AU664191B2 - Use of a 2-deoxystreptamine-type antibiotic - Google Patents

Description

F AS SUNG~TW pages 1/6-6/6, drawings, replaced by flew pages 1/8-8/8 2 oIrb- WELTORGANISATION FOJR GEISTIGES EIGENTUM i P ti Internationiales fitiro INTERNATIONALE ANMELDUNG VEROFFENTLIGHT NACH DEM VERTRAG OB3ER DIE INTERNATIONALE ZUSAMMENARBEIT AUF DEM GEBIET DES PATENTWESENS (PCT) (51) Internationale Pate ntklassi fi ktion 5 I 'II Int-nnfnnI, eVretihnsumr WO 93/00907 A61IK 31/70 Al (43) Internationaltis Verbffentlichungsdatum: 21..

lanuar 1993 (21.01.93) (21) Internationales Aktcnzeichen: (22) Internationales Anmeldedatuni: PCT/AT92/00087 8. lull 1992 (08.07.92) Prioritiitsdaten: A 1375/91 9. lull 1991 (09.07.91I) (74) AnAWijte: ITZE, Peter usw. Amerlingstr. 8, A-I1061 Wien

(AT).

(81) Bestimrnungsstaaten: AU, CA, Fl, JP, NO, europflisches Patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LU, MC, NL, SE).

Verdffentlicht Alli internafionalem Rchlirclienibricl t.

Vor Ablauf der firi,4,IderungL'n der Ansprikcle zygelasse- ,ie~ Frsi.Ve~.T/eniiicliung wvird wtiederholt falls Anderu-111 geti eihurqffen.

-F472)k,.weleflp-Erfinder: VON AHSEN, Uwe [DE/AT], Institut f. Mikrobiologie und Genetik der Universitit Wien, Althanstra~e 14, A-1090 Wien DAVIES, Julian [GB/FR]; Department of Biotechnology, Institut PasteUr, 28, rue du D,-.-Roux, F-75724 Paris C~dex 15 (FR1).

SCHROEDER, Rern~e [AT/AT]; I4u4-Mi1krobilele, gie un~d Geneti er Univers-itait-Wien, Althanstr---4-A- I OQ -l n(AT AfUeK 3IZ10ze tAVV7 Or qOhrosse Ql/4, S40c((, 4 /030 V= ,AusflS,t (70 AJ--ZYM1 Dic. 11133 bMVJS 5-rY-ET, $'LIZTF 293/ rcL-EFJJ-A qlko- 7 7 F$Vr/7hF- OF AA'E~r( A s-N L1Zf9MAO, 4 (54)Titic: USE OF A 2-DEOXYSTREPTAMINE-TYPE ANTIBIOTIC (54) Bezeichnung: VERWENDUNG EINES ANTIBIOTIKUMS DES TYPS 2-DESOXYSTREPTAMINS (57) Abstract The invention relates to the use of an antibiotic or the 2-deoxystreptamine type, especially a 4,6 or 4,5 2-deoxystreptamine subistituted with amino sugars, to inhibit the growth of organisms containing group I introns, especially in a process for making a med ica ment.

(57) Zusanumenfassung Erfindung betrifft die Verwendung eines Antibiotikums des Typs 2-Desoxystreptamin, insbesondere eines 4,6 bzw. mit Amninozuckern substituierten 2-Desoxystreptamins, zur 1-emmung des Wachistums von Gruppe I Introns enthaltenden Organismen, insbesondere in einem Verfahren zur Herstellung eines Medikaments.

*(Sictit P(T o.utte Nr. ObJl9'93, "Seetion 11") i WO 93/00907 PCT/AT92/00087 Use of an Antibiotic of the 2-deoxystrenamine type The invention relates to the use of an antibiotic of the 2deoxystrepamine type.

