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WO2005098030A2 - Procede pour identifier des composes a action fongicide sur la base de thioredoxine reductases - Google Patents

Procede pour identifier des composes a action fongicide sur la base de thioredoxine reductases Download PDF

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Publication number
WO2005098030A2
WO2005098030A2 PCT/EP2005/002951 EP2005002951W WO2005098030A2 WO 2005098030 A2 WO2005098030 A2 WO 2005098030A2 EP 2005002951 W EP2005002951 W EP 2005002951W WO 2005098030 A2 WO2005098030 A2 WO 2005098030A2
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Prior art keywords
thioredoxin reductase
thioredoxin
activity
polypeptide
sequences
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PCT/EP2005/002951
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German (de)
English (en)
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WO2005098030A3 (fr
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Edda Koopmann
Rüdiger SUELMANN
Peter Schreier
Birgitta Leuthner
Thorsten Leicher
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Bayer Cropscience Ag
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Publication of WO2005098030A2 publication Critical patent/WO2005098030A2/fr
Publication of WO2005098030A3 publication Critical patent/WO2005098030A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi

Definitions

  • the invention relates to a method for identifying fungicides, the use of fungal thioredoxin reductase to identify fungicides, and the use of inhibitors of thioredoxin reductase as fungicides.
  • fungicides are often sought in essential biosynthetic pathways. Ideal fungicides continue to be substances that inhibit gene products that are crucial
  • 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 a method for . To make available, which enables the identification of modulators of this point of attack, which can then be used as fungicides.
  • Thioredoxin reductase (EC 1.8.1.9, formerly EC 1.6.4.5), also known as NADP-thioredoxin reductase, NADPH-thioredoxin reductase or thioredoxin: NADP + -oxidoreductase, catalyzes the reaction of NADP + and thioredoxin to thio-edoxin-disulfide NADPH + ⁇ ( Figure 1).
  • the reaction of thioredoxin reductase is an essential step in the recycling of the oxidized form of thioredoxin (disulfide form), which provides the cell with reduced thioredoxin.
  • the cytoplasmic thioredoxin reductase - hereinafter also referred to as TRR - belongs to class II of pyridine nucleotide disulfide oxido eductases.
  • the enzyme recycles the oxidized form of thioredoxin and provides the cell with reduced thioredoxin. TRR is therefore important for maintaining redox homeostasis in the cell.
  • Thioredoxins themselves are small, highly conserved oxidoreductases that contain two conserved cysteine residues in the active center.
  • Thioredoxins are best characterized as antioxidants against reactive oxygen species, but their functions also include protection against reductive stress. It is believed that they also play a role in a variety of other cellular processes, such as protein folding and its regulation, the repair of oxidatively damaged proteins and in the sulfur metabolism.
  • Bacterial and fungal thioredoxin reductases are flavoenzymes and exist as homodimers. They contain a FAD cofactor and a pair of redox-active Cys groups
  • Genes for the thioredoxin reductase were cloned from various organisms, including from different fungi such as Saccharomyces cerevisiae (Swissprot Accession No .: P38816), Schizosaccharomyces pom.be (Swissprot Accession No .: Q92375), Neurospora crassa (Swissprot Accession No.
  • P51978 Pneumocystis carinii (Swissprot Accession No .: Q7Z7S3), Pneumocystis jiroveci (Swissprot Accession No .: Q8J0U0) or Penicillium chrysogenum (Swissprot Accession No .: P43496).
  • the sequence similarities are significant within the eukaryotic classes. In contrast, thioredoxin reductases from phytopathogenic fungi have so far not become known.
  • the object of the present invention was to identify new points of attack by fungicides in fungi, in particular in phytopathogenic fungi, and to make methods accessible in which inhibitors of such a point of attack or polypeptide can be identified and tested for their fungicidal properties.
  • the object was achieved by isolating the nucleic acid coding for thioredoxin reductase from a phytopathogenic fungus, the polypeptide encoded therefrom, and a method using which
  • 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 reduction of thioredoxin in its disulfide form to thioredoxin with free SH groups catalyzed by the thioredoxin reductase.
  • NADP + H + is converted to NADP + .
  • FIG. 2 Sequence alignment of the amino acid sequence of thioredoxin reductases from various fungi. The homologous areas between the thioredoxin reductases are shown. In the sequences continue to the Prosite motif amino acids were appropriate for thioredoxin reductases '' marked.
  • Yeastl and Yeast2 Saccharomyces cerevisiae.
  • PNEUCA Pneumocycstis carinii.
  • PNEUJI Pneumocystis jiroveci.
  • POMBE Saccharomyces pombe.
  • NEUCR Neurospora crassa.
  • PENCH Penicillium chrysogenum.
  • Ustilago Ustilago maydis.
