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EP1644503A1 - Procede d'identification de composes a action fongicide au moyen de mevalonate kinases provenant de champignons - Google Patents

Procede d'identification de composes a action fongicide au moyen de mevalonate kinases provenant de champignons

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Publication number
EP1644503A1
EP1644503A1 EP04740191A EP04740191A EP1644503A1 EP 1644503 A1 EP1644503 A1 EP 1644503A1 EP 04740191 A EP04740191 A EP 04740191A EP 04740191 A EP04740191 A EP 04740191A EP 1644503 A1 EP1644503 A1 EP 1644503A1
Authority
EP
European Patent Office
Prior art keywords
mevalonate kinase
mevalonate
polypeptide
kinase
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04740191A
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German (de)
English (en)
Inventor
Rüdiger SUELMANN
Edda Koopmann
Axel Trautwein
Karl-Heinz Kuck
Martin Adamczewski
Bernhard Grimmig
Rolf Kirsten
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer CropScience AG
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Bayer CropScience AG
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Filing date
Publication date
Application filed by Bayer CropScience AG filed Critical Bayer CropScience AG
Publication of EP1644503A1 publication Critical patent/EP1644503A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase

Definitions

  • the invention relates to a method for identifying fungicides, the use of mevalonate kinase to identify fungicides, and the use of inhibitors of mevalonate kinase as fungicides.
  • fungicides which are then used in the HTS process mentioned. 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 based thereon which enables the identification of modulators of this point of attack, preferably in a high-throughput method, in order to enable the identification to enable new fungicides.
  • Figure 1 Schematic representation of the reaction catalyzed by the mevalonate kinase.
  • the mevalonate kinase catalyzes the reaction of (R) -evalonate and adenosine triphosphate to (R) -5-phosphomevalonate and adenosine diphosphate.
  • Figure 2 Homology between Mevalonate kinases from different fungi. (1) Saccharomycs cerevisiae, (2) Schizosaccharomyces pombe, (3) Ustilago maydis, (4) Neurospora crassa, (5) Magnaporthe grisea. Frames represent areas with a precisely matching sequence (consensus sequence).
  • FIG. 3 SDS gel to show the course of the heterologous expression of the Mevalonat kinase in E. coli BL21 (DB3V)
  • the overexpressed GST fusion protein has a size of 74.5 kDa. Size standards were applied in lanes M. Lane 1: Pellet fraction; lane 2: cytoplasm fraction of the overexpressed mevalonate kinase (3 hours after induction with 100mM IPTG at 30 ° C); lane 3: wash fraction after application of the cytoplasm fraction to the glutathione-Sepharose column; lane 4: elution fraction with purified mevalonate kinase.
  • Figure 4 Kinetics of the conversion of mevalonate and adenosine triphosphate at different concentrations of mevalonate kinase in the assay.
  • an assay volume of 50 ⁇ l 300 ⁇ M adenosine triphosphate, 500 ⁇ M mevalonate, 300 ⁇ M NADH, 400 ⁇ M phosphoenolpyruvate, 0.2 U pyruvate kinase and 0.4 U lactate dehydrogenase as well as different amounts of mevalonate kinase were used.
  • the protein concentrations of the mevalonate kinase used can be seen in the figure.
  • the implementation of the mevalonate was monitored using the coupled reaction with pyruvate kinase and lactate dehydrogenase.
  • the implementation of the ATP by means of mevalonate is followed by the coupled decrease in absorption at 340 nm (decrease in the NADH in a coupled reaction).
  • SEQ ED NO: 1 nucleic acid sequence coding for the mevalonate kinase from Ustilago maydis. The sequence given corresponds to the genomic DNA.
  • 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, for example, 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 can also be accessed from various websites, including the IGBMC (Institut de Gen ⁇ tique 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/) as well as on all mirrored internet pages of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).
  • 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 by means of 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.
  • a protein according to the invention should therefore be understood in connection with the present invention to mean those proteins which, when using at least one of the above-described Above methods for identity determination have an identity of at least 50%, preferably of at least 60%, particularly preferably of at least 70%, more preferably of at least 80%, and in particular of at least 90%.
  • complete mevalonate kinase describes a mevalonate kinase encoded by a complete coding region of a transcription unit, comprising the ATG start codon and all information-bearing exon regions of the gene coding for mevalonate kinase present in the organism of origin, and the signals necessary for a correct termination of the transcription.
