WO2005054457A1 - Method for identifying fungicidal compounds from fungal pyruvate kinases - Google Patents

Abstract

The invention provides a method for identifying fungicides, the use of fungal pyruvate kinase for identifying fungicides and the use of inhibitors of pyruvate kinase as fungicides.

Description

Method for identifying fungicidally active compounds based on pyruvate kinases from fungi

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.

Unwanted fungal growth, which causes considerable damage in agriculture every year, can be controlled through the use of fungicides. The demands on fungicides have steadily increased in terms of their effectiveness, costs and, above all, their environmental compatibility. There is therefore a need for new substances or classes of substances that can be developed into powerful and environmentally compatible new fungicides. In general, it is common to search for such new lead structures in greenhouse tests. However, such tests are labor intensive and expensive. The number of substances that can be tested in the greenhouse is limited accordingly. An alternative to such tests is the use of high-throughput screening (HTS). A large number of individual substances are tested for their effects on cells, individual gene products or genes in an automated process. If an effect is proven for certain substances, these can be examined in conventional screening methods and, if necessary, further developed.

Favorable targets for 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. This is the second of the ATP-forming reactions (besides the phosphoglycerate kinase reaction) in glycolysis. 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). There are four different PK isoenzymes (L, R, Mi and M ₂ ) in mammals, which differ in their kinetic properties. 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. Q875M9), Yarrowia lipolytica (Swissprot Accession No. P30614), Schizosaccharomyces pombe (Swissprot Accession No. Q10208), Agaricus bispor No. 094122) and from Saccharomyces cerevisiae (PYK1, Swissprot Accession No. P00549 and PYK2, Swissprot Accession No. P52489). The sequence similarities are significant within the eukaryotic classes. Pyruvate kinase genes have also been annotated in DNA sequences of some phytopathogenic fungi originating from different sequencing projects due to sequence similarities, e.g. for Magnaporthe grisea (Locus 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).

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.

Description of the pictures

Figure 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.

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 μl 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.

SEQ ID NO: 2 amino acid sequence of Ustilago maydis pyruvate kinase.

definitions

The term "homology" or "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. As a standard, the 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 Moleculaire 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. a given reference protein and other proteins, the following parameters must be set: KTUPLE = 1, TOPDIAG = 5, WTNDOW = 5, PATRGAP = 3, GAPOPEN = 10, GAPEXTEND = 0.05, GAPDIST = 8, MAXDIV = 40, MATRLX = GONNET, ENDGAPS (OFF), NOPGAP, NOHGAP. 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. If such a database query is carried out, for example, at the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/), the standard settings that are specified for the respective comparison request should be used. For protein sequence comparisons ("blastp") these are the following settings: Limit entrez = not activated; Filter = low complexity activated; Expect value = 10; word size = 3; Matrix = BLOSUM62; Gap costs: Existence = 11, Extension = 1. As a result of such a query, the proportion of identity between the query sequence and the similar sequences found in the databases are shown in addition to other parameters. Under a protein according to the invention Therefore, in connection with the present invention, such 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%.

The term "hybridize" or "hybridization" as used herein describes the process in which a single-stranded nucleic acid molecule with a complementary strand undergoes base pairing. In this way e.g. starting from the sequence information mentioned or derivable here, for example, 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:

Melting temperature Tm = 81.5 ° C + 16.6 {log [c (Na ⁺ )]} + 0.41 (% G + C) - (500 / n) (Lottsspeich, F., Zorbas H. (ed. (1998) Bioanalytics, Spectrum Academic Publishing House, Heidelberg, Berlin). Here, c is the concentration and n is the length of the hybridizing sequence section in base pairs. The expression 500 / n is omitted for a sequence> 100 bp. It is washed with the highest stringency at a temperature of 5-15 ° C below Tm and an ionic strength of 15 mM Na ⁺ (corresponds to 0.1 x SSC). If an RNA sample is used for hybridization, the melting point is 10-15 ° C higher. 20 x SSC buffer is composed as follows: 1.5 M NaCl, 150 mM sodium citrate, pH 7.

Preferred hybridization conditions are given below:

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). In the present case, 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.

The term "complete pyruvate kinase" as used herein 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.

The term "enzymatic" 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.

The term "active fragment" as used herein 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.