  • FIG. 3 SDS gel. The expression and purification of TRR1 from U. maydis was monitored using SDS gels (A) to (C). Lane 1: non-induced E. coli cells. Lane 2: is.co/z cells after induction. Lane 3: Precipitation after cell disruption. Lane 4: supernatant after cell disruption. Lane 5: wash fraction from the Ni-NTA column. Lane 6: Elution fraction after the Ni-NTA column. Lane 7: fraction after the PD-10 column. M: 10 kDa protein standard.
  • FIG. 4 SDS gel. The heterologous expression and purification of thioredoxin, the substrate of thioredoxin reductase, was monitored using SDS gels (A) to (D). Lane 1: uninduced E. co / z ' cells. Lane 2: E. co / z cells after induction. Lane 3: Precipitation after cell disruption. Lane 4: supernatant after cell disruption. Lane 5: wash fraction from the Ni-NTA column. Lane 6: Elution fraction from the Ni-NTA column. Lane 7: fraction after the PD-10 column. M: 10 kDa protein standard. Gel (D) shows samples of the individual thioredoxin purifications.
  • FIG. 5 Graphical representation of the kinetics of the NADPH decrease in an activity test for the thioredoxin reductase.
  • the enzymatic activity of thioredoxin reductase (TRR1) is determined based on the consumption of NADPH.
  • TRR1 thioredoxin reductase
  • the decrease in NADPH was investigated in 16 batches based on the change in fluorescence (excitation at 360 nm, emission at 465 nm) as a function of time ( ⁇ ).
  • the same preparation conditions were used as a control in the absence of the enzyme ( ⁇ ).
  • the kinetics show one due to the enymatic activity of the TRR, almost linear decrease in the fluorescence yield within 35-40 min, which then changes into a plateau area.
  • Figure 6 Lineweaver-Burk plot for determining the K value of thioredoxin (A) and NADPH (B).
  • V n ⁇ ax and K M can then be read as the abscissa and ordinate sections 1 / V max and 1 / K M, respectively.
  • the K M value for E. co / z ' thioredoxin is 5 ⁇ M
  • the K M value for NADPH is between 20 and 40 ⁇ M.
  • FIG. 7 Graphical representation of the decrease in the activity of thioredoxin reductase (TRR1) in the presence of an inhibitor.
  • TRR1 thioredoxin reductase
  • the activity of TRR1 was measured by determining the NADPH concentration over time. A smaller decrease in the NADPH concentration indicates a lower or inhibited enzymatic reaction of the TRR1.
  • the measurements were carried out in the presence of different concentrations of n-ethylmaleimide. The concentrations used are given in the legend.
  • O.I. stands for the reaction in the absence of the inhibitor, i.e. the uninhibited reaction (negative control).
  • SEQ ID NO: 1 nucleic acid sequence coding for the thioredoxin reductase from Ustilago maydis.
  • identity is to be understood as the number of matching amino acids (identity) with other proteins, expressed in percent.
  • the identity is preferably determined by comparing a given sequence to other proteins with the aid of computer programs. If sequences which are compared with one another have different lengths, the identity is to be determined in such a way that the number of amino acids which the shorter sequence has in common with the longer sequence determines the percentage of identity.
  • the identity can be established by means of known computer programs available to the public, e.g. ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is e.g.
  • ClustalW can also be accessed from various websites, including at the IGBMC (Institut de Genetique et de Biologie Moleisme et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and at the EBI (ftp: //ftp.ebi .ac.uk / pub / software /) and from all mirrored websites of the EBI (European Bioinfo matics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, K). If the ClustalW computer program version 1.8 is used to establish the identity between e.g.
  • sequence database searches 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 e.g.
  • 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 methods described above for identity determination have an identity of at least 70%, preferably of at least 75%, particularly preferably of at least 80%, more preferably of at least 85%, and in particular of at least 90%.
  • Thioredoxin reductase which is 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 coding for a thioredoxin reductase present in the organism of origin, as well as the signals necessary for correct termination of the transcription.
  • biological activity of a thioredoxin reductase refers to the ability of a polypeptide to effect the reaction described above, ie the reduction of thioredoxin in its disulfide form to thioredoxin with free SH groups using NADPH + H 1 " ( Figure 1).
  • Two cysteine residues and the amino acids surrounding these cysteine residues are characteristic of thioredoxin reductases ( Figure 2).
  • active fragment no longer describes complete nucleic acids coding for thioredoxin reductase, but which still code for polypeptides with the biological activity of a thioredoxin reductase and which can catalyze a reaction characteristic of the thioredoxin reductase as described above , Such fragments are shorter than the complete nucleic acids encoding thioredoxin reductase 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 thioredoxin reductase.
  • a lower or possibly also an increased activity, which, however, still allows the characterization or use of the resulting thioredoxin reductase 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 thioredoxin reductase 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 biological activity of the enzyme, however, not being decisive is impaired.
  • the fragments can have different lengths.
