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WO1995001991A1 - Allatostatines et leur utilisation - Google Patents

Allatostatines et leur utilisation Download PDF

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Publication number
WO1995001991A1
WO1995001991A1 PCT/GB1994/001440 GB9401440W WO9501991A1 WO 1995001991 A1 WO1995001991 A1 WO 1995001991A1 GB 9401440 W GB9401440 W GB 9401440W WO 9501991 A1 WO9501991 A1 WO 9501991A1
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WO
WIPO (PCT)
Prior art keywords
peptide
conjugate
gly
peptides
conjugates
Prior art date
Application number
PCT/GB1994/001440
Other languages
English (en)
Inventor
John Patrick Edwards
Robert James Weaver
Original Assignee
The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland filed Critical The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority to AU70780/94A priority Critical patent/AU7078094A/en
Priority to GB9501907A priority patent/GB2283974A/en
Publication of WO1995001991A1 publication Critical patent/WO1995001991A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • the present invention relates to novel peptides, compositions comprising them and their use as pesticidal agents. Further provided are methods for isolating and synthesising the peptides.
  • Juvenile hormones produced by the corpora allata (CA) , play a vital role in the regulation of insect development primarily in the control of adult sexual maturation, metamorphosis and reproduction (Engelmann, F, The Physiology of Insect Reproduction, Perga-mon, Oxford, 1970 ).
  • the hormonal secretory activity of these endocrine glands is regulated by a variety of humoral and nervous factors originating, at least in part, in the insect brain (Scharrer, B, 1987, Annu Rev Entomol 32, 1-16) .
  • Peptidergic neurosecretory material from cells of the brain that project down to the CA have long been postulated to regulate the activity of these endocrine glands. These factors of cerebral origin may act either as stimulators or as inhibitors (allatotropins or allatostatins) of juvenile hormone biosynthesis and experimental evidence suggests that one of these may predominate, depending upon species at different times during development (Tobe, S.S. & Stay, B. 1 85 Adv Insect Physiol 18, 305-432).
  • All of the cockroach allatostatins are amidated neuropeptides varying from 8 to 18 amino acids in length (Woodhead A P et al,1989 Proc Natl Acad Sci USA, 86, 5997-6001; Stay, B et al, 1991. Insect Neuropeptides: Chemistry, Biology and Action, pp 164-176, Eds Menn, J J, Symp Series 453; Pratt, G E et al 1991, Proc Natl Acad Sci USA 8824l2-24l6 and Pratt, G E et al, 1989, Biochem Biophys Res Commun 163 1243-1247). They share carboxy-terminal amino acid sequence likeness, which shows some similarity to the enkephalin-related peptide [Met-enkephalin]- -Arg6-Gly7-Leu8 (met-8) , but are otherwise unique.
  • CA-active neuropeptide to be isolated and sequenced is a structurally unrelated allatostatin from heads of pharate adult moth, Manduca sexta (Kramer, S J et al, 1991. Proc Natl Acad Sci USA 889458-9462) .
  • This compound is a cysteine-containing, amino terminal blocked (pGlu) 15-amino acid peptide which bears no resemblance either to the allatotropin of this same species or to any one of the Diploptera allatostatins.
  • This compound causes rapid and potent inhibition of JH synthesis in CA from larvae and adult M.sexta but has no effect against CA from species of two other insect orders.
  • cockroach allatostatins have a weak capacity to inhibit JH synthesis by CA from adults of the more important pest species Periplaneta americana (Woodhead A P et al, 1989. Proc Natl Acad Sci USA, 86, 5997-6001). This species is related in terms of cockroach phylogeny but has a different emphasis to its mode of reproduction (Weaver R J et al, 1993. Insect Juvenile Hormone Research, eds Mauchamp B et al, I N R A Paris). However Diploptera allatostatins show at best rather weak activity spectra against the CA from different stages of developing P.americana.
  • the present invention provides novel peptides which show potent in vitro inhibition of JH synthesis in cockroach and provides methods for extraction and isolation of examples of these from the brains of adult females of P.americana.
  • Specific peptides designated Pea-AST-1 and Pea-AST-2, are amidated neuropeptides of 13 and 10 amino acids, respectively and each bear some structural similarity to members of a family of putative allatostatins identified from the cockroach D.punctata (Woodhead A P et al, 1989. Proc Natl Acad Sci USA, 86, 5997-6001). They share no sequence homology with either of the two known Lepidopteran allatoactive neuropeptides, Mas-AT and Mas-AS, isolated from pharate adults of the moth M.sexta.
  • Analogues of JH are currently being used for the control of a number of insect species in which the adult is the primary pest (Staal, G B et al, 1975, Annu Rev Entomol, 20, 417-460) but have not proved particularly useful for controlling the majority of crop-destroying insects. Those that have proved useful have been restricted in efficacy to a few species of Lepidoptera and have lacked sufficient potency for commercialisation.
  • the peptides provided by the present invention are suitable for control of insect development and thereby control of insect populations due to their ability to inhibit juvenile hormone production by CA from selected stages. Furthermore their activity, demonstrated herein on both P.americana and D.punctata provides a potential use in the control of insect populations of a range of orders of insect.
  • a first aspect of the present invention provides an isolated, enriched or purified peptide of Formula (I), equivalent to sequence ID No. 1 and 2 in the attached sequence listing
  • X is a sequence selected from S-Asp-Gly- and Q-Pro-Ser-Gly-R-Gln-; Y.Q.S and R are selected from single amino acid residues, and Z is an amidated leucine moiety; wherein Q and R are not both alanine when Y is glycine, and S is not glycine when Y is alanine.