Conventionally, pathogens or other microorganisms having a pernicious effect on the human or animal body have been treated with antibiotics. This has entailed, depending on the type of antibiotic employed, either coverage of a wider spectrum of pathogens, giving rise to the term "broadband antibiotics", or, given more specific information on the pathogen at issue, employment of antibiotics that target specific bacteria and similar organisms. All of these antibiotics have the disadvantage that their effect is very spread out, i.e. non-specific, the result of which being that damage is caused to organisms that are required by the human or animal body for life organisms that exist in symbiosis with the human organism. A particularly good example of this relEtionship involves intestinal bacteria, which humans require for the digestion of food. Even these intestinal bacteria are killed off, or at best only damaged, by use of conventionallyemployed antibiotics.

It has for many years been the goal of scientists to develop antibiotics that can be used to target specific microcrganisms.

It has now emerged that various kinds of reactions are likely to occur between antibiotics and organisms; such differences depend on the presence of Group I introns in the ribosome RNA. Group I .r L -2 introns occur primarily in prokaryotes and in some eukaryotes. This means that organisms identified normally as either pathogens or other kinds of parasite are essentially those that contain Group I introns. If, therefore, the growth of such organisms, which contain Group I introns, can be inhibited in a specific manner, harmful organisms can be specifically targeted and attacked without negatively affecting organisms that are essential for human life.

2 10 The invention seeks therefore to develop a type 2-deoxystreptamine antibiotic, more particularly a 4,6 0 or 4,5 2-deoxystreptamine substituted with amino sugars, in order to inhibit the growth of organisms containing Group I introns, and, more particularly, in a process for making medicaments.

In one aspect, the invention consists in a method of inhibiting the growth of an organism containing Group I introns, comprising the step of inhibiting self-excision of RNA introns from exons by administering an antibiotic of the 2-deoxystreptamine type.

In a preferred embodiment, the organism containing a group I intron is a fungal species.

In another preferred embodiment, the antibiotic is a 4,6- or a 4,5-2-deoxystreptamine substituted with amino sugars.

It is advantageous that the type 4,6 substituted 2-deoxystreptamine used can be gentamicin Cl, Cla, C2), kanamycin B, tobramycin or amikamycin. The a' xi' Cj -*llllyI~Y1~4~- LIIIIIPII~W Vlil 2a type 4,5 substituted 2-deoxystreptamine used can be neomycin B, paromomycin, ribostamycin, lividomycin or butirosin. Such antibiotics can, due to their bactericidal action, be used against gram-negative as well as gram-positive bacteria in clinical and general antimicrobial environments (see published articles by Davis Yagisawa, 1983; Cundliffe, 1990).

The effectiveness of 2-deoxystreptamine owes to its ability to inhibit the self-excision of Group I intron RNA from its exons. The coding region of a gene is, in fact, interrupted in many cases by non-coding regions, the introns. The introns and the coding regions, the exons, are, during transcription of DNA into I

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c f)i 3 themselves transcribed and the introns spliced onto the RNA plane.

It is essential for the following translation phase that the coding regions be ligated together again in order to permit creation of a functional gene product, a protein. From the splicing mechanism, the accepted RNA secondary or tertiary structure and their occurrence inside the organism, the group I incrons can be described as follows: They have a string of conserved sequences, whose regions are designated as P, Q, R, S, whereby the distance separating the regions can vary. The RNA can, due to pairing alternatives, exhibit a characteristic secondary structure, in which case such pairings are designated as P1 to P10. Such designations depend on membership in a given sub-group of the Group I introns. In this case, structure preservation is more important than sequence preservation (see Burke et al., 1987).