  • enzyme activity of a mevalonate kinase refers to the ability of a polypeptide to carry out the reaction described above, i.e. to catalyze the conversion of mevalonate and adenosine triphosphate to phosphomevalonate and adenosine diphosphate.
  • the reaction described here represents the reaction which is primarily catalyzed by the polypeptide.
  • active fragment no longer describes complete nucleic acids coding for mevalonate kinase, but which still code for polypeptides with the enzymatic activity of a mevalonate kinase and which are a reaction characteristic of the mevalonate kinase as described above can catalyze. Such fragments are shorter than the complete nucleic acids encoding the mevalonate kinase described above. Nucleic acids may have been removed at both the 3 'and / or 5' ends of the sequence, but parts of the sequence can also be deleted, i.e. that have not significantly affected the biological activity of the mevalonate kinase.
  • a lower or possibly also an increased activity, which, however, still allows the characterization or use of the resulting mevalonate 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 mevalonate kinase and then applies analogously to the above explanations for those polypeptides which, in comparison to the complete sequence defined above, no longer contain certain parts, the enzymatic activity of the enzyme, however, not being decisive is impaired.
  • the fragments can have different lengths.
  • mevalonate kinase inhibition test refers to a method or a test which allows the inhibition of the enzymatic activity of a polypeptide with the enzymatic activity of a mevalonate kinase by one or more to recognize chemical compounds (candidate compound (s)), whereby the chemical compound can be identified as an inhibitor of mevalonate kinase or as a fungicide.
  • 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 in the present context refers to chemical compounds which are suitable for combating fungi, in particular phytopathogenic fungi. Such phytopathogenic fungi are mentioned below, the list is not exhaustive:
  • 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, Peronospora species, such as, for example, Peronospora pisi or P.
  • Erysiphe species such as, for example, Erysiphe graminis
  • Sphaerotheca species such as, for example, Sphaerotheca fuliginea
  • Podosphaera species such as, for example, Podosphaera leucotricha
  • Venturia species such as Venturia inaequalis
  • Pyrenophora species such as Pyrenophora teres or P.
  • Drechslera conidial form: Drechslera, Syn: Hehninthosporium
  • Cochliobolus species such as Cochliobolus sativus (Konidienfonn: Drechslera, Syn: Hehninthosporium species) such as Uromyces appendiculatus
  • Puccinia species such as Puccinia recondita, 'Sclerotinia species.
  • Sclerotinia sclerotiorum such as, for example, Sclerotinia sclerotiorum, Tilletia species, such as, for example, Tületia 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 oi ⁇ zae, Fusarium species.
  • Fusarium culmorum Botrytis species, Septoria species such as Septoria nodorum, Leptosphaeria species such as Leptosphaeria nodorum, Cercospora species such as Cercospora canescens, Alternaria species such as Alternaria brassicae or Pseudocercosporella species such as for example Pseudocercosporella herpotrichoides.
  • fungicidal active ingredients which are found with the aid of the mevalonate kinase according to the invention can also interact with mevalonate kinases from fungal species which are pathogenic to humans, where the interaction with the different mevalonate kinases found in these fungi need not always be the same.
  • the present invention therefore also relates to a method for identifying antifungals, i.e. of inhibitors of mevalonate kinase from human or animal pathogenic fungi, which can be used to prepare agents for the treatment of diseases caused by human or animal pathogenic fungi.
  • 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 M., Coccidioides immitis, z.
  • Aspergillus fumigatus, A. flavus, A. niger e.g. cause bronchopulmonary aspergillosis or fungal sepsis, Mucor spec, Absidia spec, or Rhizopus spec,
  • fungicidal or “fungicide” shall apply equally to the terms “antifungal” or “antifungal” as well as for the terms “fungicidal” or “fungicide” in the conventional sense, i.e. related to phytopathogenic fungi.
  • Fungicidal active ingredients which are found with the aid of a mevalonate kinase obtained from a particular fungus, here for example from U. maydis, can therefore also interact with a mevalonate kinase from numerous other fungus species, in particular also with other phytopathogenic fungi, the interaction with the different Meva- found in these mushrooms.
  • lonat kinases do not always have to be equally strong. Among other things, this explains the observed selectivity of the substances active on this enzyme. - 1 -
  • competitive refers to the property of the compounds to compete with other, optionally identifiable compounds in order to compete for binding to the mevalonate kinase and to displace or be displaced by the enzyme.