  • Preferred peptides provided by the present invention are those of Formula I in which S and/or Y are selected from amino acids with hydrocarbon side chains and/or those in which R and Q are selected from sulphur containing amino acids.
  • Particular peptides of the present invention are those in which Y is selected from glycine and alanine and/or R is an amino acid with a sidechain comprising a thioether eg. methionine and/or Q is an amino acid with a sidechain comprising a mercapto group eg. serine.
  • Further particular peptides of the present invention are those in which Y and S are both alanine residues.
  • the present invention particularly provides an isolated, enriched or purified peptide of Formula (II), equivalent to sequence ID No. 3 and 4 in the attached sequence listings
  • X is a sequence selected from: Ala-Asp-Gly-Arg-Leu-Tyr-Ala- and Ser-Pro-Ser-Gly-Met-Gln-Arg- -Leu-Tyr-Gly- ; and Z is an amidated leucine moiety.
  • amidated leucine moiety will be understood to include sustituted leucine amide eg. N-methyl-leucine amide, substituted on the amide nitrogen, but preferred peptides of Formula (I) and Formula (II) are those in which the amidated leucine moiety is unsubstituted leucine amide.
  • conjugates of the peptides of the present invention having equivalent activity to the peptides, will also be useful as pesticides or as active agents in pesticidal compositions.
  • the peptides may be conjugated to other peptides or to proteins.
  • Particular conjugates that will have equivalent activity to the peptides of the present invention are their metabolic precursors.
  • a second aspect of the present invention provides the use of peptides for the control of insect development, particularly insects of the orders Periplaneta and/or Diploptera, more particularly insects of the species Periplaneta americana and/or Diploptera punctata.
  • compositions comprising:
  • compositions comprising:
  • compositions comprise peptides of Formula I or Formula II described as preferred above.
  • compositions contain carriers capable of holding peptides in homogenous form eg. in solution or suspension.
  • carriers will be apparent to those skilled in the art and include compounds such as eg. dimethylsulphoxide (DMSO) which are capable of solubilising peptides.
  • DMSO dimethylsulphoxide
  • vectors genetically manipulated to express the peptides or conjugates of the invention may be used to enable plants to actively synthesise them for transfer to insect populations. Suitable vectors include viruses and plasmids but other examples will occur to those skilled in the art eg. bacteria or nematodes containing plasmids or viruses.
  • the peptides of the present invention may also be formulated in an admixture with other pesticides, particularly insecticides, acaricides and/or nematocides.
  • a fourth aspect of the present invention is the preparation of peptides and conjugates described above. These may be prepared by extraction and purification from the brains of adult female pharate Periplaneta americana. by solid phase peptide synthesis or other known chemical techniques or alternatively by well known recombinant DNA techniques.
  • One suitable method for the preparation of the peptides comprises extraction from dissected, homogenised brains of adult female Periplaneta americana. using strongly acidic solvent such as 7 # ethanol in 0.2M HCl aqueous solution.
  • the resulting crude extract may subsequently be separated into its components by fast liquid chromatography, to provide fractions with complex absorption spectra at 2l4nm.
  • Providing a solvent concentration gradient during chromatography results in the peptides of the invention consistently eluting at particular solvent concentrations.
  • Fractions containing the peptides of the invention may be determined by various methods, including the measurement of any Corpus allatum inhibitory activity and/or any allatostatin immunoreactivity. Once identified the fractions containing the peptides of the invention may be subjected to further chromatography in order to purify the peptides. Further details of suitable solvents and the fractions in which the peptides elute are provided in the examples.
  • Peptides and peptide conjugates of the present invention may also be produced using recombinant DNA technology, wherein naturally occurring, eg. P.americana. or synthetic DNA encoding for the active peptide or conjugate are identified, inserted into a suitable regulatory construct, eg. in a vector, and the construct is subsequently inserted into a host organism such as to enable that organism to express the peptide or conjugate.
  • a suitable regulatory construct eg. in a vector
  • Naturally occurring gene sequences may be identified by a number of techniques known to those skilled in the art, eg. Northern, Southern or Western Blotting using antibodies or probes targeted at the peptide or theoretically encoding sequence.
  • RACE PCR may be used to rapidly identify such genes ( Frohman et al (1988) Proc Nat Acad Sci USA Vol 85, 8998-9002) using degenerate primers.
  • Synthetic or natural sequences may be identified as active peptide expressing by screening their host organisms, eg. E.coli transformed with them for production. Purified putative encoding DNA is ligated into a suitable vector,eg. pBluescript SK (Stratagene) and transformed into a host cell, eg.
  • E.coli JM109 cells (Hanahan (1985) DNA cloning, A practical approach Vol 1 (Glover Ed) pp 109-135 IRL Press Oxford) .
  • Expression may be identified by Northern blotting for RNA or by use of antibodies in Western blotting. Purification of the expressed protein or peptide may be carried out as is known in the art, eg. by affinity chromatography.
  • Suitable hosts for expression of the peptides will be apparent to those skilled in the art, eg. yeast, bacteria, mammalian and fungal cells.
  • DNA may be introduced into hosts by any suitable means eg. in the form of a plasmid or contained within a virus eg. a baculovirus.
