PL227562B1 - Application of the bacitracin antibiotic for the hydrolytic degradation of RNA - Google Patents

Abstract

The subject of the present invention is the use of bacitracin for the hydrolytic degradation of RNA, occurring at guanosine residues, preferably in single-stranded RNA regions, and occurring without the involvement of transition metal ions. According to the invention, it is possible to use bacitracin according to a mechanism dependent on the degradation of undesirable RNA, different than that stipulated to-date.

Description

The subject of the present invention is a new use of the antibiotic bacitracin, which consists in using the hitherto unknown nucleolytic properties of this drug for the degradation of ribonucleic acids (RNA). Bacitracin is a polypeptide complex with recognized antibacterial properties, it is produced by the bacterial strain Bacillus subtilis var Tracy, Bacillus Licheniformis.

This drug was discovered in the 1940s and has since been used quite widely in antibacterial therapies (Epperson, J., Ming L. Biochemistry 39, 4037-4045 (2000); Ming, L., Epperson, JJ Inoorg Biochem. 91,46-58 (2002)).

This drug is not absorbed through the alimentary tract, in therapy it is most often used as one of the ingredients of antibiotic ointments used to combat bacterial skin infections and against bacterial eye infections. It is also given intramuscularly, including even infants with bacterial pneumonia. In addition, bacitracin is used as a feed additive to prevent animal infections.

It is believed that, like other antibiotics, bacitracin should only be given when a bacterial infection is present. The probable mechanism of action of bacitracin described in the literature is that it inhibits the process of biosynthesis of gram-positive bacteria cell walls (Ming, L., Epperson, J. J. Inoorg. Biochem. 91, 46-58 (2002)).

Bacitracin is an antibiotic with strong nephrotoxic properties. Bivalent metals are the co-factor of bacitracin, while activated bacitricin has antibacterial and antiparasitic properties.

One of the advantages of using this antibiotic is that it produces relatively little strains of drug-resistant bacteria in the body.

Bacitracin is a drug approved for treatment for a long time, and therefore the issue of side effects related to its use, such as nephrotoxicity or allergenic effects, as well as the problem of drug resistance have been investigated (Podlewski, JK, Chwalibogowska-Podlewska A. Drugs of modern therapy - 20th edition) , Medical Tribune Polska, Warsaw 2010).

Bacitracin has recently been reported to degrade double-stranded DNA (Tay, W. et al., J. Am. Chem. Sci. 132, 5652-5661 (2010)). The reaction takes place only after binding some transition metal ions and proceeds according to the free radical mechanism. However, a number of other antibiotics have similar properties of inducing oxidative degradation of nucleic acids (Holmes, C. et al., Bioorg. Medicin. Chem. 5, 1235-1248 (1997); Jeżowska-Bojczuk, M. et al., Eur. J. Biochem. 269, 5547-5556 (2002); Szczepanik, W. et al., J. Inorg. Biochem. 94, 355-364 (2003a); Szczepanik, W. et al., J. Chem. Soc. Dalton Trans 8, 1488-1494 (2003b); Wrzesiński, J. et al., Biochem. Biophys. Res. Commun. 331, 267-271 (2005)). It is believed that they may be responsible for some of the side effects of antibiotic therapies used. Determining whether these processes are important from the medical point of view requires further research, because the concentrations of metal ions that could participate in them are relatively low in the cell.

The international publication WO 94/04185 describes the use of the properties of bacitracin for the inhibition of the protein disulfide isomerase (PDI) in Lara et al. (Lara, H. et al., Virol. J. 8, 137 (2011)) it has been shown that DTNB and bacitracin are able to inhibit PDI, so that viruses, e.g. HIV, RSV have a limited ability to reduce disulfide bonds in the envelope, which reduces their ability to penetrate the cells. However, the direct effect of bacitracin on viral destruction has not been proven. The effect of bacitracin on viruses in this case is only indirect. Bacitracin inhibits the PDI enzyme, thus interfering with the viral life cycle.

In publications US 4,795,740, US 5,066,783, a pharmaceutical composition effective against HSV is presented, consisting of a mixture of aciclovir and bacitracin (as a protease inhibitor), wherein bacitracin itself has no activity against HSV-1 in the control.

In the prior art, no publication has suggested the effect of bacitracin on RNA degradation.

The subject of the invention is the use of bacitracin for the production of preparations for the degradation of RNA.

According to the invention, it is possible to use the antibiotic bacitracin in a different way than before for the degradation of undesired RNA. This antibiotic could be used against viruses containing RNA as genetic material. RNA viruses include inflammation viruses

Liver, polio, or HIV virus. In topical application, it could also be used against rapidly proliferating DNA viruses such as herpes viruses by attacking viral mRNAs.

According to the invention, bacitracin can be used e.g. externally, topically to combat viral skin infections. Another possibility is the use of this antibiotic in therapies of mixed bacterial-viral and purely viral infections, taking into account a different mechanism of its action, which is the degradation of RNA, than previously thought. There is also a potential possibility of using bacitracin in the production of disinfectants.

