EP1346029A2 - Utilisation de carboxylase acetyl-coa permettant d'identifier des composants possedant un effet insecticide - Google Patents

Utilisation de carboxylase acetyl-coa permettant d'identifier des composants possedant un effet insecticide

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Publication number
EP1346029A2
EP1346029A2 EP01985351A EP01985351A EP1346029A2 EP 1346029 A2 EP1346029 A2 EP 1346029A2 EP 01985351 A EP01985351 A EP 01985351A EP 01985351 A EP01985351 A EP 01985351A EP 1346029 A2 EP1346029 A2 EP 1346029A2
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EP
European Patent Office
Prior art keywords
acetyl
insects
sequences
accase
coa carboxylase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01985351A
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German (de)
English (en)
Inventor
Reiner Fischer
Eva-Maria Franken
Ralf Nauen
Ute Teuschel
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Bayer CropScience AG
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Bayer CropScience AG
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Publication of EP1346029A2 publication Critical patent/EP1346029A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the invention relates to the use of polypeptides and enzyme preparations with the biological activity of an acetyl-CoA carboxylase for the identification of new, insecticidally active compounds, and to methods for finding modulators of these polypeptides.
  • ACCase The acetyl-CoA carboxylase (EC 6.4.1.2), hereinafter referred to as ACCase, catalyzes the biotin-dependent carboxylation of acetyl-CoA and provides the
  • the ACCase has three domains: biotin carboxyl carrier (BCC), biotin carboxylase (BCase) and carboxyl transferase (CTase).
  • BCC biotin carboxyl carrier
  • BCase biotin carboxylase
  • Case carboxyl transferase
  • the reaction catalyzed by the ACCase can be divided into two steps. In a first step, a ATP-cleavage by the BCase activity a CO 2 group from bicarbonate to a covalently to the
  • the homomeric ACCase consists of a polypeptide chain that contains all three domains and can be found in the cytosol of plants, animals and fungi (Ke J et al., 2000). In plants and vertebrates, the ACCase is activated by numerous mechanisms, e.g. Allosterically regulated by citrate, palmitoyl-CoA, by phosphorylation / dephosphorylation, by protein kinases and at the level of gene expression (Munday MR & Hemingway CJ 1999; Ke J et al. 2000). No information is available on the regulation of the enzyme from insects.
  • ACCase from Arabidopsis thaliana gene bank AAF 18638546 or for a patent registered (e.g. Haselkorn R & Gornicki P 1999; Somers DA 1999; Jenkins AR et al. 1992).
  • a patent registered e.g. Haselkorn R & Gornicki P 1999; Somers DA 1999; Jenkins AR et al. 1992.
  • an annotated sequence from insects assigned to the ACCase is not yet available.
  • Inhibitors of ACCase from plants and fungi known as herbicides or fungicides (Vahlensieck HF et al. 1994; Gronwald JW 1994).
  • Another document describes the fungicides Soraphen A and B, known as ACCase inhibitors, for controlling mites that do not belong to the order of the insects (Sutter M. et al., 1991).
  • the object of the present invention was therefore to make the ACCase from insects available, to test their suitability as a site of action for insecticides, and
  • crude extracts have now been obtained from various larval stages or adults of the peach aphid Myzus persicae by homogenization in suitable buffers. These crude extracts were pre-cleaned and the
  • the present invention further shows that, in particular, cyclic 1,3-dicarbonyl compounds and their enols of the formula (I)
  • Ar represents substituted aryl or hetaryl with at least one ortho substituent
  • R stands for H or for acyl radicals, preferably for the radicals COR 1 and CO2R 1 in which
  • R 1 represents optionally substituted alkyl, phenyl or hetaryl
  • A forms an optionally substituted 5- or 6-membered carbo- or heterocycle with the linked C atoms, N, O and / or S being suitable as hetero atoms, for example,
  • 1,3-dicarbonyl compounds can be used as inhibitors of ACCase in insects.
  • the present invention also relates to the use of the compounds of the formula (I) as inhibitors of ACCase in insects.
  • ACCase is a target protein of insecticidal substances in insects. This also shows for the first time that the ACCase is an enzyme vital for insects and is therefore particularly suitable as a target protein for the search for further and possibly improved insecticides
  • the ACCase from Drosophila melanogaster is further described for the first time on the basis of its nucleic acid sequence and thus made accessible.
  • the nucleic acid sequence of the Accession Number AAF59156 has been accessible for some time.
  • the meaning of the sequence or the polypeptide encoded by it and its biological function, however, were previously unknown, as was the coding region of this sequence section.
  • homologous polypeptides which are encoded by corresponding homologous nucleic acids, and further members of the gene family can also be used as molecular interaction partners (targets) for insecticidal active substances, in particular the compounds of the formula (I).
  • the homologous polypeptides are particularly preferably those which are used for
  • ACCase from Myzus persicae or Drosophila melanogaster have an identity of 60%, preferably 80%, particularly preferably 90% and particularly preferably 95%
  • Insecticides and / or acaricidal active ingredients which can possibly be found with the aid of the ACCases according to the invention, can accordingly also interact with ACCases from numerous other acarina or insect species, the interaction with the different ACCases occurring in the insects or acarina not always being equally strong got to. Among other things, this explains the observed selectivity of the substances active on this enzyme.
