Abstract:
The invention relates to a conjugate for treating prokaryotic infections from a transport mediator penetrating the prokaryotic cell membrane and a desired compound to be introduced into the prokaryote and directed thereagainst, which compound is preferably a peptide nucleic acid (PNA) directed against a gene of the prokaryote giving antibiotic resistance.

Description:
CROSS-REFERENCE TO RELATED APPLCATIONS 
     This application is filed under the provisions of 35 U. S.C. §371 and claims the priority of International patent application No. PCT/DE03/00124 filed Jan. 17, 2003, which in turn claims priority of German patent application No. 102 01 862.6 filed on Jan. 18, 2002. 
     FIELD OF THE INVENTION 
     The present invention relates to a conjugate for treating prokaryotic infections from a transport mediator penetrating the prokaryotic cell membrane and a desired compound to be introduced into the prokaryote and directed thereagainst, which is preferably a peptide nucleic acid (PNA) directed against a prokaryote gene giving antibiotic resistance. 
     BACKGROUND OF THE INVENTION 
     Bacterial infections which on account of a resistance formation can no longer be treated with the currently available antibiotics are advancing worldwide. The situation is critical above all in clinics, e.g. in intensive-care units, where antibiotics are administered daily and pathogens can easily develop resistances. Also, the incorrect or excessive use of antibiotics has rapidly increased the number of resistant bacterial strains. Although the problem has previously been solved by using alternative or newly developed antibiotics (e.g. cephalosporins/derivatized antibiotics) and combination therapies (e.g. cotrimoxazole/sulfonamides), new resistances also occur in this case in a relatively short period of time. For example, vancomycin, which was active against certain bacterial strains a short time ago, often can no longer be used in therapy on account of the meanwhile formed resistances. Although major efforts are made to develop new natural antibiotics of different origin from prokaryotes and eukaryotes, only temporary or partial success has been achieved with respect to the rapidly mutating bacterial strains. 
     SUMMARY OF THE INVENTION 
     Thus, the invention is substantially based on the technical problem of providing means enabling an effective therapy of diseases correlated with prokaryotes, e.g. bacteria, resistant to antibiotics. 
     This technical problem is solved by providing the embodiments characterized in the claims. 
     In order to solve this technical problem, the inventors re-sensitized the bacteria with respect to the (classical or modern) antibiotics against which they were resistant. For this sensitization a conjugate was developed comprising the following components: (a) a transport mediator for the prokaryotic cell membrane, i.e. preferably a peptide (e.g. defensin) antibacterial per se, which can penetrate, and damage, the bacterial cell wall on account of its charge and structure by pore formation, and (b) the compound to be introduced into the prokaryote, e.g. a peptide nucleic acid (PNA) which is preferably directed against a gene giving the prokaryote an antibiotic resistance. By means of the examples resulting in the present invention it was possible to show that the antibiotic resistance of bacteria ( E. coli ) resistant to ampicillin/neomyin, which were treated with specific defensin-conjugate-PNAs ampicillin/neomycin , has been overcome successfully. Thus, the conjugates according to the invention are a new class of antibiotics which can overcome the bacterial defense mechanisms or by means of which it is possible to give back the original activity to already known classical antibiotics, such as benzylpenicillins, tetracyclines, and neomycins. This differs completely from the former therapy and research strategies, yet supplements them in a very promising way. The (dual) strategy which can be carried out by means of the present invention specifically addresses to prokaryotic, e.g. bacterial, membranes and is toxic to prokaryotes, on the one hand, and compounds directed against prokaryotes, e.g. antisense nucleic acids (anti-gene nucleic acids) directed specifically against bacterial genes, e.g. PNAs, peptide domains, modified nucleotides, inhibitors of enzymes, etc., can be introduced by means of the conjugates according to the invention, on the other hand. 
     The conjugates according to the invention thus have inter alia the following advantages: (a) no expensive screening method is required for establishing them, and (b) the revitalization of classical antibiotics can be achieved in a special embodiment by blocking the prokaryotic resistance mechanisms on the nucleic acid level so that the “old” antibiotics which were no longer usable in many cases on account of their lacking activity, regain significance. 
     Thus, the present invention relates to a conjugate suited fro treating prokaryotic infections and comprising the following components:
     (a) a transport mediator passing through the prokaryotic cell membrane; and   (b) a compound to be introduced into the prokaryote and directed thereagainst.   

