JPH06503098A - Uses of antibiotics of the type 2-deoxystreptamine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Uses of antibiotics of the type 2-deoxystreptamine The present invention relates to the use of 2-deoxystreptamine Concerning the uses of antibiotics of the type of treptamine.

Traditionally, pathogens or other microorganisms with damaging effects on human or animal organs have been used. Organisms are treated with antibiotics, and depending on the type of antibiotic, relatively large In short, so-called broad-spectrum antibiotics are used to treat the exact pathogen. Antibiotics that specifically target bacteria are used when knowledge is available. these All antibiotics have a very broad spectrum, i.e. they act non-specifically. 1 As a result, the human body or the animal body needs to survive. The drawback is that the organisms that live in symbiosis with these organs are damaged.

A particularly advantageous example for this is the intestinal bacteria needed by humans to digest nutrients. . These enteric bacteria can also be eradicated by conventionally used antibiotics, or best damage even if the

Antibiotics that can be intentionally used against specific microorganisms have therefore been around for a long time. There is a scientific tendency to develop quality.

Various reactions between antibiotics and organisms may result in the formation of group I proteins in liposome-RNA. It is known that the occurrence depends on whether or not trontron is included. glue Pl introns are found in prokaryotes and uni eukaryotes, among others. That is, , a substantial group of organisms that commonly occur as pathogens or other pests It is an organism containing one intron. In short, it contains group I introns. If the growth of such organisms can be intentionally prevented, the Harmful organisms can be intentionally eradicated without damaging property.

Therefore, the present invention particularly provides methods for producing pharmaceuticals containing group I introns. 2-deoxystreptamine, especially amino Types of 2-deoxystreptamine substituted with 4,6 or 4,5 sugars The aim is to use antibiotics.

4. Antibiotics of the 6-substituted 2-deoxystreptamine type: Gentamicin (B, C1, C1a, C2), Kanamycin (A, B, C), Tobramycin or Amikam ycin) can be used advantageously. 4.54-substituted 2-deoxystreptamide Types of antibiotics include neomycin B1, paromomycin, and lipostamycin. Syn, lividomycin or butyrosin can be used. Antibiotics mentioned above The quality is clinically recognized for its bactericidal action against Gram-negative as well as Durham-positive bacteria. and in the general antimicrobial field (see Davis & Y. agisawa publications, 1983; Cundcliffe, +990)2 - The action of deoxystreptamine is to inhibit group 1 intronic RNAs from The purpose is to prevent self-excision from exons. In other words, the coding domain of the genetic element The regions are often interrupted by non-coding regions - introns. these Introns and coding regions - exons - are co-located during transcription from DNA to RNA. The intron is then split on one plane of the RNA. of subsequent translations For this purpose, the coding regions are linked together again, thus producing a functional genetic product - protein. The essence is that Due to the splitting mechanism, the reception is accepted RN A-Group 1 regarding secondary- or tertiary structure and presence in organisms The intron of Nigerloop I can be explained as follows. The region has a set of conserved sequences designated by P, Q, and RSS, and these regions The intervals between can be of different lengths. RNA has a characteristic secondary structure due to its pairability. structure, in which case these pairs are designated PI-Plo and group I depends on belonging to a specific lower group of introns. child , this depends less on sequence conservation than on structure conservation (for this, Bu (see rke et al., 1987).

The mechanism of division is introduced by an external cofactor - guanosine. in this case generally includes the guanidino ribose hydroxyl group or the last nucleo of the exon. nuclear at the phosphoester bond of the first nucleotide of the tide and of the intron. Attempts to attack, during which intronic RNA binds covalently at the 5' end . The free hydroxyl group at the 3' end of the exon above is again the last nucleotide of the intron. Attacks the phosphoester bond between the leotide and the first nucleotide of the next exon. The intron is released, splitting it open and joining the exon. this is It is involved in two types of transesterification. The only group 1 intron is this The process is carried out autocatalytically - i.e. in one tube without the addition of proteins. I am in a position to do so. For a series of group I introns, proteins are cleaved in vivo. (See Cech, 1990).