  • thioredoxin reductase 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 thioredoxin reductase recognize one or more chemical compounds (candidate compound (s)), whereby the chemical compound can be identified as an inhibitor of thioredoxin reductase.
  • gene as used herein is the term for a portion of the genome of a cell that is responsible for the synthesis of a polypeptide chain.
  • fungicide or "fungicidal” as used herein refers to chemical compounds which are suitable for combating fungi which are pathogenic to humans, animals and plants, in particular fungi which are pathogenic to plants. Such phytopathogenic fungi are mentioned below, the list is not exhaustive:
  • 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, for example, Peronospora pisi or P. brassicae, 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, for example, Venturia inaequalis
  • Pyrenophora species such as, for example, Pyrenophora teres or P.
  • graminea (conidial form: Drechslera, Syn: Helminthosobol Species, such as, for example, Cochliobolus sativus (conidial form: Drechslera, Syn: Helminthosporium), Uromyces species, such as
  • Uromyces appendiculatus Puccinia species, such as, for example, Puccinia recondita, Sclerotinia species, such as, for example, Sclerotinia sclerotiorum, Tilletia species, such as, for example, 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 - Fusa ⁇ um culmorum, Botrytis species, Septoria species, such as, for example Septoria nodorum, Leptosphaeria species such as Leptosphaeria nodorum, Cercospora species such as Cercospora canescens, Alternaria species such as Alternaria brassicae or Pseudocerco
  • Nectria hematococcus and Phytophtora species Fungicidal active ingredients which are found with the aid of the thioredoxin reductases according to the invention from plant-pathogenic fungi can also interact with thioredoxin reductases from human-pathogenic fungus species, the interaction with the different thioredoxin reductases occurring in these fungi not always having to be equally strong ,
  • the present invention therefore also relates to the use of inhibitors of
  • Thioredoxin reductase for the manufacture of agents for the treatment of diseases caused by human pathogenic fungi.
  • Dermatophytes e.g. Trichophyton spec, Microsporum spec, Epidermophyton floccosum or
  • Keratomyces ajelloi e.g. Cause foot mycoses (tinea pedis)
  • 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 Aspergiltus fumigatus, A. flavus, A. niger, e.g. bronchopulmonary
  • 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 causes Z: B. pulmonary coccidioidomycosis and sepsis, Paracoccidioides brasiliensis, which e.g.
  • Fungicidal active ingredients which are found with the aid of a thioredoxin reductase obtained from a particular fungus, here from Ustilago maydis, can therefore also " interact with thioredoxin reductases from other numerous fungal species, especially with phytopathogenic fungi, the interaction with the different ones in these Thioredoxin reductases occurring in fungi do not always have to be of the same strength, which explains, among other things, the observed selectivity of the substances active on this enzyme.
  • homologous promoter refers to a promoter which controls the expression of the gene in question in the original organism.
  • heterologous promoter refers to a promoter which has different properties than the promoter which controls the expression of the gene in question in the original organism.
  • petitor refers to the property of the compounds to compete with other, if necessary, identifiable compounds in order to compete for binding to the thioredoxin reductase and to displace or be displaced by the enzyme.
  • inhibitor or "specific inhibitor” as used herein denotes a substance that directly inhibits an enzymatic activity of the thioredoxin reductase.
  • a • Such an inhibitor preferably is “specific”, ie it inhibits specifically the thioredoxin reductase activity at a concentration which is lower than the cause concentration of an inhibitor that is required to another, so that unbonded effect.
  • the concentration is preferably two times lower, particularly preferably five times lower and very particularly preferably at least ten times or 20 times lower than the concentration of a compound which is required to produce an unspecific effect.
  • modulator represents a generic term for inhibitors and activators.
  • Modulators can be small organic chemical molecules, peptides or antibodies which bind to the polypeptides according to the invention or which influence their activity.
  • modulators can be small organic chemical molecules, peptides or antibodies which bind to a molecule which in turn binds to the polypeptides according to the invention and thereby influences their biological activity.
  • Modulators can be 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.
  • thioredoxin reductase in fungi can be a target protein (a so-called "target") of fungicidally active substances.
  • target a target protein of fungicidally active substances.
  • thioredoxin reductase 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.
  • TRR1 is localized cytoplasmic and TRR2 is suspected mitochondrial.
  • Deletion mutants of TRR2 are viable, hypersensitive to hydrogen peroxide, show temperature-sensitive growth and are auxotrophic for methionine (Machado et al., 1997; Pearson and Merril, 1998), while deletion mutants of the cytoplasmic TRR1 are not viable
  • the thioredoxin reductase has not yet been described as a target protein for fungicides. There are no known active ingredients which have a fungicidal action and whose place of action is thioredoxin reductase.
  • the thioredoxin reductase is surprisingly a target or "target” for fungicidal active substances in phytopathogenic fungi, the inhibition of the thioredoxin reductase thus leading to damage or death of the fungus.