  • agonist refers to a molecule that accelerates or enhances the activity of the mevalonate kinase.
  • antagonist refers to a molecule that slows or prevents the activity of the mevalonate kinase.
  • modulator represents the generic term for agonist or antagonist.
  • 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 mevalonate kinase (EC 2.7.1.36), hereinafter also abbreviated to MK, catalyzes the reaction of mevalonate and adenosine triphosphate to phosphomevalonate and adenosine diphosphate ( Figure 1).
  • the reaction catalyzed by the mevalonate kinase is an essential step at the beginning of the biosynthesis of ergosterol, dolichol or ubiquinone (Lees et al., 1997, Biochemistry and molecular biology of sterol synthesis in Saccharomyces cerevisiae. Biochemistry and Function of Sterols, 85-99; Mercer, 1984, The biosynthesis of ergosterol. Pestic. Sci. 15 (2), 133-55; Karst and Lacroute, 1977, Ergosterol Biosynthesis in Saccharomyces cerevisiae. Mol.Gen.Genet. 15, 269-277).
  • mevalonate kinase was also found in numerous other organisms, e.g. in Homo sapiens (Swissprot: Accession No .: AK023087), Mus musculus (Swissprot: Accession No .: BC005606) or Oryza sativa (Swissprot: Accession No .: AC091749).
  • sequence similarities between different MKs are significant within the eukaryotic classes, whereas the sequence identity to the bacterial enzymes is less significant.
  • the mevalonate kinase was isolated from various organisms, expressed. purified and also characterized (Tanaka et al., 1990, Purif ⁇ cation and regulation of mevalonate kinase from rat liver J. Biol. Chem. 265 (4), 2391-98; Chu, Xiusheng and Li, Ding, 2003, Cloning, expression, and purification of His-tagged rat mevalonate kinase. Prot. Exp. Purific. 27, 165-70; Schulte et al., 2000, Purification and characterization of mevalonate kinase from suspension-cultured cells of Catharanthus roseus (L.) G. Don Are. Biochem. Biophys.
  • the mevalonate kinase in fungi can be a target protein (a so-called "target") of fungicidally active substances.
  • the mevalonate kinase is not only an enzyme which is particularly important for fungi and is therefore particularly suitable for being used as a target protein for the search for further and improved fungicidally active compounds, but rather also that the fungal mevalonate kinase can actually be influenced in vitro and also in vivo by active substances and that these can be used as fungicides. Methods are also provided that can be used to identify such fungicides.
  • a method was developed which is suitable for determining the enzymatic activity of mevalonate kinase and the inhibition of this activity by one or more substances in a so-called inhibition test, and in this way modulators, preferably inhibitors of the enzyme, To be identified, for example, in HTS and UHTS processes. Identified inhibitors which already show an inhibiting effect on a given mevalonate kinase in vitro can then be tested in vivo for their fungicidal action.
  • the inhibitors of a fungal mevalonate kinase can be used as fungicides, in particular in crop protection or as antifungals in pharmaceutical indications.
  • the inhibition of the mevalonate kinase with a substance identified in a method according to the invention leads to the death or damage of the treated fungi in synthetic media or on the plant.
  • Mevalonate kinase can be obtained from various fungi that are pathogenic to plants, humans or animals, e.g. from fungi such as the plant pathogenic fungus U maydis.
  • the gene can e.g. recombinantly expressed in Escherichia coli and an enzyme preparation is produced from E. coli cells (Example 1).
  • Mevalonate 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 mevalonate kinases from human or animal pathogenic fungi is recommended.
  • the associated ORF open reading frame
  • UmErgl2 encoded by Umergl2 Um stands for Ustilago maydis
  • SEQ FD NO: 1 Methods known to those skilled in the art are amplified using gene-specific primers.
  • the corresponding DNA was cloned into the vector pDEST15 (Invitrogen, enables the introduction of an N-terminal GST tag) according to the manufacturer's instructions.
  • the resulting plasmid pDEST15_umergl2 contains the complete coding sequence of Umergl2 in an N-terminal fusion with a GST tag from the vector.
  • the UmErgl2 fusion protein has a calculated mass of 74.5 kDa (see Example 1 and Figure 3).
