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WO1993000907A1 - Utilisation d'un antibiotique du type 2-desoxystreptamine - Google Patents

Utilisation d'un antibiotique du type 2-desoxystreptamine Download PDF

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Publication number
WO1993000907A1
WO1993000907A1 PCT/AT1992/000087 AT9200087W WO9300907A1 WO 1993000907 A1 WO1993000907 A1 WO 1993000907A1 AT 9200087 W AT9200087 W AT 9200087W WO 9300907 A1 WO9300907 A1 WO 9300907A1
Authority
WO
WIPO (PCT)
Prior art keywords
intron
deoxystreptamine
introns
antibiotic
group
Prior art date
Application number
PCT/AT1992/000087
Other languages
German (de)
English (en)
Inventor
Uwe Von Ahsen
Julian Davies
Renée Schroeder
Original Assignee
Ahsen Uwe
Julian Davies
Schroeder Renee
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 Ahsen Uwe, Julian Davies, Schroeder Renee filed Critical Ahsen Uwe
Priority to JP5501822A priority Critical patent/JPH06503098A/ja
Priority to AU22721/92A priority patent/AU664191B2/en
Publication of WO1993000907A1 publication Critical patent/WO1993000907A1/fr
Priority to FI931018A priority patent/FI931018L/fi
Priority to NO93930834A priority patent/NO930834L/no