The splicing mechanism is introduced by means of an external cofactor, guanosine. It is generally accepted that the hydroxyl group of the ribose of the guanosine makes a nucleophilic attack on the phosphor-ester bond of the last nucleotide of the preceding exon and of the first nucleotide of the intron, thus bonding covalently to the 5' end of the intron RNA. The liberated hydroxyl radical at extremity 3' of the preceding exon in turn attacks the phosphor-ester bond located between the last nucleotide of the intron and the first nucleotide of the following exon, breaks up the latter and ligates itself to the exon, in which case the intron is liberated. What actually take place are two J .r 4 transesterifications. Individual Group I introns are capable of carrying out this process autocatalytically, in vitro i.e. without addition of proteins. It has been demonstrated in the case of a series of Group I introns that in vivo proteins are required for the splicing phase (see Cech, 1990).

Group I introns occur in the core genome of lower eukaryotes, mitochondria of lower fungi, in chloroplasts to eubacteria, in bacteriophages and archebacteria. Group I introns have not been found in higher eukaryotes. Their occurrence is not limited to genes that code for proteins; they also occur in genes for tRNA and rRNA. A recent and comprehensive compilation of their occurrence and of the splicing mechanism, can be found in the article published by Michel Westhoff, 1991.

The medicaments produced in accordance with the invention can, therefore, be used generally to inhibit the growth of organisms that contain a Group I intron and that are not sensitive to the antibiotics mentioned. The use of such medicaments should extend in particular to the therapeutic field, where the fact that the listed antibiotics act upon the intron RNA and not the ribosomal RNA, would be of interest.

The mechanism of the antibiotic that acts against Group I introns will next be described in greater detail with the aid of the attached drawings. Fig. 1 shows a number of different radiographs of introns that were broken up with the aid of guanosinetriphosphate (designated hereinafter as GTP). This serves to demonstrate the effectiveness of the antibiotic. Fig. 2 I_ I_ 5 illustrates both the process by means of which the preRNA is broken up and the ligation of both exons while the linear intron is being split off. Fig. 3 illustrates the basic structural formulae of the most important antibiotics used. Appearing beneath the individual groups of tested antibiotics are the limits of their effectiveness.

Fig. 4 shows the secondary structure of the core region, which contains the 3' splitting point of the td intron (Fig. 4a) and the sun Y intron (Fig 4b).

The procedure employed to measure the effectiveness of the antibiotics on Group I introns was carried out as follows: The gene used in this procedure is thymideylatesynthase from coliphage T4. The gene was cloned in the Vektor pTZ18U (USB Co.) with a shortened intron delta P6 (Schroeder et al. 1991). The unspliced precursor was prepared by linearizing the plasmid with EcoRI and transcribing it in vitro. The transcription conditions were as follows: 1 Mg DNA in a volume of 20 Al at 30° C for 1 hour in a buffer solution of 40 mM Tri HC1 pH 7.5, 5 mM MgCl 2 0.4 mM Spermidine, 5 mM DTT, 10 units T7 RNA polymerase (Stratagene), A Ci (Alpha-35S)-CTP (Amersham) and 10 units RNase Inhibitor (Boehringer Mannheim). The RNA precursor was then purified by means of gel-electrophoresis (5 acrylamide/7M urine in tri-borate- EDTA). For the antibiotic inhibiting test, 20,000 cpm RNA precursor was incubated in 5 Ml splicing buffer (2.5 AM GTP, 40MM Tri HC1, 8mM MgCl 2 0,4 mM Spermidine), with increasing amounts of antibiotic (as a rule 0.1 to 2 mM) for 10 minutes at 37° C.

Next, the reaction mixture was precipitated by adding 4 5pl stop 6 solution (2.5 mM EDTA, 0.1 mg/ml yeast tRNA) and 150 Al 0.3 M NaOAc/ethanol. Following resuspension in 5 Al water and heating to C for 5 minutes with a further 5 pl denaturing mix (100 formamide, 0.1 bromphenol blue, 0.1 xylene xyanol), the reaction products were, as described above, separated with 5 acrylamide gel/7M urine and rendered visible with the aid of an Xray film.

