US20050239159A1 - Gtp cyclohydrolase II as as target for fungicides - Google Patents

Gtp cyclohydrolase II as as target for fungicides Download PDF

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US20050239159A1
US20050239159A1 US10/526,207 US52620705A US2005239159A1 US 20050239159 A1 US20050239159 A1 US 20050239159A1 US 52620705 A US52620705 A US 52620705A US 2005239159 A1 US2005239159 A1 US 2005239159A1
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gtp
fungal
gtp cyclohydrolase
candidate compound
cyclohydrolase
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Annette Freund
Franz Rohl
Henning Althofer
Marvin Karos
Bruno Kaesler
Thierry Lacour
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BASF SE
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Assigned to BASF AKTIENGESELLSCHAFT reassignment BASF AKTIENGESELLSCHAFT ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ALTHOFER, HENNING, FREUND, ANNETTE, KAESLER, BRUNO, KAROS, MARVIN, LACOUR, THIERRY, ROHL, FRANZ
Publication of US20050239159A1 publication Critical patent/US20050239159A1/en
Priority to US11/477,874 priority Critical patent/US7435557B2/en
Priority to US12/205,441 priority patent/US7691596B2/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04025—GTP cyclohydrolase II (3.5.4.25)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • fungal GTP cyclohydrolase is suitable as a fungicide target.
  • the present invention comprises the use of a fungal GTP cyclohydrolase as target for the identification of antifungal agents and methods of identifying antifungal agents which inhibit fungal GTP cyclohydrolase II comprising the following steps:
  • affinity tag this denotes a peptide or polypeptide whose coding nucleic acid sequence can be fused to the sequence encoding the fungal GTP cyclohydrolase II, either directly or using a linker, by customary cloning techniques.
  • the affinity tag serves to isolate the recombinant fungal GTP cyclohydrolase II by means of affinity chromatography.
  • the abovementioned linker can optionally comprise a protease cleavage site (for example for thrombin or factor Xa), whereby the affinity tag can be cleaved off from the fungal GTP cyclohydrolase II, as required.
  • affinity tags are the “his-tag”, for example from Quiagen, Hilden, “strep-tag”, “myc-tag” (Invitrogen, Carlsberg), New England Biolab's tag which consists of a chitin-binding domain and an intein, and what is known as the CBD-tag from Novagen.
  • Enzymatic activity/activity assay the term enzymatic activity describes the ability of an enzyme to convert a substrate into a product.
  • both the natural substrate of the enzyme and a synthetic modified analog of the natural substrate can be used.
  • the enzymatic activity can be determined in what is known as an activity assay via the increase in the product, the decrease in the starting material, the decrease or increase in a specific cofactor, or a combination of at least two of the afore-mentioned parameters as a function of a defined period of time. If the enzyme catalyzes a reversible reaction, both the starting material and the product may be employed as substrate in the activity assay in question.
  • nucleotide sequences can be generated in a manner known per se by chemical synthesis from the nucleotide units such as, for example, by fragment condensation of individual overlapping complementary nucleotide units of the double helix.
  • Oligonucleotides can be synthesized chemically for example in a known manner using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897).
  • phosphoamidite method Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897.
  • a variety of DNA fragments can be manipulated to give rise to a nucleotide sequence which reads in the correct direction and is in-frame.
  • the nucleic acid fragments are linked to each other by general cloning techniques as are described, for example in T.
  • Gene describes a nucleic acid sequence which encodes a protein and which can be transcribed into RNA (mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA) and which can optionally be associated with regulatory sequences. Examples of regulatory-sequences are promoter sequences. Other elements which are optionally present are, for example, introns.
  • Genetic control sequence the term of the genetic control sequences (which is equivalent to the term “regulatory sequence”) describes sequences which have an effect on the materialization or the function of the expression cassette according to the invention and which ensure for example transcription and, if appropriate, translation in prokaryotic or eukaryotic organisms. Examples are promoters or what are known as enhancer sequences. In addition to these control sequences, or instead of these sequences, the natural regulation of these sequences before the actual structural genes may still be present and, if appropriate, may have been modified genetically in such a way that the natural regulation has been inactivated and the expression of the fungal GTP cyclohydrolase II gene increased. The choice of the control sequence depends on the host organism or starting organism.
  • Control sequences furthermore also encompass the 5′-untranslated region, introns or the noncoding 3′-region of genes. Control sequences are furthermore understood as being those which make possible a homologous recombination or insertion into the genome of a host organism or which permit the removal from the genome.
  • “Functional equivalents” in the present context describe nucleic acid sequences which hybridize under standard conditions with the nucleic acid sequence encoding the GTP cyclohydrolase II or portions of the nucleic acid sequence encoding the GTP cyclohydrolase II, and which are capable of bringing about the expression of an enzymatically active fungal GTP cyclohydrolase II in a cell or an organism.
  • oligonucleotides of a length between 10 to 50 bp, preferably 15-40 bp, for example of the conserved or other regions, which can be determined via comparisons with other related genes in a manner known to the skilled worker for the hybridization.
  • longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization are also possible. These standard conditions vary depending on the nucleic acid used, viz. oligonucleotide, longer fragment or complete sequence, or depending on which type of nucleic acid, viz. DNA or RNA, is being used for the hybridization.
  • the melting temperatures for DNA:DNA hybrids are approx. 10° C. lower than those of DNA:RNA hybrids of equal length.
  • the hybridization conditions for DNA:DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures of between approximately 20° C. and 45° C., preferably between approximately 30° C. and 45° C.