  • Particularly preferred ACCases or organisms of origin are listed by way of example and not exhaustively in Table 1 below:
  • ACCases are target proteins (targets) for insecticidal active substances and can be used to identify new, improved insecticidal active substances in suitable methods (assays).
  • the ACCases from Myzus persicae and Drosophila melanogaster are particularly suitable for identifying new insecticidal and possibly also acaricidal active ingredients.
  • the present invention therefore relates to the use of polypeptides from insects with the biological activity of an ACCase and to nucleic acids encoding them for identifying modulators of the ACCase in
  • Insects were isolated or encoded by nucleic acid sequences or fragments thereof from insects and obtained by in vivo or in vitro methods.
  • the polypeptides are particularly preferably those which form a ACCase from Myzus persicae or Drosophila melanogaster
  • the present invention particularly relates to the use of the ACCase from insects of the Aphididae and Diptera family.
  • the present invention particularly relates to the use of the ACCase from Myzus persicae and the ACCase from Drosophila melanogaster according to SEQ ID NO: 1
  • the present invention relates in particular to the use of the ACCase from Myzus persicae for identifying modulators of the ACCase
  • polypeptides according to the invention thus very particularly preferably comprise a sequence selected from
  • sequences which have an at least 60%, preferably an 80%, particularly preferably a 90% and very particularly preferably a 95% identity with the sequences mentioned under a) to d).
  • the degree of identity of the amino acid sequences is preferably determined using the GAP program from the GCG program package, version 10.0 under standard settings (Devereux et al. 1984).
  • the present use also relates to the use of for
  • ACCases encoding insect nucleic acids to identify ACCase modulators in insects and / or Akarina.
  • the present invention also relates in particular to the use of the nucleic acid coding for the ACCase from Myzus persicae and that for
  • ACCase from Drosophila melanogaster encoding nucleic acid according to SEQ ID NO: 1 for identifying ACCase modulators, as well as 60%, preferably 80%, particularly preferably 90% and particularly preferably 95% homologous nucleic acid sequences.
  • the nucleic acids according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • Preferred embodiments are fragments of genomic DNA, which may contain introns, and cDNAs.
  • cDNA denotes a single- or double-stranded copy of an RNA molecule and is therefore intron-free as a copy of a biologically active RNA, i. H. all coding regions of a gene are contained in a coherent form.
  • identity refers to the number of sequence positions that are identical in an alignment. It is usually given as a percentage of the alignment length.
  • Sequence or a specific part of this sequence is defined as the percentage of nucleotides in the nucleic acid molecule examined which is identical to the nucleotides of the specified sequence or a specific part of this sequence when the sequences are compared with one another ("alignment") and, if necessary, so-called “gaps” are introduced by the maximum percentage to obtain identical sequences, with all parameters of the program used being set to "default".
  • similarity presupposes the definition of a similarity metric, that is, a measure of how similar one would like to assume a valine to a threonine or a leucine, for example.
  • percent (%) similarity corresponds to the term “percent (%) identity” described above, the conservative amino acid substitutions in addition to the identical amino acids being included in the calculation of the% number.
  • homologous proteins have developed from a common precursor sequence. The term does not necessarily have anything to do with identity or similarity, apart from the fact that homologous sequences are usually more similar (or have more identical positions in an alignment) than non-homologous sequences.
  • the nucleic acids according to the invention are preferably DNA or DNA fragments which correspond to genomic DNA from insects, the
  • Nucleic acids preferably originate from Diptera, particularly preferably from Drosophilidae.
  • the nucleic acids according to the invention are particularly preferably DNA or DNA fragments, the genomic DNA from Myzus persicae or
  • nucleic acids according to the invention very particularly preferably comprise a sequence selected from
  • sequences which have an at least 60%, preferably an 80%, particularly preferably a 90% and very particularly a 95% identity with the sequences defined under a) and b),
  • a very particularly preferred embodiment of the nucleic acids to be used according to the invention is a cDNA molecule with the sequence coding for ACCase from Myzus persicae and the sequence coding for ACCase from Drosophila melanogaster according to SEQ ID NO: 1.
  • the nucleic acid sequence coding for the ACCase from Myzus persicae can be derived on the basis of the genetic code from the amino acid sequence, which can be isolated as described in Example 3 and defined by sequencing.
  • nucleic acid sequence To use the nucleic acid sequence to check the nucleic acid sequence actually present in Myzus persicae and, if necessary, to correct the derived sequence, as far as this seems reasonable.
  • the isolation or verification of the genomic M. persicae sequence can e.g. by using primers derived from the derived nucleic acid sequence, which can be used in PCR reactions to amplify the target sequence according to methods known to the person skilled in the art.
  • the present invention also relates to the polypeptides which are encoded by the nucleic acids according to the invention.
  • hybridize as used herein describes the process in which a single-stranded nucleic acid molecule with a complementary strand undergoes base pairing.
  • DNA fragments from insects other than Drosophila melanogaster which code for ACCases that have the same or similar properties of one of the ACCases according to the invention, can be isolated, for example, from the sequence information mentioned or derived here.
  • Hybridization conditions are approximately calculated using the following formula:
  • Hybridization solution DIG Easy Hyb (Röche, ZZ) Hybridization temperature: 37 ° C to 50 ° C, preferably 42 ° C (DNA-DNA), 50 ° C (DNA-RNA).