The terms "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.

The term "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.

The term "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. graminea (conidial form: Drechslera, Syn: Helminthosporium), Cochliobolus species, such as, for example, Cochliobolus sativus (Conidial form: Drechslera, Syn: Helminthosporium), 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 species, such as, for example, Septoria nodorum, Leptosphaeria species, such as, for example, Leptosphaeria nodorum, Cercospora species, such as, for example, Cercospora canescens, Alternaria species, such as, for example, Alternaria brassicae or Pseudocercosporella species, such as, for example, Pseudocercosporella herpotrichoides.

Of particular interest are e.g. also Magnaporthe grisea, Cochliobulus heterostrophus, Nectria hematococca and Phytophtora species.

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.

The following human pathogenic fungi are of particular interest, which can cause the following clinical pictures: 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. reticuloendothelial cytomycosis and Darling disease, 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, and 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.

The term "homologous promoter" as used in the present context refers to a promoter which controls the expression of the gene in question in the original organism. The term "heterologous promoter" as used in the present context refers to a promoter which has different properties than the promoter which controls the expression of the gene in question in the original organism.

The term “competitor” as used in the present context 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.

The term "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.

The term "modulator" as used herein 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. Furthermore, 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. The term "modulator" as used herein is preferably, however, those molecules which do not represent the natural substrates or ligands.

Description of the invention

In the present invention, 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.

Despite the extensive research on pyruvate kinase, it was previously unknown that pyruvate kinase in fungi can be a target protein (a so-called "target") of fungicidally active substances. This is the first time that the present invention shows that 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. In the context of the present invention, it has now been possible to show that 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.

It was also possible to show that the enzyme 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. Finally, it could also be shown that inhibitors of pyruvate kinase, which could be identified in such processes, can be used as fungicides.

In the context of the present invention, 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.

In the context of the present invention, it was found that 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. In the present invention it is shown, for example, that 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. For the production of pyruvate kinase from fungi, 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.

For the expression of the pyruvate kinase from U. maydis, the associated ORF was amplified by means of PCR using selected primers using methods known to the person skilled in the art. After amplification of the pyruvate kinase gene from Ustilago maydis genomic DNA by means of PCR, Subsequent restriction and ligation with the vector pET-21b (+) from Novagen (C-terminal fusion with the His tag), the transformation into E. coli DH5α MAX Efficiency Competent Cells (Gibco) took place. (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 | bp and it contains no intron Gene codes for a polypeptide of 528 amino acids in length with a molecular weight of 39000 daltons (Figure 2).

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.

These two amino acid residues, like their sequence environment, are a characteristic feature of the sequence of pyruvate kinases. This 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:

where the residue K stands for lysine in the active center and the residue E for the glutamine involved in the binding of the magnesium ion.

Pyruvate kinases with the sequence motif are preferred

[1V] -X- [IV] -I- [SPACV] -K- [IV] -E- [NS] -X- [EQ] -G- [LΓVM]

| Used. The motif can be seen in Figure 2.

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, however, 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. Using this motif, 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.

In the case of the pyruvate kinase from U. maydis, 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).

The above-mentioned 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- | rKRST] -x- [DEQHS] - [GSTA] - [LIVM]. Those polypeptides which comprise the prosite motif [IV] -x- [IV] -I- [SPACV] -K- [ΓV] -E- [NS] -X- [EQ] -G- [LIVM] are preferred ,

Because of the homologies that exist for species-specific nucleic acids coding for pyruvate kinases, 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. However, it is also conceivable to use 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. Due to the The sequence given here according to SEQ ID NO: 1 and any primers derived therefrom and, if appropriate, with the aid of the prostite motif shown above, it is possible for the person skilled in the art to obtain and identify further nucleic acids coding for pyruvate kinases from other (phytopathogenic) fungi, for example by PCR. Such nucleic acids and their use in methods for identifying fungicidal active substances are considered to be encompassed by the present 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). Preferred embodiments are fragments of genomic DNA, and in particular cDNAs.