  • the hybridization conditions for DNA:RNA hybrids are advantageously 0.1 ⁇ SSC and temperatures of between approximately 30° C. and 55° C., preferably between approximately 45° C. and 55° C.
  • the scope of the present invention also extends to, for example, those nucleotide sequences which are obtained by modification of the nucleic acid sequence of a GTP cyclohydrolase II.
  • the purpose of such a modification can be, for example, the insertion of further cleavage sites for restriction enzymes, the removal of excess DNA, or the addition of further sequences. Proteins which are encoded via said nucleic acid sequences should still maintain the desired functions, despite the deviating nucleic acid sequence.
  • GTP cyclohydrolase II activity denotes the ability of an enzyme to catalyse a reaction, wherein GTP is metabolized into 2,5-diamino-6-ribosylamino-4(H)-pyrimidinone 5′-monophosphate, pyrophosphoric acid and formic acid.
  • “Homology” between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence/polypeptide sequence by in each case the entire sequence length, which is calculated by alignment with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters: Gap weight: 8 Length weight: 2 Average match: 2,912 Average mismatch: ⁇ 2,003 matrix BLOSUM 62
  • “Mutations” comprise substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues, which may also lead to a modification of the corresponding amino acid sequence of the fungal GTP-cyclohydrolase II by substitution, insertion or deletion of one or more amino acids.
  • “Knock-out transformant” refers to a transgenic organism, in which a specific gene has been inactivated in a directed fashion by means of transformation.
  • Natural genetic environment is understood as meaning the natural chromosomal locus in the organism of origin or the presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably retained at least in part.
  • the environment flanks the nucleic acid sequence at least on the 5′ or 3′ side and has a sequence length of at least 50 bp, preferably at least 100 bp, especially preferably at least 500 bp, very especially preferably at least 1000 bp, most preferably at least 5000 bp.
  • “Operative linkage” An operative or else functional linkage is understood as meaning the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can, upon expression of the coding sequence, fulfil its function as intended.
  • Recombinant DNA technology generally known techniques from fusing DNA sequences (for example described in Sambrook et al., 1989, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press).
  • “Origins of replication” ensure the amplification of the expression cassettes or vectors according to the invention in microorganisms, for example pBR322 ori or P15A ori in E. coli (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • reporter genes encode readily quantifiable proteins. Using these genes, an assessment of transformation efficacy or of the site or time of expression can be made via growth, fluorescence, chemoluminescence, bioluminescence or resistance assay or via photometric measurement (intrinsic color) or enzyme activity. Very especially preferred in this context are reporter proteins (Schenborn E, Groskreutz D. Mol. Biotechnol. 1999; 13(1):29-44) such as the “green fluorescence protein” (GFP) (Gerdes H H and Kaether C, FEBS Lett. 1996; 389(1):44-47; Chui W L et al., Curr. Biol.
  • GFP green fluorescence protein
  • “Selection markers” impart resistance to antibiotics. Examples which may be mentioned are the neomycin-phosphotransferase-gen gene, which imparts resistance to the aminoglyciside antibiotics neomycin (G 418), kanamycin and paromycin (Deshayes A et al., EMBO J. 4 (1985) 2731-2737), the sul gene (Guerineau F et al., Plant Mol. Biol. 1990; 15(1):127-136), the hygromycin B phosphotransferase-Gen (Gen Bank Accession NO: K 01193) and the she-ble gene, which imparts resistance to the bleomycin antibiotic zeocin.
  • selection marker genes are genes which impart resistance to 2-desoxyglucose-6-phosphate (WO 98/45456) or phosphinothricin and the like, or those which impart resistance to antimetabolites, for example the dhfr gene (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994) 142-149). Also suitable are genes such as trpB or hisD (Hartman S C and Mulligan R C, Proc. Natl. Acad. Sci. U S A. 85 (1988.) 8047-8051).
  • mannose-phosphate isomerase gene WO 94/20627
  • ODC ornithin decarboxylase
  • Aspergillus terreus deaminase Tamura K et al., Biosci. Biotechnol. Biochem. 59 (1995) 2336-2338).
  • “Significant decrease” referring to the enzymatic activity, is understood as meaning the decrease in the enzymatic activity of the enzyme incubated with a candidate compound in comparison with the activity of an enzyme not incubated with the candidate compound, which lies outside an error in measurement.
  • Substrate is the compound which is recognized by the enzyme in its original function and which is converted into a product by means of a reaction catalyzed by the enzyme.
  • Transgenic Referring to a nucleic acid sequence, an expression cassette or a vector comprising said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector, transgenic describes all those constructions generated by recombinant methods in which either the nucleic acid sequence of the fungal GTP cyclohydrolase II or a genetic control sequence linked operably to the nucleic acid sequence of the fungal GTP cyclohydrolase II or a combination of both of the aforementioned nucleic acid sequences.
  • GTP cyclohydrolase II catalyses the first step in the biosynthesis of riboflavin (vitamin B2), wherein-GTP is metabolized into 2,5-diamino-6-ribosylamino-4(H)-pyrimidinone 5′-monophosphate, pyrophosphoric acid and formic acid (Ritz et. al. JBC 2001, 276: 22273-22277).
  • GTP cyclohydrolase II can be used for phonetative riboflavin synthesis in Ashbya gossypii (WO 95/26406).
  • GTP cyclohydrolase II In plants, GTP cyclohydrolase II is used as a target for herbicides (WO 00/40744). The plant enzyme, however, differs significantly from the fungal enzyme.