  • Ustilago maydis one for one Gene coding for thioredoxin reductase identified (um 140_5). Knock-out of this gene was found to be lethal (Example 1).
  • thioredoxin reductase can 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 thioredoxin reductase can therefore be used as fungicides in crop protection or as antifungals in pharmaceutical indications.
  • the inhibition of the thioredoxin reductase with a substance identified in a method according to the invention leads to the death of the treated fungi in synthetic media or on the plant.
  • Thioredoxin reductases can be obtained from various phytopathogenic or also from human or animal pathogenic fungi, 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 2).
  • Thioredoxin reductases 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 thioredoxin reductases from human or animal pathogenic fungi is recommended.
  • the associated ORF was amplified by means of PCR using selected primers using methods known to the person skilled in the art.
  • the corresponding DNA was cloned into the expression vector pTrcHis2-TOPO, so that the TRR protein is expressed starting from the plasmid ptrcTRRlO with a C-terminal His 6 tag (Example 2).
  • the plasmid ptrcTRR10 was transformed into E. coli BL21 (DE3).
  • the trrl gene from Ustilago maydis has a size of 1053 bp, es does not contain hitron.
  • UM 140_5 (trrl) codes for a polypeptide of 351 amino acids in length with a molecular weight of 39000 daltons ( Figure 3).
  • the present invention thus also provides a complete genomic sequence of a phytopathogenic fungus coding for a thioredoxin reductase, and its use or the use of the polypeptide encoded thereby for the identification of
  • the present invention therefore also relates to the nucleic acid from the fungus Ustilago maydis which codes for a polypeptide with the enzymatic function of a thioredoxin reductase.
  • Thioredoxin reductases share homologous areas. A conserved region containing two cysteines, which are involved in the redox-active disulfide bridge, is typical for thioredoxin reductases. The sequences surrounding these two cysteines are also characteristic of thioredoxin reductases.
  • cysteine and its sequence environment is a characteristic of the sequence for thioredoxin reductases.
  • Such a motif can be identified by a suitable search in the PROSITE database (Hofmann et al., 1999),
  • PROSITE enables polypeptides to be assigned a function and thus to recognize thioredoxin reductases as such.
  • the "one-letter code” is used to display the Prosite motif.
  • the symbol “x” stands for a position where every amino acid is accepted.
  • a variable position at which various specific amino acids are accepted is shown in square brackets "[...]", whereby the possible amino acids at this position are listed.
  • Amino acids that are not accepted at a certain position are enclosed in curly brackets " ⁇ ... ⁇ ”.
  • a dash “-” separates the individual elements or positions of the motif. 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 specific enzyme great.
  • this motif can be used to identify or assign further polypeptides from phytopathogenic fungi 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.
  • 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 plant pathogens
  • Mushrooms with the biological activity of a thioredoxin reductase which has the above-mentioned prostite motif Cx (2) -CD- [GA] -x (2.4) - [FY] -x (4) - [LIVM] -x- [LIVM ] (2) -G (3) - [D ⁇ ] (see also Figure 2).
  • polypeptides according to the invention with the biological activity of a thioredoxin reductase which have the motif C-AN-C-D-G-AN-P-I-F-R- ⁇ -K-P-L-xN- are particularly preferred.
  • thioredoxin reductases 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 thioredoxin reductase, which can in turn be used as fungicides in crop protection.
  • thioredoxin reductases 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 thioredoxin reductase, which can in turn be used as fungicides in crop protection.
  • another fungus which is not pathogenic to the plant, or its thioredoxin reductase or the sequence coding therefor, in order to identify fungicidal inhibitors of thioredoxin reductase.
  • nucleic acid sequence according to the invention With the aid of the nucleic acid sequence according to the invention and sequences obtained from other phytopathogenic fungi according to the methods described above, further nucleic acid sequences coding for a thioredoxin reductase from other fungi can be identified.
  • the present invention therefore relates to nucleic acids from phytopathogenic fungi which code for a polypeptide with the enzymatic activity of a thioredoxin reductase, in particular polypeptides which comprise the motifs described above.
  • the present invention preferably relates to nucleic acids from the phytopathogenic fungal species mentioned above in the definitions section, which code for a polypeptide with the enzymatic activity of a thioredoxin reductase.
  • the present invention particularly preferably relates to the nucleic acid coding for the Ustilago maydis thioredoxin reductase with the SEQ ID NO: 1 and the nucleic acids coding for the polypeptides according to SEQ ID NO: 2 or active fragments thereof.
  • 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 cDNAs.
  • nucleic acids according to the invention particularly preferably comprise a sequence of phytopathogenic fungi coding for a polypeptide with the enzymatic activity of a thioredoxin reductase selected from
  • the present invention is not only limited to the thioredoxin reductase from Ustilago maydis.