     The term “compound to be introduced into the prokaryote and directed thereagainst” as used herein relates to any compound detrimental to the prokaryote, e.g. kills it or prevents the growth and/or division thereof. Suitable compounds are known to the person skilled in the art, they comprise e.g. anti-metabolites, modified nucleotides, etc. 
     As to methods of producing the individual conjugate components and their linkage, reference is made to German patent application No. 199 33 492.7 and the below examples. The other components (e.g. spacer and/or PNA) are linked to the transport mediator by a covalent chemical bond. Where appropriate, a redox cleavage site (—S∩S—) between transport mediator and compound to be introduced can be introduced chemically by means of a redox coupling. Also, a covalent bond, preferably an acid-amide bond, exists between an optionally present spacer and the compound to be introduced, e.g. the PNA. Possible alternatives are ether or ester bonds, depending on the functional group(s) present in the compound to be conjugated. 
     The conjugate according to the invention is suited for treating all prokaryotic infections, e.g. infections with bacteria, preferably human pathogen bacteria, mycoplasms or yeasts. 
     The transport mediator of the conjugate according to the invention is preferably a peptide or protein which can penetrate the prokaryotic cell membrane and can introduce the desired compound into the cytoplasm. The length of this peptide or protein is not subject to limitation as long as it has the above property. The selected transport mediator is preferably produced biologically (purification of natural transport mediator proteins/peptide or cloning and expression of the sequence in a eukaryotic or prokaryotic expression system) and in particular synthetically, e.g. according to the Merrifield method (Merrifield, J. Am. Chem. Soc. 85 (1963), 2149). 
     This transport mediator is preferably a compound which as such is already detrimental to the prokaryote, e.g. by damaging the membranes (e.g. by forming pores or lesions). These are preferably defensins or holins (bacteriophage protein domains). 
     In a particulary preferred embodiment, the conjugate according to the invention comprises a transport mediator which includes a phage-holin protein, a phage-holin protein with one of the amino acid sequences shown in  Fig. 3  or fragments or variants thereof which can still penetrate the prokaryotic cell membrane, being even more preferred. 
     The terms “variant” and “fragment” used in the present invention comprise proteins/peptides having amino acid sequences which differ with respect to the sequences indicated in  FIG. 3  by deletion(s), insertion(s), substitution(s) of amino acid residues and/or other modifications known in the art or comprise a fragment of the original protein/peptide, the variants and/or fragments of the protein/peptide substantially having maintained the biological properties of the initial protein/peptide, i.e. may penetrate the prokaryotic cell membrane. Methods of producing the above modifications in the amino acid sequence are known to the person skilled in the art and described in standard works of molecular biology, e.g. in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd  edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. (1989)). The person skilled in the art can also determine whether such a protein or peptide still has the desired biological properties, e.g. by the methods described in the below examples. 
     In another alternative preferred embodiment of the conjugate according to the invention, the transport mediator comprises a defensin, preferably a human defensin. The defensins are polypeptides or peptides having an antimicrobial activity, which represent important factors in the innate immunity of vertebrates and non-vertebrates. Defensins have been isolated from animals (including humans), plants and insects, for example. They usually consist of 29 to 42 amino acids and contain three disulfide bridges formed of three cysteine pairs. Defensins suited for the present invention are described in Tang et al., Science 286 (1999), 498; Saido-Sakanaka et al., Biochem. J. 338 (1999), 29; Raj et al., Biochem J. 347 (2000), 633; Yu et al., J. Biol. Chem. 275 (No. 6) (2000), 3943, for example. 
     In an even more preferred embodiment of the conjugate according to the invention, the compound to be introduced into the prokaryote is a peptide nucleic acid (PNA) directed against the expression of a gene. On account of their physicochemical properties, the use of the protease and nuclease-resistant peptide nucleic acids (“PNAs”) which are oligonucleotide derivatives where the sugar phosphate backbone is preferably substituted by ethyl-amine-bound α-amino-ethyl-glycine units, permits a stable and efficient blocking of the transcription of the desired genes under physiological conditions. An anti-gene strategy based on the antisense-principle is pursued by using these PNAs, where it is not the mRNA but the gene per se that is the target. Here, the PNAs hybridize to the target DNA by forming a triple helix. The target area can be a transcribed region of the gene to be blocked, e.g. of the gene giving antibiotic resistance, on the one hand, or a regulatory region whose blocking by means of PNAs also inhibits the transcription, on the other hand. Suitable regions can be identified by the person skilled in the art by means of the known DNA sequences or the function thereof. The peptide nucleic acids preferably have a length of at least 15 base pairs, peptide-nucleic acids having a length of at least 18 bases are particularly preferred, and of at least 21 bases are even more preferred and of at least 23 base pairs are most preferred. The peptide nucleic acid can optionally be labeled, e.g. radioactively, using a dye, biotin/avidine etc. The synthesis of PNAs is known to the person skilled in the art and also described in Nielsen et al., Science 254 (1991), 1497-1500, for example. The term “gene” used herein does not only comprise genes of the prokaryote genome but also genes on extra-genomic elements, e.g. plasmids, etc. 
     In an even more preferred embodiment of the conjugate according to the invention, the peptide-nucleic acid (PNA) is directed against a gene which gives an antibiotic resistance, preferably the antibiotic resistance is a resistance to penicillin, ampicillin, kanamycin or tetracycline. 
     Furthermore, the conjugate may optionally contain a spacer which is located between the transport mediator and the compound to be transported, e.g. the peptide-nucleic acid (PNA). The spacer serves for eliminating or favorably influencing optionally existing steric interactions between the components. 
     The structure of the conjugate according to the invention is preferably as follows: transport mediator-spacer-compound to be introduced. The spacers polylysine, polyglycine or poly(glycine/lysine) are particularly preferred. The length of the spacer is preferably within a range of 2 to 6 amino acids for the purposes according to the invention. The spacer is preferably linked with the transport mediator by means of a cleavable disulfide bridge (—S∩S—). 
     In a particularly preferred embodiment of the conjugate according to the invention, the peptide-nucleic acid (PNA) comprises the following sequence: ATTGTTAGATTTCAT (SEQ ID NO: 1) (orientation: N-terminus/sequence/C-terminus). 
     The present invention finally also relates to a medicament comprising a conjugate according to the invention, optionally together with a suitable carrier. The medicament preferably comprises an antibiotic for which the prokaryote was sensitized by administering the conjugate. Suitable carriers and the formulation of such medicaments are known to the person skilled in the art. Suitable carriers are e.g. phosphate-buffered common salt solutions, water, emulsions, e.g. oil/water emulsions, wetting agents, sterile solutions, etc. The medicaments are preferably administered parenterally, transdermally or subcutaneously. The suitable dosage is determined by the attending physician and depends on various factors, e.g. on the patient&#39; age, sex and weight, the kind and stage of the infection, the kind of administration, etc. 
     Finally, the present invention relates to the use of a conjugate according to the invention for treating a prokaryotic infection, this infection being preferably caused by a prokaryote which is resistant to an antibiotic. 
    