  • the identity between fungal GTP cyclohydrolase II from Ashbya gossypii (SEQ ID NO:2) and plant GTP cyclohydrolase II from Arabidopsis thaliana (SWISS-PROT P47924) is only 31%.
  • the identity between bacterial GTP cyclohydrolase II from Escherichia coli (SWISS-PROT P25523) and SEQ ID NO:2 is 48%.
  • SGD Saccharomyces Genom Database
  • GTP cyclohydrolase II is a suitable fungicide target by demonstrating the essential role of GTP cyclohydrolase II for the pathogenic filamentous fungi Ashbya gossypii.
  • the present invention therefore provides methods of using a fungal GTP cyclohydrolase II polypeptide (used herein synonymous to fungal GTP cyclohydrolase II) encoding nucleic acid sequenence to identify inhibtors thereof, which then can be used as fungicides to suppress the growth of pathogenic fungi.
  • pathogenic fungi denotes fungi which colonize a host (a plant or a mammal) and cause disease, e.g. human pathogenens selected from the group consisting of the genera and species Candida such as Candida albicans, Candia stettatoidea, Candiala tropicatis, Candida parapsilosis, Candida krusei, Candida pseudotropicatis, Candida quittermondii, Candida rugosa, Aspergillus such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus such as Rhizopus arrhizus, Rhizopus oryzae, Absidia such as Absidia corymbifera, Absidia ramosa and Mucor such as Mucor pusiltus or phytopathogenic filamentous fungi selected from the group consisting of the genera and species Ashbya such as Ashbya gos
  • Candida
  • grisea Mycosphaerella
  • Phyllactinia such as Phyllactinia kakicola
  • Gloesporium such as Gloesporium kaki
  • Gymnosporangium such as Gymnosporangium yamadae
  • Leptotthrydium such as Leptotthrydium pomi
  • Podosphaera such as Podosphaera leucotricha
  • Pyrenophora ( P. ) such as P. graminea, P. hordei, P. japonica, P. teres, P. teres f. maculata, P. teres f. teres, P.
  • Gloedes such as Gloedes pomigena; Cladosporium such as Cladosporium carpophilum; Phomopsis; Phytopora; Phytophthora such as Phytophthora infestans; Verticillium; Glomerella such as Glomerella cingulata; Drechslera; Bipolaris; Personospora; Phaeoisariopsis such as Phaeoisariopsis vitis; Spaceloma such as Spaceloma ampelina; Pseudocercosporella such as Pseudocercosporella herpotrichoides; Pseudoperonospora; Puccinia; Typhula; Pyricularia such as Pyricularia oryzae; Rhizoctonia; Stachosporium such as Stachosporium nodorum; Uncinula such as Uncinula necator; Ustilago such as Ustilago maydis
  • pathogenic fungi denotes filamentous phytopathogenic fungi mentioned above.
  • the present invention encompasses a method for identifying antifungal agents comprising the following steps:
  • the fungal GTP cyclohydrolase II is encoded by a nucleic acid sequence comprising
  • the functional equivalent of SEQ ID NO:2 set forth in c) has an identity of at least 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% preferably at least 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70% more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% most preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,.98%, 99% homology with the SEQ ID NO:2.
  • the nucleic acid sequence originates from a fungus, wherein the term fungus denotes the above-mentioned pathogenic fungi and yeast such as Saccharomyces species (e.g. S. cerevisiae ), Pichia species (e.g. P. pastoris, P. methanolica ), Schizosaccharomyces species (e.g. Schizosaccharomyces pombe ) and Klyveromyces species (e.g. K. lactis ).
  • Saccharomyces species e.g. S. cerevisiae
  • Pichia species e.g. P. pastoris, P. methanolica
  • Schizosaccharomyces species e.g. Schizosaccharomyces pombe
  • Klyveromyces species e.g. K. lactis
  • nucleic acid sequences encoding a fungal GTP cyclohydrolase are provided, whereby said nucleic acid sequences comprise
  • the functional equivalent of SEQ ID NO:4 set forth in c) has an identity of at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, preferably at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, more preferably 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% most preferably at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the SEQ ID NO:5.
  • the selection according to step ii) can be based on binding assays detecting the protein-inhibitor interactions, wherein either the candidate compound or fungal GTP cyclohydrolase II comprises a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a candidate compound, which is bound to the fungal GTP cyclohydrolase II can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • FRET Fluorescence Energy Transfer
  • binding of a candidate compound to a fungal GTP cyclohydrolase II can be determined without labeling either of the interactants, e.g. by using a microphysiometer to detect binding of a candidate compound to the fungal GTP cyclohydrolase II.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • determining the ability of a candidate compound to bind to the fungal GTP cyclohydrolase II can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 23382345, 1991, and Szabo etal., Curr. Opin. Struct. Biol. 5,699705, 1995), a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g. BIAcore). Changes in the optical phenomenon surface plasmone resonance can be used as an indication of real-time reactions between biological molecules.
  • BIA Bimolecular Interaction Analysis
  • SELDI surface-enhanced laser desorption/ionization
  • MALDI-TOF time-of-flight mass spectrometer
  • all of the above-mentioned methods can be based on a “competition assay”, wherein a reference molecule is replaced by the candidate compound.
  • the selection according to steps iii) and iv) preferably comprises testing a candidate compound in a fungal GTP cyclohydrolase II inhibition assay.