The presence of group 1 introns is found in mitochondria of lower eukaryotes, lower fungi, and leaves. Ranging from the plastid nuclear genome to eubacteria, bacteriophages and primitive fungi. There is. Group 1 introns are not found in higher eukaryotes. Its existence is an Not limited to genes coding for white matter, but also genes for tRNA and rRNA. It also exists in Denden. The presence and cleavage mechanism has recently been extensively investigated, and Mic Edited in the publication Hel & Westhoff, 1991.

Medicaments produced according to the invention generally contain Group 1 introns. and are generally used to inhibit the growth of organisms that are not sensitive to the antibiotics listed above. can be used. In this case, the use of this medicine extends particularly to therapeutic areas. In this case, the site of action of the above antibiotics is the intronic RNA. is held, but there is no interest in liposomal RNA 0 sites.

The mechanism of action of group I intron antibiotics is shown below with the accompanying drawings. This will be explained in more detail below. Figure 1 shows guanosine-triphosphatase (hereinafter referred to as GTP). shows various radiographs of introns disassembled by Understand the effects of antibiotics. Figure 2 shows the degradation process of preRNA and the linear intron. Figure 2 illustrates the joining of two exons under splitting. Figure 3 is the most important use It shows the basic structural formulas of antibiotics tested and includes some groups related to the antibiotics tested. The effective limits are listed below the loop. Figure 4 shows the td-intron (Figure 4a) and and the secondary of the core region showing the 3′-truncated portion of the 5unY-intron (Figure 4b). It shows the structure.

In this case, the method for measuring the efficacy of antibiotics on group I introns is as follows: Do it like: The gene used in this method is timininal acid nondeterminant from Cori 1:, 'T4. ・It's Tarse. This gene was collected into the vector pTzl 8U (USB Corporation). cloned in the truncated intron-Δ-P6 (Schroeder et al. , 1991). Linearize the plasmid with EcoRT to produce uncleaved curved bodies. and then transcribed within the body. The transfer conditions are as follows. lμ in a volume of 20μm g of DNA, 40mM Tris-HCl (pH 7,5), 5mM MgCl2 ．． 0.4mM spermidine, 5mM DTT% 77-RNA poly Merase (Stratagene), 10μ Ci(a-35S)-CTP (Amersham) and IO unit glue ponuclease inhibitor (Boehr) inger, man/% im) for 1 hour at 30°C. Then RN A precursor was subjected to gel electrophoresis (5% acrylamide in Trisborate-EDTA/7 Purify by M urea). For antibiotic inhibition testing, 20. OOOcpm RNA precursors were dissolved in 5 μM cleavage buffer solution (2.5 μM GTP, 40 μM Tris). - Increase antibiotics in HCl, 8mM MgCl2.0.4mM spermidine). Add and incubate at 37°C for 10 minutes. The reaction mixture was then diluted with 45 μm stop solution (2.5mM EDTA, O, 1mg/ml yeast tPNA) or and 150 μl of 0.3 M Na0Ac/ethanol. Let it stagnate. Resuspend in 5 μl of water and add another 5 μm of the denaturation mixture [100 % formamide, 0. 1% Bromophenol Blue, 0. 1% Kishi Heat to 65°C for 2 hours with Xylenxyanol After that, the reaction product was transferred to 5% acrylamide gel/7M urea as above. Separated and made visible on x-ray film.

From Figure IA, inhibition of 2-desoxystrebtamine to group I introns. The effect can be recognized. In this case, especially from Figure IA, gentamicin or Two blocks, one being exon El and the other being compound intron E2. separation of the intron from the E2 exon and both exon It can be seen that the connection of the son is blocked. Figure] In A, there is very little rationed supply of GPT. Degradation occurs even at low gentamicin concentrations; It can be seen that degradation of the exon and ligation of both exons are inhibited.