  • 1st washing step 2x SSC, 0.1% SDS 2x5 min at room temperature;
  • 2nd washing step 1x SSC, 0.1% SDS 2x 15 min at 50 ° C; preferably 0.5x SSC, 0.1% SDS 2x 15 min at 65 ° C; particularly preferably 0.2x SSC, 2x15min at 68 ° C.
  • the degree of identity of the nucleic acids is preferably determined using the NCBI BLASTN version 2.0.4 program. (Altschul et al. 1997).
  • regulatory regions refers to untranslated regions of the gene in question, such as promoters, enhancers, repressor or activator binding sites or termination sequences that interact with cellular proteins, thereby controlling transcription.
  • the present invention also relates to DNA constructs which comprise a nucleic acid to be used according to the invention and a heterologous promoter.
  • the present invention furthermore relates to the use of such DNA constructs for identifying ACCase modulators.
  • heterologous promoter refers to a promoter which has different properties than the promoter which controls the expression of the gene in question in the original organism.
  • heterologous promoters depend on whether pro- or eukaryotic cells or cell-free systems are used for expression.
  • heterologous promoters are the early or late promoter of SV40, adenovirus or cytomegalovirus, the lac system, the trp system, the main operator and promoter regions of phage lambda, the control regions of the fd coat protein, the promoter of 3-phosphoglycerate kinase, the promoter of acids
  • Phosphatase the baculovirus immediate early promoter and the promoter of the yeast ⁇ -mating factor.
  • the invention furthermore relates to vectors which contain a nucleic acid according to the invention or a DNA construct according to the invention. All plasmids, phasmids, cosmids, YACs or artificial chromosomes used in molecular biological laboratories can be used as vectors.
  • the present invention furthermore relates to the use of vectors which contain a nucleic acid to be used according to the invention or a DNA construct to be used according to the invention in methods for identifying ACCase modulators.
  • phages All phages, plasmids, phagmids, phasmids, cosmids, YACs, BACs, artificial ones used in molecular biological laboratories can be used as vectors
  • Chromosomes or particles that are suitable for particle bombardment are used.
  • the present invention also relates to host cells which contain a nucleic acid to be used according to the invention, a DNA
  • Contain construct or a vector to be used according to the invention Contain construct or a vector to be used according to the invention.
  • the present invention also relates to the use of such host cells for identifying ACCase modulators.
  • host cell refers to cells which do not naturally contain the nucleic acids to be used according to the invention.
  • Suitable host cells are both prokaryotic cells, such as bacteria of the genera Bacillus, Pseudomonas, Streptomyces, Streptococcus, Staphylococcus, preferably E. coli, and eukaryotic cells, such as yeast, mammalian, amphibian, insect or plant cells.
  • Preferred eukaryotic host cells are HEK-293, Schneider S2, Spodoptera Sf9, Kc, CHO, COS1, COS7, HeLa, C127, 3T3 or BHK cells and in particular Xenopus oocytes.
  • polypeptides refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer amino acid chains, commonly referred to as proteins. It encompasses amino acid chains that are either natural Liehe processes, such as posttranslational processing, or by chemical methods, ⁇ the state of the art, may be modified. Such modifications can occur at various locations and multiple times in a polypeptide, such as, for example, on the peptide backbone, on the amino acid side chain, on the amino and / or on the carboxy terminus. They include, for example, acetylations, acylations, ADP ribosylations, amidations, covalent linkages with
  • the polypeptides according to the invention can be in the form of "mature" proteins or as parts of larger proteins, for example as fusion proteins.
  • 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.
  • ACCase which is coded from a complete coding region of a transcription unit, starting with the ATG start codon and comprising all information-bearing exon regions of the gene encoding the ACCase present in the organism of origin, as well as signals necessary for correct termination of the transcription.
  • gene as used herein is the designation for a section from the genome of a cell that is responsible for the synthesis of a polypeptide chain.
  • polypeptides according to the invention need not be complete ACCases, but can only be fragments thereof, as long as they have at least the biological activity of the complete ACCase.
  • Polypeptides from insects which have the same biological activity as an ACCase from Myzus persicae or Drosophila melanogaster are still considered to be according to the invention.
  • polypeptides according to the invention need not completely correspond to the ACCases from Myzus persicae or Drosophila melanogaster in terms of their sequence or catalytic activity.
  • Polypeptides which are homologous to the ACCase, for example of the following insects or to fragments thereof which can still exert the biological activity of the ACCase, are also considered as polypeptides according to the invention: From the order of the Isopoda, for example Oniscus asellus, Armadillidium vulgare, Porcellio scaber.
  • Thysanura e.g. Lepisma saccharina.
  • Orthoptera e.g. Acheta domesticus, Gryllotalpa spp., Locusta migratoria migratorioides, Melanoplus spp., Schistocerca gregaria.
  • Phthiraptera e.g. Pediculus humanus corporis, Haematopinus spp., Linognathus spp., Trichodectes spp., Damalinia spp ..
  • Thysanoptera for example Hercinothrips femoralis, Thrips tabaci, Thrips palmi, Frankliniella accidentalis.
  • Heteroptera for example Eurygaster spp., Dysdercus intermedius, Piesma quadrata, Cimex lectularius, Rhodnius prolixus, Triatoma spp.