The advantage of bacitracin and therefore its new use is the fact that this compound has been known for many years and is approved for use in humans and animals. This is extremely important in comparison with the newly synthesized therapeutics, the introduction of which for use as drugs requires long-term research and huge financial outlays.

The antiviral therapies used so far use low-molecular-weight compounds that block enzymes important for the life cycle of the virus, e.g. RNA polymerase necessary for the synthesis of this acid strand, protease involved in the generation of viral envelope proteins, etc. Virtually none of the antibiotics in use, effective in the treatment of infections bacterial infections, it is not used in antiviral therapy. On the contrary, these compounds are believed to be ineffective against viral infections.

The ability of bacitracin to degrade RNA molecules was a surprising observation, as a number of antibiotics from different therapeutic groups that had been tested previously did not show similar properties. The RNA degradation reaction takes place with relatively low specificity and takes place without the participation of transition metal ions. Therefore, it is not a free radical process. It most likely follows the hydrolytic mechanism, in a manner similar to that of protein enzymes with a molecular weight, however, several dozen times greater. It is very important that the degradation of RNA was noted even at a low concentration of the antibiotic, in the micromolar range, comparable to the concentrations determined in the blood plasma of patients treated with this drug.

The subject of the invention in the exemplary embodiments is shown in the drawing, in which:

Figure 1 shows model RNA and DNA molecules used in the study of nucleolytic properties of bacitracin: RNA-tRNAPhe, phenylalanine specific tRNA isolated from yeast; HDV fish RNA, HDV antigenomic ribozyme; RNA-R20, HDV 20 nucleotide fragment; DNA-M39, 39 nucleotide DNA oligomer; DNA-M72, 72 nucleotide DNA oligomer. The main sites of degradation in the presence of bacitracin are marked;

Fig. 2 shows an autoradiogram showing the degradation of RNA-tRNAPhe under the action of various concentrations of bacitracin (Bac), its complex with Cu (II) ions, and the complex in the presence of H2O2 (concentrations in μΜ). Reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37 ° C; time 60 minutes; total RNA concentration 2 OD / ml. K1 - control line; K2 - 50 μM Cu (II); K3 - 100 μM Cu (II) -H2O2; L - alkaline hydrolysis; T - digestion with ribonuclease T1;

Fig. 3 shows the effect of the concentration of bacitracin, its complex with Cu (II) ions and the complex in the presence of H 2 O 2 on the degree of degradation of RNA-tRNAPhe;

Fig. 4 is an autoradiogram showing the degradation of RNA-fishHDV under the influence of various concentrations of bacitracin (Bac) and its complex with Cu (II) ions (concentrations in µM), under the reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37 ° C; time 60 minutes; total RNA concentration 2 OD / ml. K1 - control line; K2 - 50 μM Cu (II); L - alkaline hydrolysis; T - digestion with ribonuclease T1;

Fig. 5 shows the effect of the concentration of bacitracin and its complex with Cu (II) ions on the degree of RNA-fish HDV degradation;

Figure 6 is an autoradiogram showing the degradation of RNA-R20 under the influence of various concentrations of bacitracin (Bac) and its complex with Cu (II) ions (concentrations in µM), under the reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37 ° C; time 60 minutes; total RNA concentration 2 OD / ml. K1 - control line; K2 - 50 μM Cu (II); L - alkaline hydrolysis; T - digestion with ribonuclease T1;

Fig. 7 shows the effect of the concentration of bacitracin and its complex with Cu (II) ions on the degree of degradation of RNA-R20;

Fig. 8 shows an autoradiogram showing the degradation of RNA-tRNAPhe in the presence of bacitracin (Bac) and its complex with Cu (II) ions at a concentration of 22 μM depending on the time (in minutes), under the reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37 ° C; time 60 minutes; total RNA concentration 2 OD / ml. K - control line; L - alkaline hydrolysis; T - digestion with ribonuclease T1;

Fig. 9 shows the progress of RNA-tRNAPhe degradation in the presence of bacitracin and its complex with 22 μ II Cu (II) ions depending on the incubation time;

Fig. 10 shows an autoradiogram showing the degradation of RNA-R20 in the presence of bacitracin (Bac) and its complex with Cu (II) ions (Bac-Cu) at a concentration of 22 μM depending on the time (in minutes), under the reaction conditions: buffer 50 mM Tris-HCl pH 7.5; temp. 37 ° C; time 60 minutes; total RNA concentration 2 OD / ml. K - control line; L - alkaline hydrolysis; T - digestion with ribonuclease T1;

Figure 11 shows the progress of RNA-R20 degradation in the presence of bacitracin and its complex with 22 µM Cu (II) ions as a function of the incubation time;

Figure 12 is an autoradiogram showing the degradation of RNA-fishHDV by the action of bacitracin at 5 and 25 µM in the presence of selected factors potentially influencing the degradation reaction under the reaction conditions: 50 mM HEPES-NaOH buffer pH 7.0; temp. 37 ° C; time 60 minutes; total RNA concentration 2 OD / ml, K - control lines;