  • Polypeptides with the activity of a thioredoxin reductase can also be obtained from other fungi, preferably from phytopathogenic fungi, in an analogous manner to those skilled in the art. can be used in a method according to the invention.
  • the thioredoxin reductase from Ustilago maydis is preferably used.
  • the present invention furthermore relates to DNA constructs which comprise a nucleic acid according to the invention and a homologous or heterologous promoter.
  • heterologous promoters depend 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 further relates to vectors which are an inventive
  • phages Contain nucleic acid, 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 e.g. the p4XXprom. Vector series (Mumberg et al, 1995) for yeast cells, pSPORT vectors (from 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 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 thioredoxin reductase, which are encoded by the nucleic acids according to the invention.
  • polypeptides according to the invention preferably comprise an amino acid sequence selected from phytopathogenic fungi
  • polypeptides refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer amino acid chains, commonly referred to as proteins. It includes
  • 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.
  • Modifications can occur at different 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
  • Carboxy terminus 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,
  • 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 secreting or "leader” sequences, pro sequences, sequences that allow easy 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 thioredoxin reductase 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 may have deletions or amino acid substitutions in comparison to the corresponding regions of naturally occurring thioredoxin reductases, as long as they at least still show the biological activity of a complete thioredoxin reductase.
  • Conservative substitutions are preferred. Such conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:
  • Aromatic residues Phe, Tyr and Trp.
  • a possible purification method for thioredoxin reductase 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 3).
  • a rapid method for isolating thioredoxin reductase, which is 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 or preferably also a histidine tag (cf. Example 3).
  • the fusion protein can then turn on
  • the fusion partner can be separated by partial proteolytic cleavage, for example on linkers between the fusion partner and the polypeptide to be purified according to the invention.
  • the protein with a His tag can be removed from the column by lnidazole-containing buffers.
  • the linker can be designed to include target amino acids, such as arginine and lysine residues, that define sites for trypsin cleavage. Standard cloning methods using oligonucleotides can be used to generate 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.
  • 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 preferred with regard to the protein content compared to a preparation from the
  • 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 activity of a thioredoxin reductase e.g. of the polypeptide TRR1 from U. maydis is characterized by
  • the present invention also relates to the use of polypeptides from fungi, preferably from phytopathogenic fungi, which have at least one biological activity
  • the thioredoxin reductase from Ustilago maydis is particularly preferably used.
  • Fungicidal active ingredients which are found with the help of a thioredoxin reductase from a certain fungal species, can also interact with thioredoxin reductase from other fungal species, although the interaction with the different thioredoxin reductase 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 thioredoxin reductase as a fungicide in other fungi can be attributed to the fact that thioredoxin reductases from various fungal species are very close and in larger ones
  • the present invention therefore also relates to a method for identifying fungicides by testing potential inhibitors or modulators of the enzymatic activity of thioredoxin reductase (candidate compound or test compound) in a thioredoxin reductase 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 determining the activity or the biological functionality of the polypeptide.
  • both whole-cell-based methods (in vivo methods) and 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.
  • test systems 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.
  • the effect of such compounds can be examined in a more targeted manner in the laboratory.
  • the inhibition or activation of the polypeptide according to the invention can be checked again in vitro in order to then test the effectiveness of the compound on the target organism, here one or more phytopathogenic fungi.
  • the compound can then optionally be used as a starting point for the further search and development of fungicidal compounds based on the original structure, but e.g. are optimized in terms of effectiveness, toxicity or selectivity.
  • modulators e.g. incubating a synthetic reaction mix (e.g. products of in vitro transcription) or a cellular component such as a membrane, a compartment or any other preparation which contains the polypeptides according to the invention together with an optionally labeled substrate or ligand of the polypeptides in the presence and absence of a candidate molecule become.
  • the maturity of the candidate molecule to inhibit the enzymatic activity of the polypeptides according to the invention is e.g. recognizable by a reduced binding of the optionally labeled ligand or by a reduced
  • Molecules that inhibit the biological activity of the polypeptides according to the invention are good antagonists or inhibitors.
  • reporter systems include, but are not limited to, colorimetric or fluorometric detectable substrates contained in one
  • 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 is determined or who have participated in an implementation, can determine exactly.
  • the binding can also be followed by means of the optionally labeled substrate, ligand or substrate analog. In this way, the effectiveness of an antagonist can be measured.
  • Test systems check both inhibitory or suppressive effects of the substances as well as stimulatory effects.
  • the effectiveness of a substance can be checked using concentration-dependent test series. Control approaches without test substances or without enzyme can be used to evaluate the effects.
  • the host cells containing nucleic acids coding for a thiorexin reductase according to the invention and 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 that modulates the activity of a thioredoxin reductase from fungi and that can be used as a fungicide in crop protection is preferably that
  • a polypeptide according to the invention or a host cell containing this polypeptide is brought into contact with a chemical compound or with a mixture of chemical compounds under conditions which allow the interaction of a chemical compound with the polypeptide,
  • the compound which specifically inhibits the activity of the polypeptide according to the invention is particularly preferably determined.