  • Homoptera e.g. Aleurodes brassicae, Bemisia tabaci, Trialeurodes vaporariorum, Aphis gossypii, Brevicoryne brassicae, Cryptomyzus ribis, Aphis fabae, Aphis pomi, Eriosoma lanigerum, Hyalopterus arundinis, Phylloxera vastatrix, Pemphigus sppe, Phros
  • Sitophilus spp. Sitophilus spp., Otiorrhynchus sulcatus, Cosmopolites sordidus, Ceuthorrhynchus assimilis, Hypera postica, Dermestes spp., Trogoderma spp., Anthrenus spp., Attagenus spp., Lyctus spp., Meligethes aeneus, Psususylbole, Ptinuslopp.
  • Tribolium spp. Tenebrio molitor, Agriotes spp., Conoderus spp., Melolontha melolontha, Amphimallon solstitialis, Costelytra zealandica, Lissorhoptrus oryzoph ⁇ lus.
  • Hymenoptera e.g. Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Nespa spp.
  • polypeptides according to the invention may have deletions or amino acid substitutions in comparison to the corresponding regions of naturally occurring ACCases, as long as they still have at least one biological activity of the complete ACCases.
  • 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.
  • the present invention therefore also relates to polypeptides which exercise at least the biological activity of an ACCase and which comprise an amino acid sequence which has at least 60% identity, preferably 80%, particularly preferably 90% identity and very particularly preferably 95% identity with the sequence from Myzus persiace or the sequence from Drosophila melanogaster encoded by the nucleic acid according to SEQ ID NO: 1, and their use for the identification of modulators of the ACCase.
  • biological activity of an ACCase means the ability to do the biotin-dependent carboxylation of acetyl-CoA catalyze. This can include all three enzyme functions, ie the ATP-dependent removal of a CO 2 group from bicarbonate, the biotin carrier function and the carboxylation of acetyl-CoA, but also only one or two of these reactions.
  • the nucleic acids according to the invention can be produced in the usual way.
  • the nucleic acid molecules can be completely chemically synthesized. It is also possible to chemically synthesize short pieces of the nucleic acids according to the invention and to label such oligonucleotides radioactively or with a fluorescent dye.
  • the labeled oligonucleotides can also be used to search cDNA libraries made from insect mRNA. Clones to which the labeled oligonucleotides hybridize are selected for the isolation of the relevant DNA fragments. After characterizing the isolated DNA, the nucleic acids according to the invention are obtained in a simple manner.
  • nucleic acids according to the invention can also be produced by means of PCR methods using chemically synthesized oligonucleotides.
  • oligonucleotide (s) as used herein means DNA-
  • Molecules consisting of 10 to 50 nucleotides, preferably 15 to 30 nucleotides. They are chemically synthesized and can be used as probes.
  • polypeptides according to the invention in particular of the polypeptide encoded by the nucleic acid sequence according to SEQ ID NO: 1, can also be carried out
  • Host cells containing nucleic acids according to the invention are cultivated under suitable conditions.
  • the desired polypeptides can then be isolated from the cells or the culture medium in a conventional manner.
  • the polypeptides can also be produced in in vitro systems.
  • To produce the ACCase from Myzus persicae according to the invention for example, larvae or adults are homogenized in a mortar. For this purpose, they can be snap frozen beforehand, for example in liquid nitrogen. The homogenate is taken up in a suitable buffer.
  • An example of the preparation of a polypeptide according to the invention is given under Example 3.
  • One possible ACCase purification method 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.
  • a rapid method for isolating the polypeptides according to the invention begins with the expression of a fusion protein, whereby the fusion partner can be easily affinity-purified.
  • the fusion partner can be, for example, glutatbion S-transferase.
  • the fusion protein can then be purified on a glutathione affinity column.
  • the fusion partner can be separated by partial proteolytic cleavage, for example on the linker between the fusion partner and the polypeptide according to the invention to be purified.
  • the linker can be designed to include target amino acids, such as arginine and lysine residues, which define sites for trypsin cleavage. Standard cloning techniques using oligonucleotides can be used to create such linkers.
  • Electrophoresis FPLC, HPLC (e.g. using gel filtration, reverse phase or slightly hydrophobic columns), gel filtration, differential precipitation, ion exchange chromatography and affinity chromatography.
  • composition containing the polypeptides according to the invention is preferably enriched at least 10-fold and particularly preferably at least 100-fold with respect to the protein content compared to a preparation from the host cells.
  • polypeptides according to the invention can also be affinity-purified without a fusion partner using antibodies which bind to the polypeptides.
  • the present invention also relates to methods of finding chemical compounds which bind to the ACCase and / or change its properties. Due to the important function of the ACCase, modulators which influence the activity represent new insecticidal and / or possibly acaricidal active ingredients. Modulators can be agonists or antagonists or inhibitors or activators.
  • the cyclic 1,3-dicarbonyl compounds of the formula (I) and their enols mentioned can also be used as optionally marked competitors in methods for finding ACCase inhibitors from insects which do not have to belong to this group of connections.
  • agonist refers to a molecule that accelerates or enhances the activity of the ACCase.
  • modulator as used herein represents the generic term for agonist or antagonist. Modulators can be small organic chemical molecules, peptides or antibodies that bind to the polypeptides according to the invention and / or their properties, for example their enzymatic Activity, change.
  • 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 / or influences their biological activity.
  • Modulators can be natural substrates and ligands or structural or functional mimetics thereof.
  • the modulators are preferably small organic chemical compounds.