  • step iii), herein after referred to as “in vitro assay”, is based on the following steps:
  • the enzymatic activity of the fungal GTP cyclohydrolase II is preferably determined in comparison to the activity of a fungal GTP cyclohydrolase II not incubated with the candidate compound.
  • step (b) candidate compounds are selected which brought about a significant decrease in the enzymatic activity corresponding to a reduction of at least 10%, advantageously at least 20%, preferably at least 30%, especially preferably by at least 50% and very especially preferably by at least 70%, or a 100% reduction (blocking) being achieved.
  • Suitable substrates added to the reaction mixture in step b) for determination of enzymatic activity are GTP or GTP comprising a detectable label, such as a fluorescent, radioisotopic or chemiluminescent label.
  • GTP-analogs These labled derivatives are hereinafter referred to as “GTP-analogs”.
  • a fungal GTP cyclohydrolase II comprising mixture (e.g. crude cell extract, partially or totally purified protein) is incubated with a suitable substrate and the conversion of the substrate or the increase in the resultant product is monitored e.g. by HPLC or by measurement of fluorescence, radioactivity or chemiluminescence of the respective sample.
  • mixture e.g. crude cell extract, partially or totally purified protein
  • the enzymatic activity can be determined by HPLC as described in Ritz et. al. Journal of Biological Chemistry 2001, 276: 22273-22277) or by monitoring radioactivity as described by Foor and Brown (1980, Meth. Enzymol. 66:303-307).
  • GTP cyclohydrolase II is preferably partially purified or purified to homogeneity.
  • GTP cyclohydrolase II activity can be successfully determined in the presence of the enzyme formate dehydrogenase (E.C. 1.2.1.2).
  • the formic acid formed by GTP cyclohydrolase can be linked to the reduction of NAD by the combination of both of these enzymes.
  • the level of formate can be determined by monitoring the formation of NADH preferaby by spectroscopy as described by Tishkov and Egorov (SU 1271873).
  • This method is not only suitable for fungal GTP cyclohydrolase II, but also for plant GTP cyclohydrolase II and GTP cyclohydrolase I [E.C. 3.5.4.16], an enzyme having the same substrate specificity as GTP cyclohydrolase I but a different physiological function: GTP cyclohydrolase I that catalyses the first step in the biosynthesis of tetrahydrofolate and tetrahydrobiopterin.
  • this method is used preferably for fungal GTP cyclohydrolase II.
  • the present invention encompasses a method for determination of GTP cyclohydrolase activity comprising the following steps:
  • the method can comprise the following steps to ensure that the candidate compound inhibits GTP cyclohydrolase II and not formate dehydrogenase:
  • the GTP cyclohydrolase II activity in step c) of the in vitro assay is determined in the presence of the enzyme formate dehydrogenase (E.C. 1.2.1.2) comprising the following steps:
  • the in vitro assay comprises the following steps:
  • This method is suitable even if unpurified cell extracts (lysates) are put in the respective assay. Furthermore, this method is applicable to high throughput screening for inhibitors of fungal GTP cyclohydrolase II. If lysates or enzyme samples are used in which both enzymes, GTP cyclohydrolase I and GTP cyclohydrolase II are present, the selected candidate compounds can be optionally further tested in another inhibition assay to confirm whether GTP cyclohydrolase I or GTP cyclohydrolase II is inhibited (e.g. according to Ritz et. al. Journal of Biological Chemistry 2001, 276: 22273-22277).
  • the fungal GTP cyclohydrolase II used for the in vitro test can be present in the lysate of the fungi or of the transgenic organism according to the invention.
  • the polypeptide according to the invention can be purified partially or fully by customary methods.
  • a general overview of customary techniques for purification of proteins is given, for example, in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6.
  • purification of the protein fused to an affinity tag may be effected by affinity chromatography.
  • the fungal GTP cyclohydrolase II used for the above-mentioned in vitro assay can either be expressed in a transgenic organism transformed with an expression cassette comprising a nucleic acid sequence encoding a fungal GTP cyclohydrolase II in enzymatically active form or be obtained by culturing fungi naturally comprising a GTP cyclohydrolase II.
  • vectors are also understood as meaning all the other vectors known to the skilled worker such as, for example, phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear DNA or circular DNA. These vectors are capable of being replicated autonomously in the host organism or replicated chromosomally, chromosomal replication being preferred.
  • Suitable expression cassette comprises fungal GTP cyclohydrolase II encoding nucleic acid sequence operatively linked to control elements, which govern the expression of the coding sequence in the host cell.
  • an expression cassette according to the invention comprises, at the 5′ end of the coding sequence, a promoter and at the 3′ end a transcription/termination signal and, if appropriate, further genetic control sequences which are linked operably to the interposed coding sequence of the fungal GTP cyclohydrolase II.
  • analogs of the above-described expression cassettes which can originate, for example, from a combination of the individual nucleic acid sequences on one polynucleotide (multiple constructs), more than one polynucleotide in a cell (cotransformation), or by sequential transformation.
  • Advantageous control sequences for the expression cassettes or vectors according to the invention are present, for example, in promoters such as the cos, tac, trp, tet, lpp, lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, 1-PR or in the 1-PL promoter, all of which can be used for expressing fungal GTP cyclohydrolase II in Gram-negative bacterial strains.
  • promoters such as the cos, tac, trp, tet, lpp, lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, 1-PR or in the 1-PL promoter, all of which can be used for expressing fungal GTP cyclohydrolase II in Gram-negative bacterial strains.