  • activity refers to the biological activity of the polypeptide according to the invention.
  • the fact is used that the thioredoxin reductase catalyzes the reduction of oxidized thioredoxin using NADPH.
  • This reaction can be carried out, for example, in the presence of insulin, which can be used as a thioredoxin-regenerating system, since thioredoxin reductase reduces thioredoxin as disulfide reductase, converting insulin to oxidized insulin and thereby converting it back into the oxidized state.
  • This oxidized thioredoxin then serves as the substrate for the thioredoxin reductase.
  • NADPH is then consumed in the reaction of the thioredoxin reductase.
  • the measurement of the enzymatic activity of the thioredoxin reductase or the inhibition of this enzymatic activity by an inhibitor is then carried out on the basis of the decreasing NADPH concentration and can be carried out photospectrometrically by absorption or fluorescence measurement (absorption decrease at 340 nm or fluorescence decrease at 340 nm (emission at 465 nm)) can be determined ( Figure 5).
  • the lower or inhibited activity of the polypeptide according to the invention is tracked based on the photospectrometric determination of the degree of decrease in the NADPH concentration relative to a control batch. If the thioredoxin reductase is inhibited, there is no enzymatic conversion of the NADPH.
  • the fluorescence intensity of the batch should correspond to that of the control batch, since there is no change in the NADPH concentration.
  • An increase in the NADPH concentration can result if the enzymatic activity of the thioredoxin reductase is inhibited so strongly that the thioredoxin oxidized by the reaction with insulin is no longer reduced and thus accumulates.
  • the amount of NADPH e.g. even after the reaction has been completed by implementing
  • Resazurin to Resorußn in the presence of PMS can be determined fluorimetrically (excitation 550 nm, emission 595 nm) compared to the amount used.
  • PMS phenazine methosulfate
  • the electrons are first transferred from NADPH to PMS.
  • the reduced PMS is then able to reduce resazurin to resorufin.
  • the concentration of resurofin can then be detected fluorimetrically (excitation 550 nm, emission 595 nm).
  • the amount of converted NADPH can then be determined by comparison with the concentrate of resorufin in the control batch. NADPH thus reduces non-fluorescent resazurin to intense red fluorescent resorufin.
  • PMS serves as a redox mediator.
  • the measurement can also be carried out in formats customary for HTS or UHTS assays, for example in microtiter plates in which, for example, a total volume of 5 to 50 ⁇ l per batch or per well is placed and the individual components are present in one of the final concentrations given above.
  • the test compound which potentially inhibits or activates the activity of the enzyme (candidate molecule), is introduced, for example, in a suitable concentration in test buffer containing the components necessary for the reaction.
  • the polypeptide according to the invention is then added in the test buffer mentioned above and the reaction is started therewith.
  • the mixture is then incubated, for example, for up to 30 minutes at a suitable temperature and, for example, the decrease in fluorescence (absorbance 340 nm; emission 465 nm) is measured.
  • 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.
  • K M value of the polypeptide according to the invention provides information about the preferred concentration of the substrate or substrates.
  • a K M of 5 ⁇ M for E. coli thioredoxin and a K M of 20-40 ⁇ M for NADPH could be determined ( Figure 6).
  • inhibitors of thioredoxin reductase could be identified with the method according to the invention.
  • Reductase are suitable, optionally in a suitable formulation, to damage or kill fungi.
  • a solution of the active substance to be tested can be 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, for example, 0J, 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. From the measurement data of the different concentrations, the dose of active substance can be determined, 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 modulators of thioredoxin reductase 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.
  • the present invention also relates to the use of modulators of thioredoxin reductase from fungi, preferably from phytopathogenic fungi as fungicides, and to methods for combating fungal attack in plants by using an inhibitor of a
  • Thioredoxin reductase affects the fungus and / or the infected plant. These are preferably inhibitors of a thioredoxin reductase from a phytopathogenic fungus.
  • the modulators are preferably small, organic-chemical molecules, but not inorganic molecules such as anions or cations.
  • the term modulators also does not include any natural effectors of the enzymes which are used in the organism in question to regulate the activity of the enzyme, such as Metabolic products of the enzymes, cofactors of the enzymes, and no non-metabolizable analogues of the cofactors or educts of the enzyme.
  • 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 mist formulations.
  • formulations are prepared in a known manner, for example by mixing the active ingredients with extenders, that is to say liquid solvents, pressurized liquefied gases and / or solid carriers, if appropriate using surface-active agents, that is to say emulsifiers and or dispersants and / or foam-generating agents.
  • 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.
  • organic solvents can, for example, also be used as auxiliary solvents.