  • Compounds or modulators that act on the ACCase can change the cellular processes in a way that leads to the death of the insects treated with them.
  • the present invention therefore also relates to modulators of
  • the present invention comprises methods for finding chemical compounds which change the expression of the polypeptides according to the invention.
  • expression modulators can also represent new insecticidal active ingredients.
  • Expression modulators can be small organic chemical molecules, peptides or antibodies that bind to the regulatory regions of the nucleic acids coding for the polypeptides according to the invention.
  • Expression modulators can also be small organic chemical molecules, peptides or Be antibodies that bind to a molecule, which in turn binds to regulatory regions of the nucleic acids coding for the polypeptides according to the invention, and thereby influences their expression.
  • Expression modulators can also be antisense molecules.
  • the present invention also relates to the use of modulators of the polypeptides according to the invention or of expression modulators as insecticides or acaracides.
  • the present invention also relates to expression modulators of
  • the methods of the invention include high throughput screening (HTS) and ultra high throughput screening (UHTS). Both host cells and cell-free preparations containing the nucleic acids according to the invention and / or the polypeptides according to the invention can be used for this.
  • HTS high throughput screening
  • UHTS ultra high throughput screening
  • a synthetic reaction mix e.g. products of in vitro transcription
  • a cellular component such as a membrane, a compartment or any other preparation which contains the polypeptides according to the invention, together with a labeled substrate or ligand of the polypeptides in the presence and In the absence of a candidate molecule, which can be an agonist or an antagonist.
  • Candidate molecule to increase or inhibit the activity of the polypeptides according to the invention is recognizable by an increased or decreased binding of the labeled ligand or by an increased or decreased conversion of the labeled substrate.
  • Molecules that bind well and lead to an increased activity of the polypeptides according to the invention are agonists.
  • Molecules that bind well and inhibit the biological activity of the polypeptides according to the invention are good antagonists. These can also be inhibitors of the above-mentioned class of insecticides, but completely new classes of substances can also have this modulatory activity.
  • Reduce expression of ACCase-encoding mRNA and / or polypeptides by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% are suitable to be used as insecticides or to be developed into such.
  • candidate molecules are then used in further tests for toxicity to vertebrate species, e.g. Mammals and checked for bioavailability.
  • the detection of the biological activity of the polypeptides according to the invention can be improved by a so-called reporter system. Reporter systems in this
  • aspects include, but are not limited to, colorimetric or radioactively labeled substrates that are converted to a product, or a reporter gene that responds to changes in the activity or expression of the polypeptides of the invention, or other known binding assays.
  • the activity of many, e.g. B. membrane-bound proteins can be advantageously measured in a further way.
  • the functional heterologous expression of such proteins in E. coli is often difficult or impossible.
  • the catalytically active part of the protein can be separated off by suitable cloning (for example using suitable PCR strategies), so that the gene product is a (better) soluble protein and can be easily purified.
  • suitable cloning for example using suitable PCR strategies
  • a wide repertoire of measurement options is available for measuring the activity of soluble proteins.
  • a particularly sensitive measurement can be carried out, for example, using a fluorescence-labeled ligand or substrate using fluorescence polarization.
  • 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 Substrate or
  • Ligand or a substrate or ligand mimetic An example of this are the compounds of formula (I) mentioned above.
  • the polypeptides of the invention themselves can be labeled, e.g. radioactive or colorimetric so that the number of polypeptides that are bound to a ligand or that have undergone a reaction can be determined exactly.
  • Potentially insecticidal compounds which are found in one of the methods according to the invention with the aid of the nucleic acids and / or polypeptides according to the invention can be supplied to the insects in various ways, e.g. orally (see also Example 4), topically or by injection.
  • Insecticides are often hydrophobic molecules and then usually have to be dissolved in organic solvents that can also evaporate (e.g. methanol or acetone) or that are added in low concentrations to facilitate absorption
  • the first step in experiments with insects is usually to determine the MLD (minimal lethal dose) after chronic insect exposure.
  • the compounds are usually diluted and added to the feed of embryos and larvae aged 0-48 hours.
  • the proportion of eggs from which larvae still hatch is determined, as is the behavior of the larvae (movement, feed intake), the number of larvae still pupating and the resulting adults.
  • the larvae can still be examined for morphological defects.
  • the acute and chronic dose can be determined.
  • the compounds are added to the food of embryos, larvae or adults and the insects are checked after 2 hours and after an overnight incubation. In the case of embryos, the number of embroyos with defects in development and the proportion which lasts until
  • Larvae are e.g. Behavioral disorders, movements during movement or molting checked. Defects in the amount or activity of enzymes as well as behavioral and / or fertility disorders are observed in adult animals.
  • the adults are e.g. placed in trays containing the compound in question for 48 hours, then transferred to a clean container and observed the fertility of the animals or the amount of activity of a particular enzyme or the death of the insects.
  • the following examples show that the ACCase is surprisingly an essential enzyme in insects, and also show that the enzyme is a suitable target protein for the identification of insecticides in methods of
  • Identification of insecticidally active compounds can be used and that the modulators of the ACCase identified in corresponding methods can be used as insecticides.