Figure IB shows similar behavior for the 5unY intron. Figure IC is glue Parallel experiments are shown with introns of step 2 and linear introns (1, intron). Antibiotics It can be clearly seen that this is done regardless of the quality concentration. , in the second case, fig. Genta intentionally suppresses group 1 introns as shown by IA and IB. Mycin was used. The process conditions are the same in all embodiments described above.

Similarly to Figure IA, Figure ID and IE are also shown, where Figure ID tobramycin instead of icin and palomycin in the case of the embodiment according to Figure E. Mycin is used.

In the case of all attached examples, the RNA tested refers to preRNA, InF3 represents the combination of intron and exon 2, L-■n represents the linear intron, and El- E2 means combined exons E1 and E2, and El means only exon 1. When explaining in 2, the thick horizontal line means both exons El and E2. In this case, the preRNA degradation mechanism is reproduced by GTP treatment. . In the first step, exon 1 is released from the intron-exon 2 of the fragment. It turns out that The first step of degradation is catalyzed by exogenous guanosine (G-OH). The solution is started, at which time guanosine attacks the G-bond side, and the nucleus is placed on the 5゛-splitting side. is cleaved by target attack and covalently linked at the first nucleotide of the intron. Ru. In this case the OH- group is added to the 3' side at exon E1. Then guanosi The guanosine attacks the 5° side of exon 2, and a linear guanosine is excised at this time. No. In two steps, both exons E1 and E2 are joined and each group is added at their ends. Introns containing anosine exist in isolation.

Figures 3A to 3C depict a series of 2-deoxystreps tested as described in Figure 1. Amine compounds are mentioned. The concentrations listed are the best effectiveness for the individual substances. It is the substance concentration.

Figure 4 shows the secondary structure of the nuclear region surrounding the 3' decomposition position, and The td-intron is shown here, and the 5unY-intron is shown at B. . This configuration shown is similar to that just before the second step of degradation, where the 5′ degradation side cleaves. The final G of the intron is present on the G-linked side. pair-element PI, P7, P9 and PIO have corresponding meanings. This model is a Mic He removed from the Tetrahymena structure as described by I et al. (9) It will be done. The upper case letters refer to introns, and the lower case letters refer to exons. Regarding residues. Other numbers for both residues indicate the nucleotide position in the intron ing. The G-residue is a guanosine-linked residue (9).

td-intron + tobramine FIG, 7D td-Δ+Baromomyson ・　・　101■l+n\l　lo心　μM　μM　Tobramycin (if not) *・No GTP additives day S2 5unY Continuation of front page (72) Inventor Fono Arseno Uwe Austria, Day 1090 Vienna, Althanstraße, 14, Institud f Microbai Ologie Land Genetik der Universität Vienna (72) Inventor: Davis Julien France, F-75724 Paris-Se Dex, 15, Ryu Doyu Doctor Ru, Instate Udoto Pastu Department of Biotechnology (72) Inventor Schrader René Austria, Day-1090 Vienna, Althanstrasse 14, Inn Stated F Microbiology Lant Genetique del Universidad -Sitate Vienna

Claims (3)

[Claims] 1. Particularly in pharmaceutical production methods, the production of organisms containing group 1 introns 2-deoxystreptamine, especially amino sugars with 4,6 or using 4,5-substituted 2-deoxystreptamine type antibiotics. To be there. 2. As an antibiotic of the type 4,6-substituted 2-deoxystreptamine , gentamicin (B, Cl, Cla, C2), kanamycin (A, B, C) , Tobramycin or Amikamycin mycin). 3. As an antibiotic of the type 4,5-substituted 2-deoxystreptamine neomycin B, paromomycin, ribostamycin, lividomycin or The use according to claim 1, wherein ptyrosine is used.