  • aromatics such as xylene, toluene or alkylnaphthalenes
  • chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chlorethylenes or methylene chloride
  • aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions
  • alcohols such as butanol or glycol, and the like 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, as well as water.
  • Liquefied gaseous extenders or carriers mean liquids which are gaseous at normal temperature and pressure, e.g. Aerosol propellants, such as halogenated hydrocarbons as well as butane, propane, nitrogen and carbon dioxide.
  • Aerosol propellants such as halogenated hydrocarbons as well as butane, propane, nitrogen and carbon dioxide.
  • solid carriers the following are possible: e.g. natural rock flours, such as kaolins, clays, talc,
  • the following are suitable as solid carriers for granules: 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 stems.
  • Possible emulsifiers and / or foaming agents are: e.g.
  • non-ionic and anionic emulsifiers such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, e.g. Alkylaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates and protein hydrolyzates.
  • Possible dispersants are: e.g. Lignin sulfite liquor and methyl cellulose.
  • Adhesives such as carboxymethyl cellulose, natural and synthetic powdery, granular or latex-shaped polymers 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 nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc can be used.
  • the formulations generally contain between 0.1 and 95 percent by weight of active compound, preferably between 0.5 and 90%.
  • the active compounds according to the invention can be used as such or in their formulations, also 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, ie the activity, are obtained the mixture is greater than the effectiveness of the individual components.
  • the application rates can be varied within wide ranges depending on the application.
  • plants 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 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 varieties which can or cannot be protected by plant breeders' rights.
  • Plants such as sprout, leaf, flower and root are understood, with 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.
  • strains were grown at 28 ° C on PD, YEPS or suitable minimal media (Holliday, 1974;
  • the primers LB2 with the sequence (5'-cacggcctgagtggccggctcggaatgatcgagctgagg-3 ') and pl5 with the sequence (5'-gatgacgcaggagggacacaagg-3') were used.
  • the primers RB1 (5'-gtgggccatctaggccggctcatctcaggtaacagcgg-3 ') and pl3 (5'-ggcgcgacgatggcatttgaagg-3') were used for the 3'-flank (1654bp).
  • the interfaces Sfi I (a) and Sfi I (b) were introduced with the primers LB2 and RB1.
  • the amplicons were restricted with S / z I and ligated with the 1884 bp Sfi I fragment from the vector pBS (hygromycin B cassette).
  • the 4742 bp trr knock-out cassette which was used in the subsequent transformation (Kämper and.), was amplified by PCR with the primers LB1 (5'-gacgggtcgattcggtcgcaagg-3 ') and RB2 (5'-ggctccgacggaaaacactttgg-3') Schreier, 2001).
  • 50 ml of a culture in YEPS medium were grown at 28 ° C. to a cell density of approx. 5 ⁇ 10 7 / ml (OD 0.6 to 1.0) and then for 7 min. centrifuged at 2500g (Hereaus, 3500 rpm) in 50 ml Falkon tubes.
  • the cell pellet was resuspended in 25 ml SCS buffer (20 mM Na citrate pH 5.8, 1.0 M sorbitol, (mix 20 mM Na citrate / 1.0 M sorbitol and 20 mM citric acid / 1.0 M sorbitol and adjust to pH 5.8 with a pH meter)) again 7 min.
  • the plating was carried out on gradient plates (lower agar: 10 ml of YEPS-1.5% agar-IM sorbitol with antibiotic; shortly before plating out, the lower agar layer was overlaid with 10 ml of YEPS-1.5% agar-IM sorbitol, which Spread protoplasts and incubate the plates for 3-4 days at 28 ° C.
  • the gene was amplified with gene-specific oligonucleotides by means of PCR and cloned into the expression vector pTrcHis2-TOPO, so that the TRR protein is expressed starting from the plasmid ptrcTRRlO with a C-terminal His 6 tag.
  • the plasmid ptrcTRR10 was transformed into E. coli BL21 (DE3).
  • 5 ml of selection medium (dYT with 100 ⁇ g / ml ampicillin) were inoculated with a single colony and incubated at 37 ° C. overnight in a shaker.
  • the main culture (dYT containing 100 ug / ml ampicillin) was inoculated 1:40 and incubated at 37 ° C with shaking to an OD 6 oo of 0.8 the culture was induced by adding 1 mM IPTG (final concentration). After an incubation period of 4 h at 37 ° C, the cells were harvested and then frozen at ⁇ 20 ° C.
  • the cells can be stored for several months at - 20 ° C without loss of activity. 2 g of cells were resuspended in 5 ml binding buffer (40 mM Tris / HCl pH 8.0, 100 mM NaCl), 600 ng lysozyme and 500, ng DNasel were added. The cell suspension was incubated with stirring on ice for 30 min. The digestion was carried out by 3
  • E. coli BL21 (DE3).