    
     
       Legends of the figures: 
         FIG. 1 : Diagram of a conjugate according to the invention using defensin as a transport mediator and a PNA directed against a resistance to ampicillin or kanamycin  FIG. 2 : Presentation of a pDNA segment of plasmid pBR322 including the sequences used for the design of a PNA conjugate 
       The sequence (SEQ ID NO: 3)of the anti-gene PNA directed against an ampicillin resistance is shown in the figure below. The underlined region of the beta-lactamase-encoding pDNA sequence of pBR322 corresponds to the target region for the PNA conjugate. 
         FIG. 3 : List of Holin-protein sequences which are suited as a transport mediator in the conjugates according to the invention 
       The amino acid sequences (SEQ ID NOs: 4-31) of the 28 individual holins are shown in the one-letter code.  FIG. 3( 1) shows SEQ ID NO: 24-31,  FIG. 3( 2) shows SEQ ID NOs: 4-11,  FIG. 3( 3) shows SEQ ID NOs: 11-19,  FIG. 3( 4) shows SEQ ID NOs: 20-22, and  FIG.3( 5) shows SEQ ID NO: 23. 
         FIG. 4 : Results of treating the competent  E. coli  resistant to ampicillin by transformation with pBR322 and/or to kanamycin by transformation with pEGFP-N 1  (Clontech, Germany, Heidelberg) with or without (control) the conjugates according to the invention 
       Top row: kanamycin culture medium+kanamycin resistance conjugate (500 nM); bottom row: ampicillin agar+ampicillin resistance conjugate (500 nM); left column: controls (without PNA conjugates). A clear effect of the conjugates according to the invention (regaining the antibiotic sensitivity) can be observed. 
         FIG. 5 : Results of the treatment of intact (non-competent)  E. coli  resistant to kanamycin with or without (control) the conjugates according to the invention 
       Top row: kanamycin culture medium+kanamycin resistance conjugate (2 μM); bottom row: kanamycin culture medium+kanamycin resistance conjugate (20 μM); left columns: controls (without PNA conjugates). A clear effect of the conjugates according to the invention (regaining the antibiotic sensitivity) can already be observed at a concentration of 2 μM. All of the bacteria are again sensitized, i.e. killed, at 20 μM. 
         FIG. 6 : Results of the treatment of intact (non-competent)  E. coli  resistant to kanamycin at different concentrations of the conjugates according to the invention to determine the optimum bacterial concentration for the purpose quantification 
       Kanamycin culture mediums were used onto which resistant bacteria were plated. In order to determine the optimum bacterial concentration, a dilution series having the kanamycin resistance conjugate were used with decreasing decimal powers. 
         FIG. 7 : Results of treating intact (non-competent)  E. coli  resistant to kanamycin or ampicillin with the conjugates according to the invention 
       Plate K1: control, plate 2: 250 nM kanamycin-resistance conjugate; plate 3: 250 nM ampicillin resistance conjugate. The results show a bacterial inhibition for ampicillin and kanamycin with 250 nM conjugate. 
         FIG. 8 : Results of treating intact (non-competent)  E. coli  resistant to ampicillin with the conjugates according to the invention 
       as in  FIG. 7 ; K2: control, plate 4: 250 nM ampicillin resistance conjugate 
         FIG. 9 : Results of treating intact (non-competent)  E. coli  resistant to kanamycin with the conjugates according to the invention 
       as in  FIG. 6 ; K1: control; plate 1: 2.5 μM kanamycin resistance conjugate; plate 2: 250 nM kanamycin resistance conjugate. 
         FIG. 10 : Structure of defensin used for the conjugates according to the invention following cyclization (top) and following the formation of three disulfide bridges (left bottom) 
       The hypothetical spatial conformation is shown on the right bottom. 
         FIG. 11 : Results of treating HeLa cells with a conjugate according to the invention
     A: untreated control   B: cells treated with the conjugate according to the invention   
     