  • promoters amy and SPO2 both of which can be used for expressing fungal GTP cyclohydrolase II in Gram-positive bacterial strains, and in the yeast or fungal promoters AUG1-, ADC1 GPD-1-, PX6-, TEF-, CUP1-, PGK-, GAP1-, TPI, PHO5-, AOX1, GAL10/CYC-1, CYC1, OliC-, ADH-, TDH-, Kex2-, MFa-, rp28- or the NMT-promotor or combinations of the aforementioned promotors (Degryse et al., Yeast Jun.
  • Luo X. Gene Sep. 22, 1995; 163(1):127-31; Nacken et al., Gene Oct. 10 1996; 175(1-2): 253-60; Turgeon et al., Mol Cell Biol September 1987; 7(9):3297-305) all of which can be used for expressing fungal GTP cyclohydrolase II in yeast strains.
  • suitable terminators are the NMT-, Gcy1-, TrpC-, AOX1-, nos-, the PGK- or the CYC1-terminator, preferably the nos-terminator (Degryse et al., Yeast Jun. 15, 1995; 11(7):629-40; Brunelli et al. Yeast Dec.
  • Control elements which may be mentioned as being suitable for expression in insect cells are, for example, the polyhedrin promoter and the p10 promoter (Luckow, V. A. and Summers, M. D. (1988) Bio/Techn. 6, 47-55).
  • Examples of advantageous control sequences for expressing fungal GTP cyclohydrolase II in cell culture are, besides polyadenylation sequences, the following eukaryotic promoters of viral origin, such as, for example, promoters of the polyoma virus, adenovirus 2, cytomegalovirus or simian virus 40.
  • the expression cassettes according to the invention and the vectors derived from them may also comprise further functional elements, in addition to the abovementioned promoters.
  • the following may be mentioned by way of example, but not by limitation: reporter genes, origins of replication, selection markers and/or affinity tags, fused to fungal GTP cyclohydrolase II either directly or by means of a linker optionally comprising a protease cleavage site.
  • the expression cassette and the vectors derived from them can be employed for the transformation of bacteria, cyanobacteria, yeasts, filamentous fungi and algae and eukaryotic cells (for example insect cells) with the purpose of recombinantly producing fungal GTP cyclohydrolase II, the generation of a suitable expression cassette depending on the organism in which the gene is to be expressed.
  • the nucleic acid encoding a fungal GTP cyclohydrolase II may advantageously also be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA may consist of a linearized plasmid or else only of the nucleic acid construct as vector or the nucleic acid sequences used.
  • the nucleic acid sequences used in the method according to the invention may also be introduced into an organism by themselves.
  • genes may be introduced together into the organism in a single vector or each individual gene may be introduced into the organism in one vector each, it being possible to introduce the various vectors simultaneously or in succession.
  • transgenic organisms generated by transformation can be used for recombinant expression of fungal GTP cyclohydrolase II.
  • microorganisms for the recombinant expression are, besides bacteria, yeasts and fungi, and eukaryotic cell lines.
  • bacteria Preferred within the bacteria are bacteria of the genus Escherichia, Erwinia, Flavobacterium, Alcaligenes or cyanobacteria, for example of the genus Synechocystis or Anabena.
  • Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.
  • Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria, Mortierella, Saprolegnia, Pythium, or other fungi described in Indian Chem Engr. Section B. Vol 37, No 1, 2 (1995).
  • transgenic animals are also suitable as host organisms, for example C. elegans.
  • pGEX Pharmacose Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40
  • pMAL New England Biolabs, Beverly, Mass.
  • pRIT5 Pharmacia, Piscataway, N.J.
  • GST glutathion S-transferase
  • pTrc vectors Amann et al., (1988) Gene 69:301-315
  • pKK233-2 from CLONTECH, Palo Alto, Calif.
  • pET and “pBAD” vector series from Stratagene, La Jolla.
  • vectors for use in yeast are pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES derivatives, PGAPZ derivatives, pPICZ derivatives, and the vectors of the “Pichia expression kit” (Invitrogen Corporation, San Diego, Calif.). Vectors for use in filamentous fungi are described in: van den Hondel, C. A. M. J. J. & Punt, P. J.
  • insect cell expression vectors may also be used advantageously, for example for expression in Sf 9 cells.
  • These are for example the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • Others which may be mentioned are the baculovirus expression systems “MaxBac 2.0 kit” from Invitrogen, Carlsbad, or “BacPAK baculovirus expression system” from CLONTECH, Palo Alto, Calif.
  • the fungal GTP cyclohydrolase II can be expressed in mammalian cells.
  • expression vectors are pCDM8 and pMT2P, which are mentioned in: Seed, B. (1987) Nature 329:840 or Kaufman et al. (1987) EMBO J. 6:187-195).
  • promoters to be used by preference are of viral origin such as, for example, promoters of polyoma virus, adenovirus 2, cytomegalovirus or simian virus 40.
  • prokaryotic and eukaryotic expression systems are mentioned in Chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • transgenic organism transformed with at least one of the above-mentioned expression cassettes or vectors are herein below termed as “transgenic organism according to the invention”.
  • the fungal GTP cyclohydrolase II can be isolated from an organism naturally comprising a fungal GTP cyclohydrolase II, for example from the pathogenic fungi mentioned above and for example from fungi selected from the group consisting of the genera and species, e.g. Pichia such as Pichia pastoris and Pichia methanolica, Saccharomyces such as Saccharomyces cerevisiae, Hansenula such as Hansenula poymorpha; Trichoderma, Ashbya such as Ashbya gossipii, Neurospora such as Neurospora crassa, Beauveria, Mortierella, Saprolegnia, Pythium, or other fungi described in Indian Chem Engr. Section B. Vol 37, No 1, 2 (1995).