Particularly preferably, 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

a) a sequence according to SEQ ID NO: 1,

b) sequences which code for a polypeptide which comprises the amino acid sequence according to SEQ TD NO: 2,

c) Sequences coding for a polypeptide which has the motif [LrVAC] -x- [LTVM] (2) - [SAPCV] -K- [LIV] -E- [NKRST] -x- [DEQHS] - [GSTA ] - [LIVM] includes

d) sequences which hybridize to the sequences defined under a) and b) at a hybridization temperature of 42-65 ° C., and e) sequences which have at least 80%, preferably at least 85% and particularly preferably at least 90% identity with the sequences defined under a) and b).

As already stated above, the present invention is not restricted only to the pyruvate kinase from Ustilago maydis. In an analogous manner known to those skilled in the art, 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.

The selection of heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression. Examples of 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. For insect cells, 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. Further advantageous 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.

The term "host cell" as used in the present context 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.

The polypeptides which can be used in accordance with the invention preferably comprise an amino acid sequence selected from phytopathogenic fungi

(a) the sequence according to SEQ ID NO: 2,

(b) sequences which have at least 70%, preferably at least 80%, particularly preferably 90% and particularly preferably 95% identity with the sequence defined under a),

(c) the sequences given under b) which contain the prosite motif [LIVAC] -x- [LιVM] (2) - [SAPCV] -K- [LJN] -E- [NKRST] -x- [DEQHS] - [ GSTA] - [LIVM] include, and

(d) Fragments of the sequences given under a) to c), which have the same biological activity as the sequence defined under a).

The term "polypeptides" as used herein 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. They 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.

The 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.

The 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:

1. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and

5. 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.

Further possible 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.

The terms "isolation or purification" as used in the present context mean that the polypeptides according to the invention are separated from other proteins or other macromolecules of the cell or the tissue. A 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.

The polypeptides according to the invention can also be affinity-purified without a fusion partner using antibodies which bind to the polypeptides.

The method of making polypeptides with pyruvate mase activity, e.g. the pyruvate kinase from U. maydis is characterized by

(a) culturing a host cell containing at least one expressible nucleic acid sequence coding for a polypeptide from fungi with the biological activity of a pyruvate kinase under conditions which ensure the expression of this nucleic acid, or (b) expressing an expressible nucleic acid sequence coding for a polypeptide from fungi with the biological activity of a pyruvate kinase in an in vitro system, and

(c) obtaining the polypeptide from the cell, the culture medium or the in vitro system.

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

Activity or the biological functionality of the polypeptide. In principle, methods based on whole cells (in vivo methods) as well as methods based on are based on the use of the polypeptide isolated from the cells, which can be present in purified or partially purified form or as a crude extract. These cell-free in vitro methods can be used just like in vivo methods on a laboratory scale, but preferably also in HTS or UHTS methods. Following the in vivo or in vitro identification of modulators of the polypeptide, tests can be carried out on fungal cultures in order to test the fungicidal activity of the compounds found.

Many 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. In a first step, 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.

To find 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.

The detection of the biological activity of the polypeptides according to the invention can be improved by a so-called reporter system. Reporter systems in this regard 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. However, 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.

Effects such as cell toxicity are usually ignored in these in vitro systems. The 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) 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,

b) comparing the activity of the polypeptide according to the invention in the absence of a chemical compound with the activity of the polypeptide according to the invention in the presence of a chemical compound or a mixture of chemical compounds, and

c) selects the chemical compound which modulates the activity of the polypeptide according to the invention, and, if appropriate

d) tests the fungicidal activity of the selected compound in vivo.

The compound which specifically inhibits the activity of the polypeptide according to the invention is particularly preferably determined. The term "activity" as used here refers to the biological activity of the polypeptide according to the invention.

In a preferred embodiment, 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. The reaction can be shown schematically as follows:

PK phosphoenolpyruvate + ADP + H + - pyruvate + ATP LDH pyruvate + NADH + ET * ^" -> lactate + NAD ⁺

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. When pyruvate kinase is inhibited, less or no pyruvate is formed, so the LDH reaction takes place to a lesser extent and less or no NADH is consumed.

Since an inhibition of LDH would also lead to a reduced decrease in NADH, inhibitors found in the test described above must be checked for any inhibition of LDH.

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 (candidate molecule) is e.g. in a suitable concentration in test buffer containing ATP, phosphoenol pyruvate, and NADH and the auxiliary enzyme lactate dehydrogenase. Then 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.