    
    
     The invention is further described by means of the below examples. 
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLE 1 
     General Methods 
     (A) Cell Culture 
     The bacteria were plated onto agar with LB broth (and the corresponding antibiotics) and incubated at 37° C. overnight. HeLa cells were cultured in a liquid culture under common conditions. 
     (B) PNA Synthesis 
     Peptide nucleic acid (PNA) imitates a DNA and was originally developed as a reagent for the sequence-specific recognition of double-stranded DNA via a conventional triple helix formation. For the solid phase synthesis the Fmoc strategy was used by means of a fully automated synthesis device (Syro II, Multisyntech, Witten, Germany). The synthesis was carried out on a 0.05 mmol Fmoc-AS polystyrene resin (1% cross-linked). The coupling reagent used was 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU). The side chain-protecting groups were Lys(Boc), Asp(OBut), Ser(But), Cys(Trt) and Asn(Trt). The protected peptidyl resin was treated with 20% piperidine in dimethylformamide. The cleavage and separation of the protecting groups were obtained by treatment with 90% trifluoroacetic acid, 5% ethane dithiol, 2.5% thioanisole and 2.5% phenol (v/v/v) at room temperature for 2.5 hours. All of the products were precipitated in ether and purified by preparative HPLC (Shimazu LC-8A, Shimazu, Duisburg, Germany) on a YMC ODS-A 7A S-7 μm reverse phase HPLC column (20×250 mm) using 0.1% trifluoroacetic acid in water (A) and 60% acetonitrile in water (B) as an eluting agent. The peptides were eluted with a linear gradient of 25% B to 60% B at a flow rate of 10 ml/min within 40 minutes. The fractions corresponding to the purified conjugate were lyophilized. Sequences of individual molecules and the complete bimodular construct were characterized by analytical HPLC (Shimadzu LC-10) and laser desorption mass spectroscopy (Finnigan Vision 2000, Finnigan MAT, San Jose, Calif., U.S.A.) as described below. 
     The sequence of the PNA directed against an ampicillin resistance was as follows: H 2 N -ATTGTTAGATTTCAT-COOH (SEQ ID NO: 1). This is a sequence which can hybridize with the region of position 86 to position 100 of the pDNA of pBR322 (GeneBank accession number J01749). The sequence of the PNA used against the kanamycin resistance was H 2 N -TCTTGTTCAATCAT-COOH (SEQ ID NO: 2). 
     (C) Chemical Synthesis of Defensin 
     For the solid phase synthesis of defensin (cf.  FIG. 10  as regards the amino acid sequence and structure) the Fmoc strategy (Merrifield, J. Amer. Chem. Soc. 85 (1963), 2149-2154; Ruegg and Rudinger, Methods Enzymol. 47 (1977), 111-126) was used with a fully automated synthesis device (ABI 431; Applied Biosystems, Germany, Darmstadt). The synthesis was carried out on a 0.05 mmol Fmoc-Arg(Pbf) polystyrene resin (1% cross-linked). 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) was used as the coupling reagent. The side chain-protecting groups were: Thr(But), Arg(Pbf). Three different selectively cleavable protecting groups were used for Cys. t-Butylthio was used for Cys(3,15), acetamidomethyl was used for Cys(5,13) and a trityl group was employed for Cys(7,11). 
     In the first step, the t-butylthio protecting group was cleaved using tris(2-carboxyethyl)phosphine (TCEP) and the sulfur bridge was cleaved with 20% DMSO in water. In the second step, the acetamidomethyl. protecting group was cleaved and at the same time, the second sulfur bridge was oxidized with a 0.01 mole iodine solution. The protected peptidyl resin was treated with 20% piperidine in formamide for 12 min. and then thoroughly washed with dimethylformamide. The protecting groups were cleaved and removed from the peptide resin by treatment with 90% trifluoroacetic acid, 5% ethane dithiol, 2.5% thioisanole and 2.5% phenol (v/v/v/v) at room temperature for 2.5 hours. The product was precipitated in ether. The raw material was purified on preparative HPLC (Shimazu LC-8A, Shimazu, Duisburg, Germany) on a YMC-Pack ODS-A,S-5 μm reverse phase HPLC column (20×150 mm) using 0.1% trifluoroacetic acid in water (A) and 60% acetonitrile in water (B) as an eluting agent. The peptide was eluted with a linear gradient of 25% B to 60% B at a flow rate of 20 ml/min for 40 min. The fractions corresponding to the purified peptide were lyophilized. 
     As the last step, a head/tail/cyclization was carried out with propane phosphonic acid anhydride (T3P), and the purification method was repeated. The purified material was characterized by analytical HPLC (Shimadzu LC-10) and laser desorption mass spectroscopy (Finnigan Vision 2000, Finnigan MAT, San Jose, Calif., U.S.A.). 
     Peptide Purification: 
     