  • Transgenic plants comprising DNA encoding the peptides or conjugates of the invention may also be produced by inserting that DNA into a suitable . construct, and inserting that into an organism vector eg. Agrobacterium, (eg.
  • a further aspect of the present invention provides recombinant DNA encoding a peptide or peptide conjugate as described above and transgenic cells comprising such DNA.
  • Transgenic cells may suitably be microbial cells, mammalian cells or plant cells. This aspect also provides transgenic plants comprising DNA encoding for a peptide or conjugate as discussed above, per se.
  • EXAMPLE 1 EXTRACTION AND PURIFICATION OF ALLATOSTATINS FROM ADULT FEMALE P.AMERICANA
  • the brains of adult female Periplaneta americana were dissected, homogenised and extracted with a strongly acidic solvent (75# ethanol in 0.2M HCl aqueous solution).
  • the homogenates were centrifuged (4,000 x g, 20 min, 4°C) , the supernatants separated and the pellets re-extracted. After centrifugation the resultant acidic solution was diluted 6-fold with 0.1 TFA.
  • An array of disposable reversed-phase extraction cartridges (Sep-Pak C l8 ) was then used to remove fat from the crude extracts. Pilot studies revealed that up to 100 brain equivalents at a time could be applied to a single cartridge without significant washout or lack of retention of active materials.
  • the eluted materials were detected by their absorbance at 214 nm.
  • 2-(methylthio)ethanol 0.1%) was added to the fractions at each purification step and the collection vessels were pre-loaded with 1 ⁇ g of HPLC purified oxidised insulin ⁇ -chain (Sigma) .
  • the first FPLC separation was performed under the following operating conditions: 100 solvent A for 5 min, a linear gradient to 10% B for 5 min, followed over the next 35 mins by a linear gradient (1%/min) from 10% B to 45% B; Solvent A, 0.1% trifluoroacetic acid (TFA) in water; Solvent B, 0.1% TFA in acetonitrile. Fractions were collected at one minute intervals and twenty six of these separations were required to process 4,400 brain equivalents of material. Allatostatin bioactivity had previously been shown to elute from such columns over a fairly broad range of increasing organic solvent concentrations (Weaver, R J et al, 1993. Insect Juvenile Hormone Research).
  • the second FPLC separation was performed using a gradient of 1-propanol with 0.01% HCl.
  • the pooled biologically active fractions from the first step were diluted 3 _ fold with new solvent A, 0.01% HCl.
  • Approximately 1000 brain equivalents at a time were pumped directly into the column, re-equilibrated with new solvent A.
  • the carrier peptide B-insulin was the only major contaminant at this stage, at times almost co-running with Pea-AST-1. The presence of this "contaminant” was utilised in order to monitor the repetitive recovery by virtue of the fact the amount of added insulin was accurately known for each sample. It is to be assumed that the added insulin afforded some degree of loss prevention through otherwise unregulated and largely irreversible adsorptive processes.
  • the final FPLC separation step was a repeat of the first stage purification. Pooled active fractions from each the previous steps (ca.l ml) were diluted 1:10 with 0.1% aqueous TFA and pumped 2 mis at a time into the same FPLC column, re-equilibrated with 0.1% TFA. The column was eluted with the same solvent B (acetonitrile/0.1% TFA) and gradient conditions (10-45% B over 35 mi , 1 ml/min) as specified in the first step. In the first such run pure Pea-AST-1 was recovered in a single peak at 29 min. Two identical runs were required to purify Pea-AST-2, which finally eluted as a single peak at 28.5 min.
  • solvent B acetonitrile/0.1% TFA
  • the peptides were sequenced with an Applied Biosystems model 477A pulsed liquid-phase protein sequencer coupled to an online phenylthiohydantoin analyser (Applied Biosystems model 120A) . Full sequence determination required 200-500 pmol of each peptide. Sequence analysis was undertaken at the Protein Sequencing Unit of the Department of Biochemistry at Royal Holloway and Bedford New College, Egham, Surrey. The molecular masses of the peptides were determined by M-Scan Ltd, Sunningdale, Ascot, Berkshire using positive ion fast atom bombardment mass spectrometry (FAB-MS) on a VG AutospecE double focusing mass spectrometer (VG Analytical, Manchester, UK). Amino acid analyses were conducted by M-Scan S.A., Geneva, Switzerland.
  • FAB-MS positive ion fast atom bombardment mass spectrometry
  • a peptide having the sequence Ser-Pro-Ser-Gly-Met-Gln-Arg-Leu- -Tyr-Gly-Phe-Gly-Leu was synthesised with the carboxyl terminus in both amidated and free acid forms using standard automated solid-phase techniques (see eg. Barany, G et al, 1979. The peptides, eds Gross et al, Academic press, New York, Vol 2, pp 1-284). An Applied Biosystems model 430A automatic solid-phase peptide synthesiser (Charing Cross and Riverside Hospital Medical School Protein Synthesis Core Facility) was used and the synthetic peptides were purified by HPLC on preparative C l8 reverse-phase columns.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Pest Control & Pesticides (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouvelles allatostatines, leur utilisation en tant qu'agents pesticides, des compositions les comprenant, ainsi que des procédés servant à leur préparation. Les peptides peuvent se préparer de façon appropriée par extraction à partir d'insectes, par synthèse chimique ou au moyen de techniques d'ADN recombinant.
PCT/GB1994/001440 1993-07-05 1994-07-04 Allatostatines et leur utilisation WO1995001991A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU70780/94A AU7078094A (en) 1993-07-05 1994-07-04 Allatostatins and their use
GB9501907A GB2283974A (en) 1993-07-05 1994-07-04 Allatostatins and their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB939313891A GB9313891D0 (en) 1993-07-05 1993-07-05 Pesticides
GB9313891.5 1993-07-05