In order to determine optimal conditions for a method for identifying inhibitors of pyruvate kinase or for determining the activity of the polypeptides according to the invention, it may be advantageous to determine the respective K _M value of the polypeptide according to the invention used. This provides information about the preferred concentration of the substrate or substrates. In the case of U. maydis pyruvate kinase, a K _M of 0.1 mM for phosphoenolpyruvate and a K _M of 0.22 mM for ADP could be determined.

In this way, 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.

Table I

The compounds 1 and 2 are known cyclopentabenzofuran derivatives which have been described in connection with the therapy of NF-κB-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.

It goes without saying that in addition to the methods mentioned for determining the enzymatic activity of a pyruvate kinase or the inhibition of this activity and for identifying fungicides, others, e.g. Already known methods or inhibition tests can be used as long as these methods make it possible to determine the activity of a pyruvate kinase and to recognize an inhibition of this activity by a candidate compound.

In the context of the present invention, it was also found that the 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.

In order to determine ED ₅₀ values, 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 μm. 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 π 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

It is also possible to test the fungicidal activity of the compounds found by a method according to the invention on the phytophthora infestans fungus which is pathogenic to plants.

To do this, the active ingredient to be tested is mixed with a solvent and emulsifier and the concentrate is diluted with water to the desired concentration. To test 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.

Table HI

Based on these results, it is shown by way of example that 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.

Compounds which are identified with the aid of a method according to the invention and which have a fungicidal action due to the inhibition of the fungal pyruvate kinase can thus be used for the preparation of fungicidal compositions.

Depending on their respective physical and / or chemical properties, 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.

These 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. The following are essentially suitable as liquid 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. The following are suitable as 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. broken and fractionated natural rocks such as calcite, marble, pumice, sepiolite, dolomite as well as synthetic granules from inorganic and organic flours as well as granules from organic material such as sawdust, coconut shells, corn cobs and tobacco stalks. 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, Feπocyanblau 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, as such or in their formulations, 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. In many cases, synergistic effects are obtained, i.e. the effectiveness of the mixture is greater than the effectiveness of the individual components.

When using the compounds according to the invention as fungicides, the application rates can be varied within wide ranges depending on the application.

According to the invention, all plants and parts of plants can be treated. 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 following examples illustrate various aspects of the present invention and are not to be interpreted as limiting.

Examples

example 1

Cloning and expression of U. maydis pyruvate kinase

For the heterologous expression of the pyruvate kinase gene, 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 ebensoovagen. The resulting plasmid pEB017 thus codes for a pyruvate kinase with a C-terminal His ₆ tag

To express the PK, the plasmid pEB017 is transformed into E. coli AD494 (DE3). An overnight culture (ÜΝK) is set up by inoculating 25 ml LB-ampicillin medium (c [ampicillin]: 100 μg / 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 next morning, 1000 ml LB-ampicillin medium is divided into 5 100 ml Erlenmeyer flasks, each with 200 ml and inoculated with ~ 3 ml each of the ÜNK. A further incubation at 37 ° C. and 200 rpm follows until the cultures calibrate an optical density of OD ₆₀ o ~ 1 (approx. 3-4 h). Then, for induction, 200 ml of culture are added to 2 ml of EPTG (100 mM stock solution) (final concentration: 1 mM) and the cultures are shaken overnight at 18 ° C. and 200-220 rpm.

The next morning, 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.

Example 2

Purification of pyruvate kinase from U. maydis

cell disruption

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 μl 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. 10 x ~ 15 sec, while stirring in an ice-water bath, until the suspension looks opaque. Since the PK is unstable to cold, the sample should only be cooled slightly for ultrasonic digestion. The cell suspension sonicated in this way is distributed over 2 ml of Eppendorf tubes and centrifuged for 25 min at 10,000 × g. The supernatant is then divided into 2 x 50 ml Falcon tubes and 0.45 ml of resuspended Ni-NTA agarose per ml of supernatant is added to each tube. The mixture is now <60 min. incubated at room temperature.

chromatography

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.

Concentrate / desalt (dialysis of hnidazole

After checking the activity, 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. Ultimately, approximately 0.25 ml of concentrated enzyme solution remain, which are combined and filled with fresh storage buffer, pure glycerol and BSA in such a way that the enzyme solution has a final concentration of 50% glycerol, 20 μg / ml BSA and an enzyme concentration of approx. 42 mg / ml.