         
         Gradient: analytical 5%→80% (in a period of 35 min.); preparative: 5%→80% (in a period of 40 min.); 
         Purity: &gt;90%.
 
(B) Linkage Reactions
 
       
    
     The linkage reactions were effected as described in German patent application no. 199 33 492.7 under mild oxidative conditions (DMSO/H 2 O). For this purpose, cysteine groups of defensin and the spacer H—S-Gly and the PNA were oxidized in a region of 2 mg/ml in a 20% DMSO/water solution. The reaction was complete after about 5 hours. The course of the oxidation was monitored by analytical C18 reverse phase HPLC (Tam et al., J. Amer. Chem. Soc. 113 (1991)). The components were linked according to the Merrifield method (Merrifield, J. Americ. Chem. Soc. 85 (1963), 2149). 
     The thus synthesized PNA module has the following structure:
 
DEFENSIN human -S∩S-(Gly)X-PNA AmpR/NeoR 
 
 X= 1-5
 
     The purification was made by means of reverse phase HPLC, following by lyophilization. Having determined the mass by means of MS, the lyophilizate was dissolved in a defined volume of physiological common salt solution to give a stock solution of 10 μM. 
     EXAMPLE 2 
     Determining the Activity of the Conjugates According to the Invention in the Case of Antibiotic-Resistant  E. coli  Strains 
     Resistant bacteria were confluently plated onto common agar plates with and without antibiotic and partially treated with the conjugates according to the invention. In a preliminary experiment, already competent  E. coli  (i.e. having already perforated membranes) were used; see  FIG. 4 ; top row: kanamycin culture medium+kanamycin resistance conjugate (500 nM); bottom row: ampicillin agar+ampicillin resistance conjugate (500 nM); left columns: controls. A marked effect of the conjugates according to the invention (regaining the antibiotic sensitivity) can be observed. 
     In the then following experiments (shown in  FIGS. 5 to 9 ), intact (non-competent)  E. coli  were used, and the conjugates directed against an antibiotic resistance were tested at various dilutions. As to the employed bacteria, antibiotics and concentrations, reference is made to the legend of the figures. In any case, the results of these studies clearly show that the conjugates according to the invention effect another sensitization of the bacterium for the corresponding antibiotic by means of a PNA directed against a gene giving antibiotic resistance and that they can thus be combated by means of this antibiotic again. 
     The non-toxic effect of this conjugate on eukaryotic cells (HeLa cells) was detected by incubation of the cells with/without conjugate (at concentrations as for the experiments with bacteria); see FIGS.  11 A+ 11 B.