Publications (1)

Publication Number Publication Date
WO1995001991A1 true WO1995001991A1 (fr) 1995-01-19

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AU (1) AU7078094A (fr)
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WO (1) WO1995001991A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997035981A1 (fr) * 1996-03-26 1997-10-02 Btg International Limited Genes de neuropeptides d'insectes et peptides
CN116250548A (zh) * 2022-08-30 2023-06-13 华南师范大学 抑咽侧体神经肽在防治美洲大蠊中的应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648486A (en) * 1992-07-13 1997-07-15 Cytomed, Inc. Compounds and methods for the treatment of inflammatory and immune disorders

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A.P.WOODHEAD ET AL: "Primary structure of four allatostatins:Neuropeptide inhibitors of juvenile hormone synthesis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA., vol. 86, August 1989 (1989-08-01), WASHINGTON US, pages 5997 - 6001 *
R.J.WEAVER ET AL: "INSECT JUVENILE HORMONE RESEARCH", 1992, MAUCHAMPS, PARIS *
R.J.WEAVER: "Identification of two allatostatins from the CNS of the cockroach Periplaneta americana", COMP.BIOCHEM.PHYSIOL., vol. 107C, no. 1, 1994, pages 119-127 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997035981A1 (fr) * 1996-03-26 1997-10-02 Btg International Limited Genes de neuropeptides d'insectes et peptides
CN116250548A (zh) * 2022-08-30 2023-06-13 华南师范大学 抑咽侧体神经肽在防治美洲大蠊中的应用

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Publication number Publication date
GB9313891D0 (en) 1993-08-18
AU7078094A (en) 1995-02-06
GB2283974A (en) 1995-05-24
GB9501907D0 (en) 1995-03-22

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WO1995001991A1 (fr) Allatostatines et leur utilisation

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