  • step iv) is based on an in vivo assay. In a preferred embodiment this comprises the following steps:
  • step (c) compounds are selected which brought about a reduction in growth, viability or infectivity of at least 10%, advantageously at least 20%, preferably at least 30%, especially preferably by at least 50% and very especially preferably by at least 70%, or a 100% reduction (blocking) being achieved.
  • An analogous untransformed organism is to be understood as the fungi which has been used for generating the transgenic organism according to the invention in step a).
  • Suitable organisms are the fungi defined above, preferably those, which can be easily genetically manipulated by the skilled artisan, e.g. Saccharomyces species, Pichia species, Fusarium species, Ashbya species, Schizosaccharomyces species, Magnaporte species, Ustilago species, Neurospora species and Klyveromyces species.
  • sample comprising-an antifungal agent
  • the sample can be divided into different groups, for example when it consists of a multiplicity of different components, in order to reduce the number of different substances per sample and then to repeat the method according to the invention with such a “subsample” of the original sample.
  • the above-described steps can be repeated several times, preferably until the sample identified in accordance with the method according to the invention only encompasses a small number of substances or only one substance.
  • the substance identified in accordance with the method according to the invention, or derivatives thereof is preferably formulated further so that it is suitable for use in plant breeding or in plant cell or plant tissue culture.
  • the substance in question is incubated with a culture of a pathogenic fungus, preferably a culture of a phytopathogenic fungus, especially preferably a culture of a filamentous phytopathogenic fungus, it being possible to determine the fungicidal action for example on the basis of limited growth.
  • the above-mentioned embodiments of the method for identifying antifungal agents are preferably realized in a high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of candidate compounds can be quickly screened.
  • microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l, preferably 200 ⁇ l.
  • assay volumes typically range from 50 to 500 ⁇ l, preferably 200 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the respective well format.
  • free format assays or assays that have no physical barrier between samples can be used as described in Jayaickreme et al. (Proc. Natl. Acad. Sci U.S.A. 19 (1994) 161418), Chelsky (“Strategies for Screening Combinatorial Libaries”, First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (November 710, 1995)) and Salmon et al. (Molecular Diversity 2 (1996), 5763). Additionally, a high throughput screening method as described in U.S. Pat. No.
  • 5,976,813 can be used based on a porous matrix, in which test samples are placed; one or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • either the fungal GTP cyclohydrolase II or the candidate compound is preferably bound to a solid support.
  • suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach the fungal GTP cyclohydrolase II or candidate compound to a solid support, including the use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the fungal GTP cyclohydrolase II or candidate compound and the solid support.
  • Candidate compounds are preferably bound to the solid support in an array, so that the location of individual candidate compounds can be tracked. Binding of a candidate compound to a fungal GTP cyclohydrolase II can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • antifungal agents identified by the abovementioned methods further designated as “identified compounds” are subject matter of the present invention.
  • they Preferably, they have a molecular weight below 1000 g/mol, preferably 500 g/mol, more preferably 400 g/mol and most preferably 300 g/mol.
  • the identified compounds further exhibit a Ki value below 1 mM, preferably 1 ⁇ M, more preferably 0.1 ⁇ M and most preferably 0.01 ⁇ M.
  • the identified compounds may be: expression libraries, for example cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic substances, hormones, PNA(s) or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein). They may be chemically synthesized substances or substances produced by microorganisms and can be present for example in cell extracts or, for example, plants, animals or microorganisms.
  • the reaction mixture can be a cell-free extract or comprise a cell or cell culture.
  • Suitable methods are known to the skilled worker and are described generally for example in Alberts, Molecular Biology the cell, 3 rd Edition (1994), for example Chapter 17.
  • the substances mentioned can be added to the reaction mixture or the culture medium or injected into the cells or sprayed onto a plant.
  • the identified compounds may also be present in the form of their agriculturally useful salts.
  • Suitable salts among agriculturally useful salts are mainly the salts of those cations or the acid addition salts of those acids whose cations, or anions, respectively, do not adversely affect the fungicidal action of the identified compound.
  • the invention therefore furthermore relates to processes for the preparation of the fungicidal composition, which comprises
  • the identified compounds according to the invention in step a) can be formulated for example in the form of directly sprayable aqueous solutions, powders, suspensions, also highly concentrated aqueous, oily or other suspensions or suspoemulsions or dispersions, emulsions, oil dispersions, pastes, dusts, compositions for spreading, or granules, and applied by spraying, atomizing, dusting, spreading or pouring.
  • the use forms depend on the intended purposes and the nature of the identified compound used; in any case, they should ensure the finest possible distribution of the identified compounds according to the invention.
  • the identified compounds as such can be dissolved or dispersed in an oil or solvent, it being possible to add further formulation auxiliaries for homogenization.
  • liquid or solid concentrates which are composed of identified compound and, if appropriate, solvent or oil and optionally further auxiliaries, and these concentrates are suitable for dilution with water.
  • Materials which may be mentioned in this context are emulsion concentrates (EC, EW), suspensions (SC), soluble concentrates (SL), pastes, pellets, wettable powders or granules, it being possible for the solid formulations to be either soluble or dispersible (wettable) in water.