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention relates to the use of an antibiotic of the type 2-deoxystreptamine. So far, pathogens or other microorganisms which have a damaging effect on the human or animal organism have been treated by antibiotics, depending on the type of antibiotics either a larger spectrum being covered, that is to say so-called broad-spectrum antibiotics, or with a precise knowledge of the patient -unit antibiotics are used, which are specifically based on bacteria and the like. are directed. All these antibiotics have the disadvantage that they are very widely spread, i.e. act unspecifically, so that those organisms are damaged which the human or animal body needs to live, i.e. which live in symbiosis with the human organism. A particularly good example of this are the intestinal bacteria that humans need to digest food. These intestinal bacteria are also killed or at best only damaged by the antibiotics used up to now.
  • Group 1 introns are mainly found in prokaryotes and individual eukaryotes. That that is to say that the organisms which usually appear as pathogens or other pests are essentially those which contain group 1 introns. Thus, if those organisms that contain group 1 introns can be prevented from growing in a targeted manner, harmful organisms can be targeted in a targeted manner without damaging the organisms necessary for human life.
  • the invention therefore lies in the use of an antibiotic of the type 2-deoxystreptamine, in particular a 4,6 or 4,5, 2-deoxystreptamine substituted with amino sugar to inhibit the growth of group 1 introns. tendency organisms, in particular in a method for producing a medicament.
  • an antibiotic of the 4,6-substituted 2-deoxy streptamine type can be gentamicin (B, Cl, Cla, C2), kanamycin (A, B, C), Tobra-5 mycin or amikamycin.
  • Neomycin B, paromomycin, ribostamycin, lividomycin or butirosin can be used as the antibiotic of the 4,5-substituted 2-deoxystreptamine type. Because of their bactericidal activity, the antibiotics mentioned can be used against gram-negative and against gram-positive bacteria in the clinical and generally antimicrobial field (see publication by Davis & Yagisawa, 1983; Cundcliffe, 1990).
  • 2-deoxystreptamine inhibits the self-excision of Group 1 intron RNA from their exons.
  • the coding region of a gene is interrupted by non-coding regions, the introns. These introns and the coding areas, the exons, are overwritten during transcription from the DNA into the RNA and the introns are now spliced out at the RNA level.
  • the coding regions are ligated together again so that a functional gene product, the protein, is formed.
  • the group 1 introns can be described as follows:
  • RNA can have a characteristic secondary structure, these pairings being denoted from P1 to P10, depending on the affiliation to a specific subgroup within group 1 introns. Sequence preservation is less important than structural preservation (see Burke et al., 1987).
  • the splicing mechanism is initiated by an external cofactor, guanosine. It is generally believed that the hydroxyl group of the ribose of guanosine is attached to the phosphorus ester bond of the last NuMeotide of the previous one Exons and the first nucleotide of the intron makes a nucleophilic attack and is covalently bound to the 5 'end of the intron RNA. The released hydroxyl at the 3 'end of the previous exon in turn attacks the phosphorus ester bond between the last nucleotide of the intron and the first nucleotide of the subsequent exon, breaks it open and ligates to the exon, the intron is released. There are two reesterifications.
  • Group 1 introns are able to carry out this process autocatalytically, ie without the addition of proteins, in vitro.
  • a number of Group I introns have been shown to require proteins in vivo for the splicing process (see Cech, 1990).
  • the occurrence of group 1 introns extends from the nuclear genome of never eukaryotes, mitochondria from lower fungi, chloroplasts to eubacteria, bacteriophages and archaebacteria.
  • Group 1 introns have not been found in higher eukaryotes. Their occurrence is not limited to genes coding for proteins, they also occur in genes for tRNA and rRNA. A recent and comprehensive compilation of the occurrence and the splicing mechanism can be found in the publication by Michel & Westhoff, 1991.
  • the medicaments produced according to the invention can thus generally be used to inhibit the growth of organisms which contain a group 1 intron and are normally not sensitive to the antibiotics mentioned.
  • the use of these drugs is said to extend particularly to the therapeutic field, the site of action of the antibiotics mentioned being of interest to the intron RNA and not to the ribosomal RNA.
  • FIG. 1 shows various radiographs of introns which have been cleaved by means of guanosine triphosphate (hereinafter referred to as GTP), the effect of the antibiotic being shown.
  • GTP guanosine triphosphate
  • 2 illustrates the cleavage process of the preRNA and the ligation of the two exons with cleavage of the linear intron.
  • 3 shows the basic structural formulas of the most important antibiotics used, the effectiveness limit being given below the individual groups with regard to the antibiotics tested.
  • FIG. 4a shows the secondary structure of the core region, which shows the 3 'interface of the td intron (FIG. 4a) and the sunY intron (FIG. 4b).
  • the procedure for measuring the effect of antibiotics on group 1 introns was carried out as follows:
  • the gene used for this procedure is the thymidylate synthase from the
  • Coliphagen T4 The gene was cloned into the vector pTZ18U (company USB) with a truncated intron delta P6 (Schroeder et al. 1991). To produce the non-spliced precursor, the plasmid is linearized with EcoRI and transcribed in vitro. The transcription conditions are as follows: 1 ⁇ g DNA in a volume of 20 ⁇ l at 30 ° C.
  • RNA precursor is then purified by gel electrophoresis (5% acrylamide / 7M urea in Tris-Borate-EDTA).
  • RNA precursor For the antibiotic inhibition test, 20,000 cpm of RNA precursor are placed in 5 ⁇ l of splicing buffer (2.5 ⁇ M GTP, 40 / M Tris-HCl, 8mM MgCl 2 , 0.4 mM spermidine), with increasing amounts of antibiotic (usually 0.1 ⁇ M to 2 mM) incubated at 37 ° C for 10 minutes.
  • the reaction mixture is then precipitated by adding 45 ⁇ l stop solution (2.5 mM EDTA, 0.1 mg / ml yeast tRNA) and 150 ⁇ l 0.3 M NaOAc / ethanol. After resuspension in 5 ⁇ l of water and heating to 65 ° C.
  • FIG. 1A shows the analogous relationships with respect to sunY intron.
  • FIG. IC shows a parallel experiment with a group 2 intron, and it can clearly be seen that the preRNA is split here into the linear intron (L intron) and into the ligated E1, E2 exons, regardless of the Antibiotic concentration.
  • gentamicin was used, which, as can be seen from FIGS. 1A and 1B, specifically inhibits group 1 introns.
  • FIGS. ID and 1E also show, a tobramycin being used instead of the gentamicin in FIG. 1 and paromomycin in the embodiment according to FIG. 1E.
  • preRNA means the RNA tested, In-E2 the connection of intron with exon 2, L-In the linear intron, E1-E2 the ligated exons El and E2 and El alone exon 1.
  • the two exons E1 and E2 are indicated by the strong bars, the splitting mechanism of the preRNA being represented by the treatment with GTP. It can be seen that in the first step exon 1 is split off from the intron exon 2 fragment.
  • the cleavage is initiated by the exogenous guanosine (G-OH), the guanosine attacking the G bond side, cutting open on the 5 'cleavage side by nucleophilic attack and covalently connecting to the first nucleotide of the intron .
  • G-OH exogenous guanosine
  • the OH group attaches itself to the exon El on the 3 'side.
  • the guanosine attacks the 5 'side of exon 2, the linear guanosine being cut out.
  • the two exons E1 and E2 are then ligated, and the intron, which contains the guanosine at its ends, is separated.
  • 3A to 3C show a series of 2-deoxystreptamine compounds with which the tests shown in FIG. 1 were carried out. Both Concentrations were given the best concentration of activity for the individual substances.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne l'utilisation d'un antibiotique du type 2-désoxystreptamine, notamment d'une 2-désoxystreptamine 4,6 ou 4,5 substituée par des amino-sucres, pour l'inhibition du développement d'organismes renfermant des introns du groupe I, en particulier dans un procédé de fabrication d'un médicament.
PCT/AT1992/000087 1991-07-09 1992-07-08 Utilisation d'un antibiotique du type 2-desoxystreptamine WO1993000907A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP5501822A JPH06503098A (ja) 1991-07-09 1992-07-08 ２−デオキシストレプトアミンのタイプの抗生物質の用途
AU22721/92A AU664191B2 (en) 1991-07-09 1992-07-08 Use of a 2-deoxystreptamine-type antibiotic
FI931018A FI931018L (fi) 1991-07-09 1993-03-08 Anvaendning av ett 2-deoxistreptamin-typ antibioticum
NO93930834A NO930834L (no) 1991-07-09 1993-03-08 Anvendelse av et 2-deoksystreptamin-type antibiotikum