  • 5 ml selection medium dYT (double yeast tryptone) with 100 ⁇ g / ml ampicillin
  • the main culture dYT with 100 ⁇ g / ml ampicillin was inoculated 1:40 and incubated at 37 ° C. with shaking.
  • the culture was induced by adding 1 mM IPTG (final concentration). After an incubation period of 4 h at 37 ° C, the cells were harvested and then frozen at -20 ° C. The cells can be stored for several months at -20 ° C without loss of activity. 6 g cells were resuspended in 94 ml binding buffer (40 mM Tris HCl pH 8.0, 100 mM NaCl) and lysozyme and DNasel were added. The cell suspension was incubated with stirring on ice for 30 min. The disruption was carried out by means of ultrasound in a rosette rim vessel with 8 cycles of 90 seconds and a break of 90 seconds each (cooled with ice water).
  • the protein solution was frozen at -20 ° C.
  • the enzyme isolated in this way can be stored at -20 ° C for several months without loss of activity.
  • the K M value was determined by means of the initial decrease in fluorescence at 360 nm / 465 nm. Due to the strong fluctuations in very small thioredoxin
  • the test was carried out with different concentrations of NADPH.
  • the consumption of NADPH is measured depending on the NADPH concentration used.
  • the TRR1 concentration was 300 ng in a reaction volume of 50 ⁇ l.
  • the application was carried out as a lineweaver burk plot ( Figure 6 (B)).
  • the K M value of the thioredoxin reductase for NADPH from E. coli is 3-8 ⁇ M according to the literature. Since the NADPH fluorescence also serves as a measurement parameter, the K M value determination was carried out in parallel by detecting the free SH groups of the thioredoxin formed with DTNB and the change in absorption was measured.
  • a K M value for NADPH of 16-40 ⁇ M resulted from the measurement with NADPH.
  • a screening method or an inhibition test is therefore carried out, for example, with 30 ⁇ M NADPH in order to obtain an adequate read-out.
  • N-ethylmaleimide M r 125Jg / mol
  • the literature describes N-ethylmaleimide (M r 125Jg / mol) as an inhibitor of thioredoxin reductase.
  • the method was tested using this known inhibitor. The test was carried out in the presence of different concentrations of N-ethylmaleimide. It was shown that the NADPH fluorescence initially decreases very strongly in the presence of high concentrations of inhibitor.
  • N-ethylmaleimide can also inhibit thioredoxin itself as an "SH group reagent".
  • the negative control was pipetted into columns 1 and 2 of the MTP. This was composed of 5 ⁇ l 5% (v / v) DMSO, 20 ⁇ l substrate solution (18.75mM potassium phosphate buffer pH
  • test substances in a concentration of 10 ⁇ M in DMSO were placed in the remaining columns, H 2 0 being used to dilute the substance.
  • 20 ml substrate solution (18J5mM potassium phosphate buffer pH 7.4; l, 25mM EDTA, 75 ⁇ M NADPH)
  • 25 ⁇ l enzyme solution 15mM potassium phosphate buffer pH 7.4; ImM EDTA, 256 ⁇ M insulin, 40nM / ⁇ l thioredoxin and 4.8 ng / ⁇ l
  • a resazurin PMS mix 300 mM sodium acetate pH 5.8, 1% (v / v) DMSO, 20 mM PMS, 10 ⁇ M resazurin
  • the NADPH not reacted during the reaction reacts with resazurin in the presence of PMS to form fluorescent resorufin.
  • the resorufin was detected by determining the decrease in the fluorescence intensity at 595 nm (excitation 550 nm) in a suitable SPECTRAFluor Plus reader from Tecan.
  • a methanolic solution of the active substance identified by means of a method according to the invention (Example 4), mixed with an emulsifier, is pipetted into the cavities of microtiter plates. After the solvent has evaporated, 200 ⁇ l of potato dextrose medium are added to each cavity. Suitable concentrations of spores or mycelia of the fungus to be tested are added to the medium beforehand.
  • the resulting concentrations of the active ingredient are e.g. 0J, 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 photometrically at a wavelength of 620 nm. From the measurement of the different concentrations, the dose of active ingredient that results in a 50% inhibition of fungal growth compared to the untreated control (ED 5 o) was calculated.

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Abstract

La présente invention concerne un procédé pour identifier des fongicides, l'utilisation de thiorédoxine réductase fongique pour identifier des fongicides, et l'utilisation d'inhibiteurs de la thiorédoxine réductase comme fongicides.
PCT/EP2005/002951 2004-04-02 2005-03-19 Procede pour identifier des composes a action fongicide sur la base de thioredoxine reductases WO2005098030A2 (fr)

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DE200410016252 DE102004016252A1 (de) 2004-04-02 2004-04-02 Verfahren zum Indentifizieren von fungizid wirksamen Verbindungen basierend auf Thioredoxin Reduktasen
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