prepared (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgSO 4 ; 30% (v / v) glycerol; 1, 5mM pyruvate; 6mM DTT; 20 ⁇ g / ml BSA).
  • This solution is stored at room temperature. 5 ⁇ l of the substances to be tested are placed in 5% (v / v) DMSO in test buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgSO) in the cavities of a 384-well microtiter plate.
  • the concentration of the substances is measured so that the final concentration is between 2 ⁇ M and 10 ⁇ M.
  • 10 ⁇ l of the pyruvate solution are added and the reaction is carried out by adding 35 ⁇ l of substrate mix for LDH measurement (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4 ; 0.36 mM ADP; 0.36 mM NADH; 1.43 mM fructose 1,6-bisphosphate and 4 U LDH / ml; stored on ice) started.
  • the microtiter plate is incubated at room temperature.
  • the decrease in optical density at 340 nm is measured over 15-30 minutes.
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • test buffer 50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 4
  • a methanolic solution of the active substance identified by an inventive method (Example 3), piped with an emulsifier, is pipetted into the cavities of microtiter plates. After the solvent has evaporated, 200 ⁇ l of potato dextrose medium are added to each cavity. Suitable concentrations of spores or mycelia of the fungus to be tested are added to the medium beforehand.
  • the resulting concentrations of the active ingredient are 0.1, 1, 10 and 100 ppm.
  • the resulting concentration of the emulsifier is 300 ppm.
  • the plates are then incubated on a shaker at a temperature of 22 ° C. until sufficient growth can be determined in the untreated control.
  • the evaluation is carried out photometrically at a wavelength of 620 nm.
  • the dose of active substance which leads to a 50% inhibition of fungal growth compared to the untreated control (ED 50 ) is calculated from the measurement data of the different concentrations.
  • active compound 1 part by weight of active compound is mixed with 49 parts by weight of N, N-dimethylformamide (solvent) and 1 part by weight of alkylaryl polyglycol ether (emulsifier) and the concentrate is diluted with water to the desired concentration (see Table ID).
  • solvent N, N-dimethylformamide
  • alkylaryl polyglycol ether emulsifier
  • young tomato plants are sprayed with the active substance preparation in the stated application rate. 1 day after the treatment, the plants are inoculated with a spore suspension of Phytophthora infestans and then remain at 100% rel. Humidity and 20 ° C. The plants are then placed in a climatic cell at approx. 96% relative air humidity and a temperature of approx. 20 ° C.
  • Evaluation is carried out 7 days after the inoculation. 0% means an efficiency that corresponds to that of the control, while an efficiency of 100% means that no infection is observed.
  • Wang polystyrene solid phase linker (“Wang resin”; Rapp-Polymer, Tübingen; 1.08 mmol / g) are swollen in DMF.
  • the solvent is filtered off with suction and a solution of 360 mg of bromoacetic acid (acid reagent) in 8 ml of dimethylformamide is added and shaken Room temperature for 15 minutes and then add 345 ⁇ l of pyridine and 543 mg of 2,6-dichlorobenzyl chloride to the solution. It is shaken for 2 h at room temperature. It is then suctioned off and the solid phase linker is washed with DMF.
  • the solid phase linker from step (a) is allowed to swell in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the solvent is filtered off with suction and treated with a solution of 1.5 g of l, l-dimemoxypropan-2-amine in 8.5 ml of DMSO and shaken overnight.
  • the solid phase linker is washed with DMF, methanol, tetrahydrofuran (THF) and dichloromethane.
  • step (b) 70 mg of the solid phase linker from step (b) are allowed to swell in DMF.
  • the solvent is filtered off with suction and a solution of 138 mg (2E) -3- (1,3-benzodioxol-5-yl) acrylic acid in 0.865 ml DMF and a solution of 118 mg diisopropylcarbodiimide in 0.21 ml DMF are added.
  • the mixture is stirred for 3 h at room temperature, the solvent is filtered off and washed with DMF.
  • a solution of 4.4 g of bromoacetic acid in 50 ml of DMF and a solution of 5.2 g of diisopropylcarbodiimide in 6 ml of DMF are then added.
  • the mixture is stirred at room temperature for 2 h, the solvent is filtered off with suction and washed with DMF.
  • a solution of 4.4 g of bromoacetic acid in 50 ml of DMF and a solution of 5.2 g of diisopropylcarbodiimide in 6 ml of DMF are then added again.
  • the mixture is again stirred at room temperature for 2 h, the solvent is filtered off and washed with DMF.
  • the solvent is filtered off with suction, a solution of 7.1 gl, lDimethoxypropan-2-amine in 30 ml DMSO is added and shaken overnight. It is then washed with DMF, methanol, THF and dichloromethane.
  • step (c) A solution of 77 mg of N-hydroxy-1-phenylmethanamine hydrochloride in 1.2 ml of ethanol / toluene (2:10) is added to the solid phase linker from step (c). The mixture is stirred at 70 ° C. for 6 h and then allowed to cool to room temperature. The solvent is suctioned off and the solid phase linker is washed with THF / methanol / water, THF and dichloromethane.
PCT/EP2004/013323 2003-12-03 2004-11-24 Verfahren zum identifizieren von fungizid wirksamen verbindungen basierend auf pyruvat-kinasen aus pilzen WO2005054457A1 (de)

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