  • the extraction of this enzyme from Myzus persicae is described by way of example and finally the applicability of the present invention for the
  • Example 1 Preparation of an ACCase enzyme preparation from Myzus persicae
  • the following buffer is preferably used as the extraction buffer: 0.25 M sucrose,
  • the ACCase enzyme preparation was used to find modulators of the ACCase in a biochemical test as follows: First, an aliquot of the enzyme preparation from Example 1 was mixed with the reaction buffer and the radioactively labeled substrate and incubated. To demonstrate the incorporation of CO 2 , smoking HC1 was pipetted in, an aliquot of the reaction mixture on Watman
  • reaction buffer 50 mM Tris / HCl pH 7.4, 15 mM MgCl 2 , 2.5 mM ATP, 1 ⁇ g / ⁇ l bovine serum albumin, 10 mM potassium citrate, 84 mM sodium bicarbonate (all from Sigma, St. Louis) were used as reaction buffer.
  • a sub-step of the ACCase-catalyzed reaction is the fixation of the carbonate group to the co-factor biotin. This fixation takes place under ATP
  • Homogenization buffer 250 mM sucrose, 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 10 mM Na citrate, proteinase inhibitor mix (SIGMA P-8340) crushed (e.g. by mortar or using a homogenizing stick). The homogenized material is then centrifuged for clarification. The supernatant containing the ACCase is then removed using a 4 PD-10 column (Pharmacia Corporation, Peapack,
  • ACCase solution Fractions are tested for their ACCase activity as described under (b). The fractions with the highest specific ACCase activities are pooled and represent the starting material for the inhibitor measurements described under (c) (hereinafter referred to as ACCase solution).
  • reaction buffer 50 mM Tris-HCl pH 7.4, 20 mM MgCl 2 , 1 mg / ml bovine serum albumin, 20 mM Na citrate, 5 mM NaHCO 3 , 1 mM ATP, 200 ⁇ M Acetyl-CoA
  • reaction buffer 50 mM Tris-HCl pH 7.4, 20 mM MgCl 2 , 1 mg / ml bovine serum albumin, 20 mM Na citrate, 5 mM NaHCO 3 , 1 mM ATP, 200 ⁇ M Acetyl-CoA
  • 2 vol of the staining reagent BiomolGreen (Biomol Research Laboratories Inc., Plymouth Meeting, PA, USA), prepared as described by the manufacturer
  • reaction batches are also measured in the absence of the substrate acetyl-CoA and deducted accordingly when calculating the specific ACCase activity.
  • aliquots of the ACCase solution from (a) are mixed with the reaction buffer and the inhibitors to be tested and incubated at physiological temperatures (24 ° C to 37 ° C). After the desired reaction time, the detection reaction is carried out as described under (b). A control measurement without the addition of inhibitor is carried out in parallel. The inhibition of the ACCase is then calculated in comparison to this control.
  • Larvae or adults of the peach aphid Myzus persicae were fed by the sachet method (Nauen et al. 1996) with nutrient solutions with and without one of the identified inhibitors of ACCase for two days.
  • the animals were then collected and homogenized in organic solvent using a mortar.
  • the organic phase was cleaned of water-soluble constituents by repeated shaking with aqueous solution and then the solvent was evaporated.
  • the remaining pellet was taken up in a little solvent and separated by means of thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the separated lipids were stained with amido black.
  • 14 C acetate incorporated in lipids was detected by autoradiography after 2-3 days of exposure. To quantify the 14 C incorporation in lipids, the corresponding bands were scraped out after the staining, dissolved and the activity determined in the scintillation counter.
  • Figure la shows the lipid status after acetate feeding without (lanes 1-3) and with the active ingredient of the formula (IA) (lanes 4-6) using a separated lipid extract from the peach aphid Myzus persicae. There are no significant differences in lipid composition and lipid content.
  • Figure lb shows the autoradiography of the same DC plate. While in tracks 1-3 the blackening shows those lipids into which radioactively labeled acetate was incorporated in the control aphids during de novo synthesis, no labeled lipids are present in the active ingredient-treated peach aphids. Thus no de novo lipid synthesis from acetate has taken place.
  • Figure 1c shows again schematically how much acetate was present in the presence of an ACCase inhibitor (0.01 ppm to 100 ppm) in the de novo synthesis compared to the control without ACCase inhibitor. It can clearly be seen that de novo lipid biosynthesis comes to a standstill as the concentration of the inhibitor increases.
  • N-Hexane diethyl ether: glacial acetic acid (60: 45: 1) was used as the eluent for TLC chromatography.
  • Lanes 1-3 are recognizable from the blackening of those lipids into which radioactively labeled acetate was incorporated in the control aphids during the de novo synthesis, no labeled lipids are present in the active ingredient-treated peach aphids.

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Abstract

L'invention concerne des acides nucléiques qui codent des polypeptides d'insectes possédant l'activité biologique de carboxylases acétyl-CoA, des polypeptides ainsi codés et leur utilisation afin d'identifier de nouveaux composants présentant un effet insecticide. L'invention concerne également des procédés de découverte de modulateurs de ces polypeptides et l'utilisation de ces composants comme inhibiteurs de ACCase provenant d'insectes.