  • such powders or granules or tablets may additionally be provided with a solid coating which prevents abrasion or an unduly early release of the identified compound.
  • auxiliaries is understood as meaning, in principle, the following classes of substances: antifoams, thickeners, wetters, stickers, dispersants or emulsifiers, bactericides and thixotropic agents.
  • antifoams thickeners
  • wetters stickers
  • dispersants or emulsifiers bactericides
  • thixotropic agents bactericides and thixotropic agents.
  • the skilled worked is familiar with the meaning of the abovementioned agents.
  • SLs, EWs and ECs can be prepared by simply mixing the constituents in question; powders can be prepared via mixing or grinding in specific types of mills (for example hammer mills).
  • SCs and SEs are usually prepared by wet milling, it being possible to prepare an SE from an SC by adding an organic phase comprising further auxiliaries or identified compounds. The preparation is known.
  • Granules for example coating granules, impregnated granules and homogeneous granules, can be prepared by binding the identified compounds to solid carriers.
  • solid carriers which are suitable for granules according to the invention, for example mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, and products of vegetable origin such as cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders or other solid carriers.
  • mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphat
  • inert liquid and/or solid carriers which are suitable for the formulations according to the invention, such as, for example, liquid additives such as mineral oil fractions of medium to high boiling point, such as kerosene or diesel oil, furthermore coal tar oils and oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene, alkylated naphthalenes or their derivatives, alkylated benzenes or their derivatives, alcohols such as methanol, ethanol, propanol, butanol, cyclohexanol, ketones such as cyclohexanone, or strongly polar solvents, for example amines such as N-methylpyrrolidone, or water.
  • liquid additives such as mineral oil fractions of medium to high boiling point, such as kerosene or diesel oil, furthermore coal tar oils and oils of vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for
  • surfactants which are suitable for the formulations according to the invention, such as, for example, the alkali, alkaline earth or ammonium salts of aromatic sulfonic acids, for example lignin sulfonic acid, phenol sulfonic acid, naphthalene sulfonic acid and dibutylnaphthalenesulfonic acid, and of fatty acids, alkyl sulfonates, alkylaryl sulfonates, alkyl sulfates, lauryl ether sulfates and fatty alcohol sulfates, and salts of sulfated hexadecanols, heptadecanols and octadecanols, and of fatty alcohol glycol ethers; condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of na
  • aromatic sulfonic acids for example lignin
  • Powders, dusts and materials for spreading being solid carriers, can be prepared advantageously by mixing or concomitantly grinding the active substances with a solid carrier.
  • concentrations of the identified compounds in the ready-to-use preparations can be varied within wide limits and depend on the nature of the formulation in question.
  • the fungicidal compositions, or the identified compounds, can be applied in curative.
  • the applications of identified compounds amount to from 0.001 to 3.0, preferably 0.01 to 1.0 kg/ha active substance, depending on the aim of the control measures, the season, the target plants and the stage of growth.
  • the present invention furthermore relates to a method of controlling harmful fungi, which comprises treating the fungi or the materials, plants, soils or seeds to be protected from fungal infection, with an effective amount of an antifungal agent according to the invention or of a fungicidal composition according to the invention. Harmful fungi are understood as meaning the pathogenic fungi mentioned at the outset.
  • Another object of the present invention is the use of the identified compounds for preparation of drugs, preferably pharmaceutical compositions comprising at least an identified compound.
  • the identified compounds according to the invention can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly and intraperitoneally) in a conventional way. Administration may also take place with vapors or sprays through the nasopharyngeal space.
  • the dosage depends on the age, condition and weight of the patient and on the mode of administration.
  • the daily dose of active substance is about 0.5-50 mg/kg of bodyweight on oral administration and about 0.1-10 mg/kg of bodyweight on parenteral administration.
  • the identified compounds can be used in conventional solid or liquid pharmaceutical forms, eg. as uncoated or (film-) coated tablets, capsules, powders, granules, suppositories, solutions, ointments, creams or sprays. These are produced in a conventional way.
  • the active substances can be processed with conventional pharmaceutical excipients such as tablet binders, bulking agents, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, release-slowing agents, antioxidants and/or propellant gases (cf. H. Sucker et al.: Pharmazeutician Technologie, Thieme-Verlag, Stuttgart, 1991).
  • the administration forms obtained in this way normally contain from 0.1 to 90% by weight of active substance.
  • Cloning methods such as, for example, restriction cleavages, DNA isolation, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, bacterial cultures, sequence analysis of recombinant DNA and Southern and Western Blots were carried out as described by Sambrook et al., Cold Spring Harbor Laboratory Press (1989) and Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6.
  • the bacterial strains used hereinbelow ( E. coli XL1-blue) were obtained from BRL Gibco or Invitrogen, Carlsberg, Calif.
  • the Ashbya gossypii wild-type strain has the ATTC number ATTC 10895.
  • Recombinant DNA molecules were sequenced using an ABI laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467(1977)). Fragments resulting from a polymerase chain reaction were sequenced and verified in order to avoid polymerase errors in constructs to be expressed.
  • DNA-modifying enzymes were obtained from AGS (Heidelberg), Amersham (Brunswick), Biometra (Gottingen), Roche (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach/Taunus), Novagen (Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise specified, they were used following the manufacturer's instructions.