  • the plasmid pDEST15_umergl2 was then used for the recombinant expression of UmErgl2 in E. coli BL21 (DE3) (cf. Example 1).
  • the present invention thus also provides a further complete genomic sequence of a phytopathogenic fungus coding for a mevalonate kinase, and the use thereof, or the use of the polypeptide encoded thereby, for identifying inhibitors of the enzyme and the use thereof as fungicides.
  • the present invention therefore also relates to the nucleic acid from the Ustilago maydis fungus which codes for a polypeptide with the enzymatic function of a mevalonate kinase.
  • Mevalonate kinases share homologous areas. Typical of mevalonate kinases is a conserved region that is involved in the binding of ATP. This ATP binding site is a characteristic feature of the sequence for mevalonate kinases. Such a motif can be identified by a suitable search in the PROSITE database (Hofmann K., Bucher P., Falquet L., Bairoch A. (1999) "The PROSITE database, its Status in 1999". Nucleic Acids Res. 27, 215). It can be represented as follows: [LIVM] - [PK] -x- [GSTA] -x (0, l) -G- [LM] - [GS] -SS- [GSA] - [GSTAC],
  • PROSITE enables polypeptides to be assigned a function and thus to recognize mevalonate 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 braces “ ⁇ ... ⁇ ”.
  • a dash “-” separates the individual elements or positions of the motif. Repeats a certain position, e.g. "x”, several times in succession, this can be represented by specifying the number of repetitions in a subsequent bracket, e.g. "x (3)” which stands for "x-x-x".
  • 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.
  • PIGAGLGSSA 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 enzymatic activity of a mevalonate kinase which have the above-mentioned prostite motif [LIVM] - [PK] -x- [GSTA] -x (0, 1) -G- [LM ] - [GS] -SS- [GSA] - [GSTAC], preferably those polypeptides which comprise the above-mentioned motif PxGxGLGSSA, and particularly preferably those polypeptides which comprise the consensus sequence PIGAGLGSSA.
  • a mevalonate kinase which have the above-mentioned prostite motif [LIVM] - [PK] -x- [GSTA] -x (0, 1) -G- [LM ] - [GS] -SS- [GSA] - [GSTAC], preferably those polypeptides which comprise the above-mentioned motif PxGxGLGSSA, and particularly preferably those
  • mevalonate kinases from other phytopathogenic fungi can also be identified and used to solve the above-mentioned problem, i.e. they can also be used to identify inhibitors of a mevalonate kinase, which in turn can be used as fungicides in crop protection.
  • mevalonate kinases from other phytopathogenic fungi can also be identified and used to solve the above-mentioned problem, i.e. they can also be used to identify inhibitors of a mevalonate kinase, which in turn can be used as fungicides in crop protection.
  • another fungus which is not pathogenic to the plant, or its mevalonate kinase or the sequence coding therefor, in order to identify fungicidal inhibitors of the mevalonate kinase.
  • nucleic acids coding for mevalonate kinases from other (phytopathogenic) fungi by means of PCR.
  • nucleic acids and their use in methods for identifying fungicidal active substances are considered to be encompassed by the present invention.
  • the present invention particularly preferably relates to the nucleic acids coding for the Ustilago maydis mevalonate 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 cDNAs.
  • the nucleic acids according to the invention particularly preferably comprise a sequence 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 and c) sequences which have at least 90% identity with the sequences defined under a) and b) and for the sequence motif [LIVM ] - [PK] -x- [GSTA] -x (0, l) -G- [LM] - [GS] -SS- [GSA] - [GSTAC].
  • the present invention is not limited to the use of U. maydis mevalonate kinase.
  • polypeptides with the enzymatic activity of a mevalonate kinase can also be used or obtained from other fungi, preferably from phytopathogenic fungi, which can then be used in a process according to the invention.
  • the Mevalonate kinase from U. 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.
  • 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.
  • heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression.
  • heterologous promoters are the 35S promoter of the 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.
  • 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 p4XXprom.
  • Vector series Moberg, D., Müller, R., Funk, M.,. 1995.
  • Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Genes 156, 119-122) for yeast cells, pSPORT vectors (Fa. Life Technologies) for bacterial cells, or the gateway vectors (from 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 naturally do not 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 enzymatic activity of a mevalonate kinase, which are encoded by the nucleic acids according to the invention.