EP01985351A 2000-12-14 2001-12-03 Utilisation de carboxylase acetyl-coa permettant d'identifier des composants possedant un effet insecticide Withdrawn EP1346029A2 (fr)

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DE10062422 2000-12-14
DE10062422A DE10062422A1 (de) 2000-12-14 2000-12-14 Verwendung von Acetyl-CoA Carboxylase zum Identifizieren von insektizid wirksamen Verwendung
PCT/EP2001/014108 WO2002048321A2 (fr) 2000-12-14 2001-12-03 Utilisation de carboxylase acetyl-coa permettant d'identifier des composants possedant un effet insecticide

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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60326640D1 (de) 2002-08-05 2009-04-23 Cropsolution Inc Rekombinante biotincarboxylasedomänen zur identifizierung von acetyl-coa-carboxylase-inhibitoren
US20060195938A1 (en) * 2004-06-15 2006-08-31 Eva-Maria Franken Polynucleotides encoding insect acetyl coenzyme-A carboxylase and uses thereof
DE102005003076A1 (de) * 2005-01-22 2006-07-27 Bayer Cropscience Ag Verwendung von Tetramsäurederivaten zur Bekämpfung von Insekten aus der Gattung der Pflanzenläuse (Sternorrhyncha)
DE102006022821A1 (de) * 2006-05-12 2007-11-15 Bayer Cropscience Ag Verwendung von Tetramsäurederivaten zur Bekämpfung von Insekten aus der Ordnung der Käfer (Coleoptera), Thrips (Tysanoptera), Wanzen (Hemiptera), Fliegen (Diptera) und Zikaden (Auchenorrhynchae)
DE102011080406A1 (de) * 2011-08-04 2013-02-07 Bayer Pharma AG Substituierte 3-(Biphenyl-3-yl)-4-hydroxy-8-methoxy-1-azaspiro8[4.5]dec-3-en-2-one
CN110607378B (zh) * 2019-10-31 2022-11-29 贵州省烟草科学研究院 检测烟蚜对吡虫啉抗药性的引物对及检测方法

Family Cites Families (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5142065A (en) * 1988-08-20 1992-08-25 Bayer Aktiengesellschaft 3-aryl-pyrrolidine-2,4-diones
US4985063A (en) * 1988-08-20 1991-01-15 Bayer Aktiengesellschaft 3-aryl-pyrrolidine-2,4-diones
DE3913682A1 (de) * 1989-04-26 1990-10-31 Bayer Ag 3-aryl-pyrrolidin-2,4-dione
ES2063108T3 (es) * 1989-01-07 1995-01-01 Bayer Ag Derivados de 3-aril-pirrolidin-2,4-diona.
US5186737A (en) * 1989-01-07 1993-02-16 Bayer Aktiengesellschaft Pesticidal 3-aryl-pyrrolidine-2,4-diones
EP0412937A1 (fr) * 1989-08-10 1991-02-13 Ciba-Geigy Ag Acaricide pour les plantes
DE3929087A1 (de) * 1989-09-01 1991-03-07 Bayer Ag 3-aryl-pyrrolidin-2,4-dion-derivate
DE4032090A1 (de) * 1990-02-13 1991-08-14 Bayer Ag Polycyclische 3-aryl-pyrrolidin-2,4-dion-derivate
DE4102339A1 (de) * 1991-01-26 1992-07-30 Bayer Ag Substituierte 3-phenyl-4-hydroxy-(delta)(pfeil hoch)3(pfeil hoch)-pyrrolin-2-one
US5585384A (en) * 1991-01-31 1996-12-17 Bayer Aktiengesellschaft Substituted bicyclic 3-aryl-pyrrolidine-2,4-dione derivatives
DE4102778A1 (de) * 1991-01-31 1992-08-06 Bayer Ag Substituierte bicyclische 3-aryl-pyrrolidin-2,4-dion-derivate
DE4109208A1 (de) * 1991-03-21 1992-09-24 Bayer Ag 3-hydroxy-4-aryl-5-oxo-pyrazolin-derivate
US5358924A (en) * 1991-03-21 1994-10-25 Bayer Aktiengesellschaft 3-hydroxy-4-aryl-5-oxo-pyrozoline derivatives, compositions and use
DE4121365A1 (de) * 1991-06-28 1993-01-14 Bayer Ag Substituierte 1-h-3-aryl-pyrrolidin-2,4-dion-derivate
DE4216814A1 (de) * 1991-07-16 1993-01-21 Bayer Ag 3-aryl-4-hydroxy-(delta)(pfeil hoch)3(pfeil hoch)-dihydrofuranon- und 3-aryl-4-hydroxy-(delta)(pfeil hoch)3(pfeil hoch)-dihydrothiophenon-derivate
GB9125330D0 (en) * 1991-11-28 1992-01-29 Commw Scient Ind Res Org Novel dna clones and uses thereof
DE4308451A1 (de) * 1992-09-10 1994-04-14 Bayer Ag 3-Aryl-pyron-Derivate
US5539092A (en) * 1992-10-02 1996-07-23 Arch Development Corporation Cyanobacterial and plant acetyl-CoA carboxylase
AU666040B2 (en) * 1992-10-28 1996-01-25 Bayer Aktiengesellschaft Substituted 1-H-3-aryl-pyrrolidine-2,4-dione derivatives
DE4243818A1 (de) * 1992-12-23 1994-06-30 Bayer Ag 5-Aryl-1,3-thiazin-Derivate