  • the KO plasmid pDeltarib1G418 was obtained from pJR765 (WO95/26406) by inserting the G418R expression cassette (Degryse et al., Yeast 1995, 11(7):629-40) into pJR765 so that the GTP cyclohydrolase II gene (rib 1) that is set forth in SEQ ID NO:3 is deleted from 220 bp upstream the ATG codon to 739 bp downstream the ATG codon. 6 mg plasmid DNA of pDeltarib1G418 were linearized with the restriction enzyme Asp 700 and purified by classical gel elution for subsequent transformation.
  • A. goosypii spores were cultured for 12 h in MA2 medium (peptone 10 g/l, yeast extract 1 g/l, myoinositol 0.3 g/l, pH7) at 28° C. and 250 rpm.
  • the cells were pelleted by classical centrifugation and suspended with phosphate buffer 50 mM, DTT 25 mM and incubated at 28° C. with low agitation for 30 min.
  • the cells were collected by centrifugation and suspended with 25 ml cold phosphate buffer 50 mM pH 7.5; 150 ⁇ l of the cell suspension were mixed with 6 ⁇ g pDeltarib1G418 treated with Asp 700.
  • the mixture was electroporated with a Gene Pulser II electroporator (Bio-Rad; parameters: 200 ohms; 1.5 Kv; 25 ⁇ F)
  • the cells were mixed with 1 ml MA2 medium and spread on fresh Petri dishes containing MA2-agar supplemented with 200 mg vitamin b2. The plates were incubated at 28° C. for 6 h. Then, a fine layer of Top-agar (1% LMP agarose) containing G418 antibiotic 50 mg/ml was loaded at the top of the plates. The incubation was further conducted at 28° C. for 5 days. Several colonies capable of growth on selective medium were isolated for subsequent characterization.
  • Top-agar 1% LMP agarose
  • the KO mutants were grown on Petri dishes containing MA2 agar, G418 50 mg/ml and vitamin b2 200 mg/ml and then replicated on the same medium in the absence of vitamin b2. In the latter case, the KO mutants were not able to grow at all. This convincingly demonstrates that GTP cyclohydrolase II is essential for the life of the fungi.
  • Ashbya gossypii cells may be obtained after 2 days from a culture in 3% (w/v) malt extract+0.3% (w/v) difco-soyton, pH 5.6 at 28° C.
  • a cell-free extract can be obtained by mechanical breakage of the cells with a kitchen blender in 50 mM imidazole buffer pH 7 containing 1 mM EDTA-Na salt, 5 mM MgSO4, 10 mM KCl, 5 mM dithiothreitol and 30% (v/v) glycerol and separation of unbroken cells and debris by centrifugation.
  • the assay is performed in a suitable buffer e.g. Tris/HCl, pH 7.8 including 1 mM Mg.Cl 2 , 1 mM DTT, 0.5 mM NAD, 2 units/ml formate dehydrogenase and 2.5 mM GTP Li salt.
  • a suitable buffer e.g. Tris/HCl, pH 7.8 including 1 mM Mg.Cl 2 , 1 mM DTT, 0.5 mM NAD, 2 units/ml formate dehydrogenase and 2.5 mM GTP Li salt.
  • a suitable solvent e.g. DMSO
  • an aliquot of the afore made solution is added to the reaction mixture.
  • the enzyme activity of this sample is compared with the activity of a control comprising the pure solvent instead of the candidate compound.
  • the resulting relative activity was calculated as percent inhibition in relation to the sample without the candidate compound.
  • FIG. 1 the GTP dependant formation of NADH in the reaction mixture and inhibition by 3-Butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione is shown.
  • the subtrate GTP is replaced with formic acid.
  • An inhibitor of GTP cyclohydrolase shows inhibition in the presence of GTP and no inhibition in the presence of formic acid (see e.g. FIG. 2 ).
  • a stock solution of 3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione is prepared in DMSO at a concentration of 10,000 ppm a.i. For the test, this is diluted with sterile deionized water to a concentration of 125 ppm; the DMSO concentration is constant at all test concentrations.
  • Spore suspensions of the fungi employed in the test ( Phytophthora infestans, Pyricularia oryzae and Septoria tritici ) are made in double strength growth medium (4% (w/v) malt extract in water).
  • 3 (three) wells are prepared: 50 ⁇ l of compound solution are pipetted to each well, were to this are added 50 ⁇ l of spore suspension.
  • the prepared plates are then incubated at 18° C. for 7 days after which the density of developed fungal mycelium is measured in a photometer at a wavelength of 405 nm.
  • FIG. 1 shows the GTP dependent formation of NADH in the reaction mixture and inhibition by 3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione.
  • ⁇ designates values measured in the presence of GTP, ⁇ values measured without GTP and ⁇ values measured in the presence of GTP and 3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione.
  • FIG. 2 shows the specificity of the inhibition by 3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione.
  • No inhibition of formate dehydrogenase alone is observed. Therefore, the inhibition of the GTP dependent NADH formation is a consequence of the inhibition of GTP cyclohydrolase.
  • ⁇ designates values measured in the presence of formate, * ⁇ values measured without formate and ⁇ values measured in the presence of formate and 3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione.

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DE60314258D1 (de) 2007-07-19
AU2003270098A8 (en) 2004-03-29
US7435557B2 (en) 2008-10-14
EP1537233A2 (de) 2005-06-08
WO2004022776A2 (en) 2004-03-18
WO2004022776A3 (en) 2004-07-08
EP1537233B1 (de) 2007-06-06
ATE364095T1 (de) 2007-06-15
US20090023172A1 (en) 2009-01-22
AU2003270098A1 (en) 2004-03-29
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