1.5 ml aliquots are filled from this solution and frozen at -85 ° C.

Example 3

Determination of the enzymatic activity of pyruvate kinase by coupling the reaction with the reaction of lactate dehydrogenase 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

phosphoenolpyruvate; 6mM DTT; 20 μg / ml BSA) depending on the activity of the enzyme preparation to 0.5 -

Diluted 2 μg / ml protein concentration. This enzyme solution is stored at room temperature. In the wells of a 96-well microtiter plate, 20 ul 5% (v / v) DMSO in test buffer (50 mM

MES, pH 6.2; 100 mM KC1; 15 mM MgS0 ₄ ) submitted, 40 ul of the enzyme solution added and the reaction by adding 140 ul substrate mix (50 mM MES, pH 6.2; 100 mM KC1; 15 mM

MgS0 ₄ ; 0.36 mM ADP; 0.25 mM phosphoenol pyruvate; 0.36 mM NADH; 1.43 mM fructose 1,6-bisphosphate and various concentrations of lactate dehydrogenase; stored on ice) started. The concentrations of the LDH in the substrate mix were chosen so that the total

Test batch (200 μl) 0.2; 1; 5 and 10 U LDH (corresponding to final concentrations of

1, 5, 25 and 50 U LDH / ml). The microtiter plate is incubated at room temperature. The decrease in optical density at 340 nm is measured over 15-30 minutes.

Example 4

Identification of modulators of pyruvate kinase in an inhibition test

The inhibition test for finding modulators is carried out in 384-well microtiter plates. The volume of the batches is 50 μl.

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 μg / ml BSA) depending on the activity of the enzyme preparation diluted to 0.5 - 2 μg / ml protein concentration. This enzyme solution is stored at room temperature. 5 μl 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 ₄ ) in the cavities of a 384-well microtiter plate. The concentration of the substances is measured so that the final concentration is between 2 μM and 10 μM. 10 μl of the enzyme solution are added and the reaction by adding 35 μl 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). 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 μl 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 μg / ml BSA) without Pyruvate kinase is added. Otherwise, this approach is treated like the approaches with substances to be tested.

A measurement in which no substance to be tested but 5 μl of 0.5% DMSO in test buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 ₄ ) and the same enzyme solution as in is added to the batches with substances to be tested. This approach is also treated like the approaches with substances to be tested.

Negative and positive control result in the comparative values for the batches with substances to be tested.

Test for modulation of the LDH

Substances that caused an increase or decrease in enzyme activity in the test described are checked in a second test for the modulation of the LDH:

In this test, 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.

Instead of the enzyme solution described in Example 2, 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; 20µg / ml BSA). This solution is stored at room temperature. 5 μl 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 μM and 10 μM. 10 μl of the pyruvate solution are added and the reaction is carried out by adding 35 μl 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.

A measurement in which no substance to be tested but 5 μl of 0.5% DMSO in test buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 ₄ ) is used as a negative control and from the substrate mix for the LDH measurement the enzyme is omitted. Otherwise, this approach is treated like the approaches with substances to be tested. A measurement in which no substance to be tested, but 5 μl of 0.5% DMSO in test buffer (50 mM MES, pH 6.2; 100 mM KC1; 15 mM MgS0 ₄ ) and the same solutions as in be added to the batches with substances to be tested. This approach is also treated like the approaches with substances to be tested.

Negative and positive control give the comparison values for the batches with substances to be tested.

Example 6

Detection of the fungicidal activity of the identified inhibitors of pyruvate kinase in microtiter plates

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 μl 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.

Example 7

Evidence of the fungicidal activity of the identified inhibitors of pyruvate kinase in the greenhouse (Phytophthora Υest, protective)

To produce a suitable preparation of 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).

To test the protective effectiveness of the identified compounds, 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.

Example 8

Unless otherwise stated, all quantitative information is percent by weight. The retention time is given in minutes and was determined by means of HPLC with an RC column (Eurospher 100, C18, ID 4 mm) and by means of UV absorption. Eluent: 0.1% acetonitrile / water (0 min = 10% acetonitrile; 13 min = 80% acetonitrile; 15 min = 80% acetonitrile; 17 min = 10% acetonitrile).