  • polypeptides according to the invention preferably comprise an amino acid sequence selected from
  • polypeptides refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, and 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 formations, formylations, gamma-carboxylations, glycosylations, hydroxylations, iodinations, methylations, myristoylations, oxidations, proteolytic processes, selylations, phosphorylations and tRNA-mediated 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 mevalonate 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 to the corresponding regions of naturally occurring mevalonate kinases, as long as they at least still show the enzymatic activity of a complete mevalonate 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:
  • Mevalonate Kinase is based on preparative electrophoresis, FPLC, HPLC (e.g. using gel filtration, reverse phase or slightly hydrophobic columns), gel filtration, differential precipitation, ion exchange chromatography or affinity chromatography (see .. Example 1).
  • a rapid method for isolating mevalonate kinases that are 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, a GST tag (see example 1).
  • the fusion protein can then be purified on glutathione-Sepharose column.
  • the fusion partner can be separated by partial proteolytic cleavage, for example on a link 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, which 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.
  • the method for producing polypeptides with the enzymatic activity of a mevalonate kinase is thus characterized by
  • the cells obtained in this way, containing the polypeptide according to the invention or the purified polypeptide obtained in this way, are suitable for use in methods for identifying modulators or inhibitors of mevalonate kinase.
  • the present invention also relates to the use of polypeptides from fungi, preferably from phytopathogenic or human or animal pathogenic fungi, which have the enzymatic activity of a mevalonate kinase, in methods for identifying inhibitors of these polypeptides, and the use of these inhibitors of mevalonate kinase as fungicides.
  • Fungicidal active ingredients that are found with the help of a mevalonate kinase from a particular fungal species can also interact with mevalonate kinases from other fungal species, although the interaction with the different mevalonate kinases 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 active ingredients which have been found with a mevalonate kinase of a particular fungal species as fungicides in other fungal species can be attributed to the fact that mevalonate kinases from different fungal species are relatively close and show pronounced homology in larger areas. Figure 2 clearly shows that such homology exists over considerable sequence sections between U. maydis, S.
  • Methods which are suitable for identifying modulators, in particular inhibitors or antagonists, of the polypeptides according to the invention are generally based on determining the activity or the biological functionality of the polypeptide.
  • both whole-cell-based methods in vivo methods
  • methods 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, are also possible.
  • 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.
  • tests can be carried out on fungal cultures in order to test the fungicidal activity of the compounds found.
  • 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 an initial check has been made and one or more compounds, extracts etc found, the effect of such compounds can be investigated even more specifically 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 which are based on the original structure, but which are optimized, for example, in terms of effectiveness, toxicity or selectivity.
  • modulators e.g. a synthetic reaction mix (eg 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, which can be an antagonist, are incubated.
  • the ability of the candidate molecule to inhibit the 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.
  • An 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 substrate or ligands or a substrate or ligand mimetic.
  • the polypeptides of the invention themselves can be labeled, e.g. fluorimetric or colorimetric, so that the number of polypeptides that are bound to a ligand or that 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 antagonists 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 olme test substances or without enzyme can be used to evaluate the effects.
  • the host cells which are available with the aid of the present invention and contain nucleic acids coding for a 'mevalonate kinase according to the invention. Development of test systems based on cells enables the identification of substances that 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 mevalonate kinase from fungi and which can be used as a fungicide in crop protection is therefore preferably that
  • a polypeptide with the enzymatic activity of a mevalonate kinase preferably from fungi and particularly preferably from phytopathogenic fungi, or a host cell containing such a polypeptide in contact with a chemical compound or with a mixture of chemical compounds under conditions which bring about the interaction of the allow chemical connections to 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 a molecule of adenosine diphosphate (ADP) is released in the reaction of the mevalonate kinase.
  • ADP adenosine diphosphate
  • the activity or the increase or decrease in the activity of the polypeptide according to the invention can therefore be determined by detecting the resulting ADP.
  • the lower or inhibited activity of the polypeptide according to the invention is tracked on the basis of the detection of the resulting ADP by coupling to the subsequent reaction of the pyruvate kinase and lactate dehydrogenase.
  • the pyruvate kinase converts phosphoenolpyruvate to pyruvate using ADP, which in turn is used by the lactate dehydrogenase to oxidize NADH to NAD.
  • the increasing NAD concentration or decreasing NADH concentration due to the coupled reaction concentration can be determined by spectrometry by absorption or fluorescence measurement (at 340 nm or an excitation wavelength of 360nm and an emission wavelength of 465nm).