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT0137591A AT397102B (de) 1991-07-09 1991-07-09 Verwendung eines antibiotikums des typs 2-desoxystreptamin
ATA1375/91 1991-07-09

Publications (1)

Publication Number Publication Date
WO1993000907A1 true WO1993000907A1 (fr) 1993-01-21

Family

ID=3512526

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AT1992/000087 WO1993000907A1 (fr) 1991-07-09 1992-07-08 Utilisation d'un antibiotique du type 2-desoxystreptamine

Country Status (7)

Country Link
EP (1) EP0548314A1 (fr)
JP (1) JPH06503098A (fr)
AT (1) AT397102B (fr)
AU (1) AU664191B2 (fr)
CA (1) CA2091264A1 (fr)
FI (1) FI931018L (fr)
WO (1) WO1993000907A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009792A1 (fr) * 1992-10-23 1994-05-11 University Of Massachusetts Medical Center Inhibition de la fixation arn/ligand au moyen de petites molecules
US5433291A (en) * 1994-12-07 1995-07-18 Shoestock, Sr.; Richard F. Combination tree stand and wheeled game carrier
US6150134A (en) * 1994-07-29 2000-11-21 Innogenetics, N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
EP1302543A1 (fr) * 2001-10-15 2003-04-16 Bayer CropScience AG Procédé d'épissage comme cible pour l'identification de préparations pharmaceutiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BURGER A., Medicinal Chemistry Part 1, (WILEY-INTERSCIENCE), 1970, "Antibiotics", pages 333-340. *
NATURE, Vol. 327, June 1987, DANESH MOAZED et al., "Interaction of Antibiotics With Functional Sites in 16S Ribosomal RNA", pages 389-394. *
NATURE, Vol. 353, September 1991, UWE VON AHSEN et al., "Antibiotic Inhibition of Group I Ribozyme Function", pages 368-370. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009792A1 (fr) * 1992-10-23 1994-05-11 University Of Massachusetts Medical Center Inhibition de la fixation arn/ligand au moyen de petites molecules
US6150134A (en) * 1994-07-29 2000-11-21 Innogenetics, N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US5433291A (en) * 1994-12-07 1995-07-18 Shoestock, Sr.; Richard F. Combination tree stand and wheeled game carrier
EP1302543A1 (fr) * 2001-10-15 2003-04-16 Bayer CropScience AG Procédé d'épissage comme cible pour l'identification de préparations pharmaceutiques
WO2003033711A1 (fr) * 2001-10-15 2003-04-24 Bayer Cropscience Ag Epissage en tant que cible pour l'identification de nouveaux principes actifs

Also Published As

Publication number Publication date
FI931018L (fi) 1993-04-06
FI931018A0 (fi) 1993-03-08
AU664191B2 (en) 1995-11-09
AT397102B (de) 1994-02-25
CA2091264A1 (fr) 1993-01-10
ATA137591A (de) 1993-06-15
EP0548314A1 (fr) 1993-06-30
AU2272192A (en) 1993-02-11
JPH06503098A (ja) 1994-04-07

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