Preparation of compound 3 according to Table I

Compound 3 can be synthesized according to the following reaction scheme:

Wang Resin

Connection 3

Step (a):

1.2 g of 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 μl 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. A further 360 mg of bromoacetic acid in 8 ml of DMF are added and the mixture is shaken for 15 minutes. 345 μl of pyridine and 543 mg of 2,6-dichlorobenzyl chloride are then added. The mixture is shaken overnight at room temperature. The solid phase linker is finally washed with DMF, methanol and dichloromethane.

Step (b):

The solid phase linker from step (a) is allowed to swell in dimethyl sulfoxide (DMSO). 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 (c):

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 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 once more and the mixture is added for 3 h stirred at room temperature. It is then suctioned off and the solid phase linker is washed with methanol / THF, THF and dichloromethane.

Step (ά):

A solution of 173 mg of N-hydroxy-l- [3- (trifluoromethyl) phenyl] propan-2-amine 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.

Steps):

The solid phase linker from step (d) is stirred in 1 ml of trifluoroacetic acid / dichloromethane (1: 1) for 1 h, filtered off and the filtrate is concentrated in vacuo. 18 mg of compound 3 are obtained. MS (ESI): 507 [M + H0]. Retention time (HPLC): R _t = 8.8. Preparation of compound 4 according to Table I

The synthesis of compound 4 can be carried out according to the following reaction scheme:

Connection 4

Step (a):

4.2 g of a Wang solid phase linker loaded with Fmoc-L-proline (Rapp-Polymer Tübingen; 0.75 mmol / g) are allowed to swell in DMF. The solvent is suctioned off and the solid phase linker is left for 5 min. treated with 32 ml of a piperidine solution (20% piperidine in DMF), the solvent is suctioned off and a further 32 ml of the piperidine solution are added. The mixture is stirred for 30 min. at room temperature, sucks off the solvent and washes 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.

Step (b): The solid phase linker from step (a) is allowed to swell in DMSO. 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.

Steps):

70 mg of the solid phase linker from step (b) are allowed to swell in DMF. After the solvent has been suctioned off, a solution of 98 mg (2E) -3- (1,3-benzodioxol-5-yl) acrylic acid in 0.865 ml DMF and a solution of 84 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 98 mg (2E) -3- (1,3-benzodioxol-5-yl) acrylic acid in 0.865 ml DMF and a solution of 84 mg diisopropylcarbodiimide in 0.21 ml DMF are added once more and the mixture is stirred at room temperature for 3 h. It is then suctioned off and the solid phase linker is washed with methanol / THF, THF and dichloromethane.

Step (ä):

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.

Step (egg:

The solid phase linker from step (d) is stirred in 1 ml of trifluoroacetic acid / dichloromethane (1: 1) for 1 h, filtered off and the filtrate is concentrated in vacuo. 17 mg of compound 4 are obtained. MS (ESI): 508 [M + H ^]. Retention time (HPLC): R _t = 8.6.

literature

1. Pyruvate kinase. Muirhead, Hilary. Dep. Biochem., Univ. Bristol, Bristol, UK. Biological Macromolecules and Assemblies (1987), 3, 143-86.

2. The structure of cat muscle pyruvate kinase. Muirhead, H .; Clayden, D.A .; Barford, D .; Lorimer, C.G .; Fothergill-Gilmore, L.A .; Sagittarius.; Schmitt, W. Dep. Biochem., Univ. Bristol, Bristol, UK. EMBO Journal (1986), 5 (3), 475-81.

3. Pyruvate kinase in yeast sugar metabolism. Boles, Eckhard. Institute for Microbiology, Technical University Darmstadt, Germany. Editor (s): Zimmermann, Friedrich K .; Entian, Karl-Dieter. Yeast Sugar Metabolism (1997), 171-186.

4. The tumor metabolome. Mazurek, Sybille; Eigenbrodt, Erich. Institute for Biochemistry and Endocrinology, Veterinary Faculty, Justus-Liebig-University of Giessen, Giessen, Germany. Anticancer Research (2003), 23 (2A), 1149-1154.