  • the measurement can also be carried out in formats common to HTS or UHTS assays, e.g. in microtiter plates in which e.g. a total volume of 5 to 50 ⁇ l per batch or per well is presented and the individual components are present in the desired final concentrations (cf. Example 2).
  • 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 mevalonate, adenosine triphosphate, phosphoenol pyruvate and the coupled auxiliary enzymes pyruvate kinase and lactate dehydrogenase.
  • the polypeptide according to the invention is then added in test buffer and the reaction is started thereby.
  • the mixture is then incubated at a suitable temperature and the decrease in absorption is measured at 340 nm.
  • the incubation period can be varied over a longer period. Incubation is preferably carried out for 5 to 60 minutes, preferably for 15 to 45 minutes, ie on average about 30 minutes.
  • the coupled enzyme test is e.g. in Tanaka et al., 1990.
  • a further measurement is carried out in a corresponding approach, but without the addition of a candidate molecule and without the addition of 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 controls thus give the comparison values for the batches in the presence of a candidate molecule.
  • Table I shows an example of a compound that could be identified as an inhibitor of mevalonate kinase using a method according to the invention.
  • inhibitors of a mevalonate kinase according to the invention identified with the aid of a method according to the invention are suitable for damaging or killing fungi.
  • a solution of the active substance to be tested is pipetted into the cavities of microtiter plates. 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 e.g. 0.1, 1, 10 and 100 ppm.
  • the plates are then incubated on a shaker at a temperature of approximately 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 ingredient can be determined from the measurement data of the various concentrations, which leads to a 50% inhibition of fungal growth compared to the untreated control (ED 50 ).
  • ED 50 the untreated control
  • Table TJ shows the results of such a test as ED 50 values for a compound found in an inventive procedure (see Table I). The compound has a fungicidal effect on various fungi.
  • the present invention therefore also relates to the use of modulators of mevalonate kinase from fungi, preferably from phytopathogenic fungi as fungicides, and to methods for combating fungal attack in plants by applying an inhibitor of the fungal mevalonate kinase to the fungus or from the fungus infested plant.
  • 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 usual formulations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, very fine encapsulations in polymeric substances and in coating compositions for seeds, and ULV cold and warm fog formulations.
  • formulations are made in a known manner, e.g. 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,
  • 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, chlorethylenes or methylene chloride, aliphatic hydrocarbons substances such as cyclohexane or paraffins, for example petroleum fractions, alcohols, such as butanol or glycol, and their 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 foam-generating 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 polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol, polyvinyl acetate, and natural phospholipids such as cephalins and lecithins, and synthetic phospholipids can be used in the formulations.
  • Other additives can be mineral and vegetable oils.
  • Dyes such as inorganic pigments, e.g. Iron oxide, titanium oxide, ferrocyan blue and organic dyes such as alizarin, azo and metal phthalocyanine dyes and trace non-alcoholic substances 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 to spread 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 may vary. can be varied within a wide range after application.
  • 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 the 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 surroundings, living space or storage space using the customary treatment methods, e.g. by dipping, spraying, vaporizing, atomizing, scattering, spreading and, in the case of propagation material, in particular in the case of seeds, furthermore by coating in one or more layers.
  • the ORF was amplified from genomic DNA from Ustilago maydis using gene-specific primers.
  • the corresponding DNA an amplifier of 1059 bp in length, was intercloned into the vector pDON201 from Invitrogen and then cloned into the vector pDEST15 (Invitrogen) by recombination.
  • the resulting plasmid pDEST15_umergl2 contains the complete coding sequence of umergl2 in an N-terminal fusion with the GST tag, which is part of the vector.
  • the UmErgl2 fusion protein has a calculated mass of 74.5 kDa.
  • the plasmid pDEST15__umergl2 was transformed into E. coli BL21 (DE3).
  • a preculture was inoculated into the transformants in 50 ml of selection medium. These cells were incubated overnight at 37 ° C. and then diluted 1:20 in selection medium (LB medium with 100 ⁇ g / ml ampicillin). Induction was carried out at an OD ⁇ oo nm of 0.8 with 0.1 mM IPTG (final concentration) at 30 ° C. After 3 h induction, the cells were harvested and worked up directly.