Figs. 1A to IE show the inhibiting action of the 2deoxystreptamine on Group I introns, whereby it can be seen from Fig. 1 A in particular that gentamicin permits separation into two pieces, one being exon El and the other the intron compound E2, but prevents both the intron from splitting away from exon E2 and the ligation of both exons. It can be seen in Fig. 1A that, even given very low doses of GTP, break-up occurs, in which event however, even in the presence of low concentrations of gentamicin, the splitting away of the introns and the ligation of both exons is prevented. Fig. 1B shows analogous relationships for the sun Y intron. Fig. 1C describes a parallel procedure involving a Group 2 intron, and it will be readily seen that in this case the preRNA breaks off into linear intron (L intron) and into the ligated El, E2 exons. This process occurs independently of the concentration level of the antibiotic. In the present case, gentamicin was employed, which, as can be appreciated from Figs. 1A and 1B, inhibits growth of the group 1 introns specifically. The procedure conditions were identical in all embodiments. Analogously to Fig.

1A, Fig. 1D shows, instead of gentamicin, a tobramycin and the V -LLFi~I~llR"~; __111111111111~--- 1~ 1111.

7 embodiment example in accordance with Fig. 1E illustrates the use of paromomycin.

In all examples used, preRNA means the tested RNA, In-E2 the bonding of intron with exon 2, L-In the linear intron, E1-E2 the ligated exons El and E2 and El exon 1 alone.

In Fig. 2, the thick lines represent the two exons El and E2, whereby the splitting mechanism of the preRNA is shown in the process involving GTP. It can be seen that in the first step, the exon 1 is split off from the broken piece intron-exon 2. In the first step of the breaking-up process, the breaking-up process is introduced by means of the exogene guanosine the guanosine attacks the G-bonding side, incises on the 5' splitting side with a nucleophile attack, and is bonded covalently with the first nucleotide of the intron. The OH group attaches itself in this case to the 3' side of exon El. Next, the guanosine attacks the side of exon 2, the result being that the linear guanosine is excised. In a second step, both exons El and E2 are ligated, and the intron containing the guanosine at both of its ends is already split off.

In Figs. 3A to 3C are described a series of 2-deoxystreptamine compounds, with which the tests, illustrated in Fig. 1, were carried out. Of the concentrations, the concentration having the best effect was added to the individual substances.

Fig. 4 shows the secondary structure of the core region surrounding the 3' splitting side, whereby A designates the td intron and B the sun Y intron. The configuration shown depicts the V j c Q _J I 8 situation shortly prior to the second stage of breaking up (the splitting side is split away, and the last G of the intron is located on the G-bonding side. Pairing elements PI, P7, P9 and are similarly indicated. This model was taken from the tetrahymena structure as described by Michel et al. The letters appearing at the top refer to the intron and the letters appearing at the bottom the exon residue. The numbers appearing beside both residues represent the position of the nucleotide inside the intron. The G residue is the guanosine bonding side s_ .1

Claims (6)

1. A method of inhibiting the growth of an organism containing Group I introns, comprising the step of inhibiting self-excision of RNA introns from exons by administering an antibiotic of the 2-deoxystreptamine type.

2. A method according to claim 1 wherein the antibiotic is a 4,6- or a 4,5-2-deoxystreptamine substituted with amino sugars.

3. A method according to claims 1 or 2 wherein the antibiotic is 4,6 substituted 2-deoxystreptamine selected from the group gentamicin Cl, Cla, C2), Kanamycin tobramycin and amikamycin.

4. A method according to claims 1 or 2 wherein the antibiotic is 4,5 substituted 2-deoxystreptamine selected from the group neomycin B, paramomycin, ribostamycin, lividomycin and butirosin.

A method according to any one of the preceding claims, wherein the organism containing a group I intron is a fungal species.

6. A method according to claims 1 to substantially as hereinbefore described, with reference to the Figures. DATED this 31st day of August, 1995 NZYM, INC Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS Y,: I L ul i; t- i