5. The structure of pyruvate kinase from Leishmania mexicana reveals details of the allosteric transition and unusual effector specificity. Date in: J. Mol. Biol. (1999) Oct 29; 293 (3): 145-749. Rigden D J; Phillips S E; Michels P A; Fothergill-Gilmore L A, J. Mol. Biol. (1999), 291 (3), 615-35.

6. The allosteric regulation of pyruvate kinase by fructose-l, 6-bisphosphate. Jurica, Melissa S .; Mesecar, Andrew; Heath, Patrick J .; Shi, Wuxian; Nowak, Thomas; Stoddard, Barry L. Molecular and Cellular Biology Program, Fred Hutchinson. Structure (1998), 6 (2), 195-210.

7. Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition. Mattevi A; Valentini G; Rizzi M; Speranza M L; Bolognesi M; Coda A. Structure (1995), 3 (7), 729-41.

8. The PROSITE database, its status in 1999. Hofmann K., Bucher P., Falquet L., Bairoch A. NucleicAcids Res. (1999), 27, 215.

9. US 2001/0046997 AI

10. WO 01/96535 A2

11. WO 02/095063 AI

12. WO 00/08007 A2

Claims

claims

1. A method of identifying fungicides, characterized in that one

(a) contacting a fungal polypeptide with the enzymatic activity of a pyruvate kinase with a chemical compound or a mixture of chemical compounds under conditions which allow the chemical compound to interact with the polypeptide,

(b) compares the activity of the pyruvate kinase in the absence of a chemical compound with the activity of the pyruvate kinase in the presence of a chemical compound or a mixture of chemical compounds, and (c) selects the chemical compound that specifically inhibits the pyruvate kinase.

2. The method according to claim 1, characterized in that the polypeptide is selected from

(a) the sequence according to SEQ ID NO: 2,

(b) sequences which have at least 70%, preferably at least 80%, particularly preferably 90% and particularly preferably 95% identity with the sequence defined under a),

(c) the sequences given under b), which contain the prosite motif [LIVAC] -x- [LIVM] (2) - [SAPCV] -K- | TIV] -E- | NKRST] -x- [DEQHS] - [ GSTA] - [LIVM], and (d) fragments of the sequences given under a) to c), which have the same enzymatic activity as the sequence defined under a).

3. The method according to claim 1 or 2, characterized in that the activity of the pyruvate kinase is determined by

(a) the pyruvate formed in the reaction of the pyruvate kinase is converted to lactate and NAD ⁺ by lactate dehydrogenase using NADH, and

(b) the decrease in the NADH concentration in the absence of a chemical compound with the decrease in the NADH concentration in the presence of a chemical compound or a mixture of chemical compounds, and

(c) selects the chemical compound in the presence of which a smaller decrease in NADH concentration is observed.

4. The method according to any one of claims 1 to 3, characterized in that in a further step the fungicidal activity of the identified compound is tested by bringing it into contact with a fungus.

5. The method according to any one of claims 1 to 4, characterized in that one uses a pyruvate kinase from a phytopathogenic fungus.

6. Use of polypeptides with the activity of a pyruvate kinase to identify fungicides.

7. Use of inhibitors of polypeptides with the activity of a pyruvate kinase as fungicides.

8. A method for controlling phytopathogenic fungi, characterized in that an inhibitor of the fungal pyruvate kinase is allowed to act on the fungus and / or its surroundings.

9. Fungicidal agents, characterized by a content of an inhibitor of fungal pyruvate kinase and conventional extenders and / or surface-active substances.

10. Nucleic acid coding for a pyruvate kinase from a phytopathogenic fungus, characterized in that it comprises a sequence which is selected from: a) a sequence according to SEQ ID NO: 1, b) sequences which code for a polypeptide which comprises the amino acid sequence according to SEQ ID NO: 2.

11. DNA construct comprising a nucleic acid according to claim 10 and a heterologous promoter.

12. Vector comprising a nucleic acid according to claim 10 or a DNA construct according to claim 11.

13. Vector according to claim 12, characterized in that the nucleic acid is functionally linked to regulatory sequences which ensure the expression of the nucleic acid in pro- or eukaryotic cells.

14. Host cell containing a nucleic acid according to claim 10, a DNA construct according to claim 11 or a vector according to claim 12 or 13.

15. Pyruvate kinase from phytopathogenic fungi, characterized in that it comprises a sequence according to SEQ ID NO: 2.