  • the digestion was carried out by sonication in lysis buffer (100 mM Tris-HCl, pH 8, 5 M DTT, 5 mM MgCl 2 , 0.1% Triton, 10 ⁇ M ATP) after previous lysozyme treatment (15 minutes, 30 ° C, 1 mg / ml final in lysis buffer).
  • lysis buffer 100 mM Tris-HCl, pH 8, 5 M DTT, 5 mM MgCl 2 , 0.1% Triton, 10 ⁇ M ATP
  • 384-well microtiter plates from Greiner were used to identify modulators of the Mevalonate kinase.
  • the negative control was pipetted into the first and second column. This was composed of 5 ⁇ l of solution (5% DMSO in H 2 0), 20 ⁇ l of solution 2 (100 mM Tris / HCl pH 7.5, 15 mM MgCl 2 , 12.5 mM glutathione, 0.25% BSA) and 25 ⁇ l solution 3 (100 mM Tris / HCl pH 7.5, 15 mM MgCl 2 , 20 mM KCl, 0.6 mM ATP, 0.8 mM PEP, 0.6 mM NADH, 20 mM DTT, 0.02% Tween 20) with 200 mU pyruvate kinase, 400 mU lactate dehydrogenase.
  • solution 3 100 mM Tris / HCl pH 7.5, 15 mM MgCl 2 , 20 mM KCl, 0.6 mM ATP, 0.8 mM PEP, 0.6 mM
  • the positive control was pipetted into the third and fourth column. This was composed of 5 ⁇ l solution (5% DMSO in H 2 0), 20 ⁇ l solution 4 (100 mM Tris / HCl pH 7.5, 15 mM MgCl 2 , 12.5 mM glutathione, 0.25% BSA, 0, 1 ⁇ g mevalonate kinase) and 25 ⁇ l solution 3 (100 mM Tris / HCl pH 7.5, 15 mM MgCl 2 , 20 mM KCl, 0.6 mM ATP, 0.8 mM PEP, 0.6 mM NADH, 20 mM DTT, 0.02% Tween 20) with 200 mU pyruvate kinase, 400 mU lactate dehydrogenase.
  • 5 ⁇ l solution 5% DMSO in H 2 0
  • 20 ⁇ l solution 4 100 mM Tris / HCl pH 7.5, 15 mM Mg
  • a test substance at a concentration of 2 ⁇ M in DMSO was placed in the remaining columns, H 2 0 being used to dilute the substance.
  • 20 ⁇ l of solution 4 100 mM Tris / HCl pH 7.5, 15 mM MgCl 2 , 12.5 mM glutathione, 0.25% BSA, 0.1 ⁇ g mevalonate kinase
  • 25 ⁇ l solution were added to start the reaction 3 (100mM Tris / HCl pH 7.5, 15mM MgCl 2 , 20mM KCl, 0.6mM ATP, 0.8mM PEP, 0.6mM NADH, 20mM DTT, 0.02% Tween 20) with 200 mU pyruvate kinase and 400 mU lactate dehydrogenase added.
  • the subsequent measurement was carried out by determining the absorption at 340 nm in a SPECTRA
  • the resulting concentration of the emulsifier was 300 ppm.
  • the plates were then incubated on a shaker at a temperature of 22 ° C. until sufficient growth was found in the untreated control.
  • the expansion was carried out photometrically at a wavelength of 620 nm.
  • the dose of active ingredient 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.

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Abstract

L'invention concerne un procédé d'identification de fongicides, l'utilisation de mévalonate kinase pour identifier des fongicides, ainsi que l'utilisation d'inhibiteurs de la mévalonate kinase comme fongicides.
EP04740191A 2003-07-04 2004-06-23 Procede d'identification de composes a action fongicide au moyen de mevalonate kinases provenant de champignons Withdrawn EP1644503A1 (fr)

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DE2003130234 DE10330234A1 (de) 2003-07-04 2003-07-04 Verfahren zum Identifizieren von fungizid wirksamen Verbindungen basierend auf Mevalonat Kinasen aus Pilzen
PCT/EP2004/006766 WO2005003355A1 (fr) 2003-07-04 2004-06-23 Procede d'identification de composes a action fongicide au moyen de mevalonate kinases provenant de champignons

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US5165931A (en) * 1989-11-15 1992-11-24 E. R. Squibb & Sons, Inc. Peptifluorin and neopeptifluorin
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