DE4306259A1 (de) * 1993-03-01 1994-09-08 Bayer Ag Dialkyl-1-H-3-(2,4-dimethylphenyl)-pyrrolidin-2,4-dione, ihre Herstellung und ihre Verwendung
DE4306257A1 (de) * 1993-03-01 1994-09-08 Bayer Ag Substituierte 1-H-3-Phenyl-5-cycloalkylpyrrolidin-2,4-dione, ihre Herstellung und ihre Verwendung
EP0706527B1 (fr) * 1993-07-02 2001-11-14 Bayer Ag Derives substitues spiro-heterocycliques de la 1h-3-arylpyrrolidine-2,4-dione, leur procede de preparation et leur utilisation comme insecticides
EP0647637B1 (fr) * 1993-09-17 1999-01-27 Bayer Ag Dérivés de 3-aryle-4-hydroxy-3-dihydrofuranone
DE4425617A1 (de) * 1994-01-28 1995-08-03 Bayer Ag 1-H-3-Aryl-pyrrolidin-2,4-dion-Derivate
DE4431730A1 (de) * 1994-02-09 1995-08-10 Bayer Ag Substituierte 1H-3-Aryl-pyrrolidin-2,4-dion-Derivate
DE4410420A1 (de) * 1994-03-25 1995-09-28 Bayer Ag 3-Aryl-4-hydroxy- DELTA·3·-dihydrothiophenon-Derivate
AU2072695A (en) * 1994-04-05 1995-10-23 Bayer Aktiengesellschaft Alkoxy-alkyl-substituted 1-h-3-aryl-pyrrolidine-2,4-diones used as herbicides and pesticides
AU2925195A (en) * 1994-07-07 1996-02-09 Bayer Aktiengesellschaft 2-aryl cyclopentane-1,3-dione derivatives
BR9509793A (pt) * 1994-11-17 1997-09-30 Bayer Ag Derivados de tiofeneno
US6153374A (en) * 1994-12-13 2000-11-28 Novartis Finance Corporation Method for identifying inhibitors of soraphen A resistant acetyl-coenzyme a carboxylase
CN1079798C (zh) * 1994-12-23 2002-02-27 拜尔公司 3－芳基－季酮酸衍生物、其生产和作为杀虫剂的应用和中间体
AU4715896A (en) * 1995-02-13 1996-09-04 Bayer Aktiengesellschaft 2-phenyl-substituted heterocyclic 1,3-ketonols as herbicides and pesticides
AU5762696A (en) * 1995-05-09 1996-11-29 Bayer Aktiengesellschaft Alkyl dihalogenated phenyl-substituted ketoenols useful as p esticides and herbicides
JP4082724B2 (ja) * 1995-06-28 2008-04-30 バイエル・アクチエンゲゼルシヤフト 有害生物防除剤及び除草剤として使用される２，４，５−三置換されたフェニルケト−エノール
EP0835243B1 (fr) * 1995-06-30 2003-01-29 Bayer CropScience AG Cetoenols a substitution dialkyle-halogenure de phenyle s'utilisant comme herbicides et comme pesticides
DE19540080A1 (de) * 1995-10-27 1997-04-30 Bayer Ag 3-Aryl-5-halogen-pyron-Derivate
DE19544457A1 (de) * 1995-11-29 1997-06-05 Bayer Ag Oxymethoxy-3-aryl-pyron-Derivate
BR9708425B1 (pt) * 1996-04-02 2012-02-22 fenilcetoenóis substituìdos, processo para preparação e uso dos mesmos, bem como pesticidas ou herbicidas e método para controle de pragas e ervas daninhas.
JP4306799B2 (ja) * 1996-05-10 2009-08-05 バイエル アクチェンゲゼルシャフト 新規な置換ピリジルケトエノール
JP4202423B2 (ja) * 1996-08-05 2008-12-24 バイエル・アクチエンゲゼルシヤフト ２―および２，５―置換フェニルケトエノール
DE19632126A1 (de) * 1996-08-09 1998-02-12 Bayer Ag Phenylsubstituierte cyclische Ketoenole
US6391912B1 (en) * 1996-12-12 2002-05-21 Bayer Aktiengesellschaft Substituted phenylketoenols
DE19651686A1 (de) * 1996-12-12 1998-06-18 Bayer Ag Neue substituierte Phenylketoenole
AU9307098A (en) * 1997-09-08 1999-03-29 Arqule, Inc. Spiro{pyrrolidine-2,3'-oxindole} compounds and methods of use
DE19742492A1 (de) * 1997-09-26 1999-04-01 Bayer Ag Spirocyclische Phenylketoenole
DE19749720A1 (de) * 1997-11-11 1999-05-12 Bayer Ag Neue substituierte Phenylketoenole
DE19808261A1 (de) * 1998-02-27 1999-10-28 Bayer Ag Arylphenylsubstituierte cyclische Ketoenole
DE19813354A1 (de) * 1998-03-26 1999-09-30 Bayer Ag Arylphenylsubstituierte cyclische Ketoenole
DE19818732A1 (de) * 1998-04-27 1999-10-28 Bayer Ag Arylphenylsubstituierte cyclische Ketoenole
DE19846517A1 (de) * 1998-10-09 2000-04-20 Bayer Ag 3-Phenyl-pyrone
DE19939395A1 (de) * 1998-10-23 2000-04-27 Bayer Ag Wirkstoffkombinationen mit insektiziden und akariziden Eigenschaften
US20030100572A1 (en) * 2001-06-21 2003-05-29 Ariad Pharmaceuticals,Inc. Novel pyridopyrimidones and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0248321A3 *

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JP2005500810A (ja) 2005-01-13
WO2002048321A2 (fr) 2002-06-20

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