Figure 13 shows the progress of fish-HDV RNA degradation under the influence of 5 and 25 µM bacitracin in the presence of selected factors potentially influencing the degradation response;

Figure 14 is an autoradiogram showing the degradation of DNA-M39 under the influence of various concentrations of bacitracin (2.5, 25, 250 and 2500 µM) in the presence of selected factors potentially influencing the degradation reaction, under the reaction conditions: 37 ° C; time 60 minutes; total DNA concentration 0.7 OD / ml. K - control lines;

Fig. 15 is an autoradiogram showing the degradation of DNA-M39 under the influence of various concentrations of bacitracin (Bac) (concentrations in µM) in the presence of selected factors potentially influencing the degradation reaction, under the reaction conditions: 37 ° C; time 60 minutes; total DNA concentration 0.7 OD / ml, K - control lines;

Figure 16 is an autoradiogram showing DNA-M72 degradation under the action of various concentrations of bacitracin (25, 250 and 2500 µM) in the presence of selected factors potentially influencing the degradation reaction under the reaction conditions: 50 mM HEPES-NaOH buffer pH 7.0; temp. 37 ° C; time 60 minutes; total DNA concentration 0.2 OD / ml. K - control lines; G + A, C + A - sequential lines;

Fig. 17 shows the autoradiogram showing the degradation of the RNA-R20 molecule incubated in the presence of bacitracin in 50 mM HEPES-NaOH buffer pH 7.0, at 37 ° C for 2 minutes, Panel Bac: 25 and 50 μM bacitracin, RNA concentration 8 μg / ml, Panel tRNA carrier: 25 μM bacitracin, RNA concentration 8, 0.8, 0.08 and 0.01 μg / ml (-). K - control line; L - alkaline hydrolysis; S1, T1 - digestion with S1 nuclease and T1 ribonuclease;

Figure 18 is an autoradiogram showing the degradation products of DNA-M39 when exposed to various concentrations of bacitracin (250 and 2500 µM) under the reaction conditions: 50 mM HEPESNaOH buffer pH 7.0; temp. 37 ° C; time 60 minutes; total DNA concentration 0.2 OD / ml. K - control line; S1 - digestion with S1 nuclease, C + A - sequence line; the image of the short and long electrophoretic run is shown;

Fig. 19 is an autoradiogram showing the degradation products of RNA-R20 when exposed to different concentrations of bacitracin (concentrations in µM), under the reaction conditions: RNA concentration 0.01 µg / ml, 50 mM HEPES-NaOH buffer pH 7.0; temp. 37 ° C; time 2 minutes, K - control line; L - alkaline hydrolysis; S1, T1 - digestion with S1 nuclease and T1 ribonuclease.

Examples

According to the invention, studies on the nucleolytic properties of bacitracin were carried out with the aim of answering the following questions:

1) what is the specificity of bacitracin for various types of phosphodiester bonding, i.e. whether it degrades RNA or DNA molecules, or whether it exhibits the activity of phosphatase or pyrophosphatase,

2) according to what mechanism the reaction of degradation of nucleic acids takes place,

3) whether RNA / DNA degradation occurs preferentially at specific nucleotide sites or sections of the nucleotide sequence and whether the reaction is influenced by the secondary structure of molecules,

4) taking into account the possibility of practical use of bacitracin, how the degradation conditions affect its effectiveness, i.e. the minimum active concentration of bacitracin, dependence on the concentration of RNA / DNA, influence on the reaction of divalent and monovalent metal ions,

5) whether the addition of ribonuclease inhibitors to the reaction using a commercial preparation of bacitracin, or a pre-incubation at high temperature, will affect the properties of bacitracin.

A commercial preparation of bacitracin purchased from Sigma-Aldrich Co. was used in the experiments. As model RNA molecules: phenylalanine-specific tRNA isolated from yeastPL 227 562 B1, 76 nucleotides long (RNA-tRNAPhe) and two RNA fragments of hepatitis D virus (HDV), antigenomic HDV ribozyme 72 nucleotides long (RNA- fish HDV) and a 20-nucleotide viral fragment (RNA-R20). On the other hand, the following were used as model DNA molecules: a 39-nucleotide DNA oligomer (DNA-M39) and a 72-nucleotide DNA oligomer (DNA-M72) (Fig. 1).

RNA and DNA molecules were labeled with the ³² P isotope at the 5 'end using T4 polynucleotide kinase and [γ- ³² Ρ] ΑΤΡ according to the standard procedure, the products after the reaction with bacitracin were separated by electrophoresis in high-resolution polyacrylamide gels, visualized using imaging screens and a computer scanner FLA-5100 (Fujifilm) radioactivity, and the Multi Gauge program was used for quantification.

P r z k ł a d 1

The reaction of bacitracin with RNA-tRNAPhe at the concentration of 2 OD / ml was carried out under the following conditions: 60-minute incubation at 37 ° C in the presence of 5 μΜ of the antibiotic.

Approx. 40% of the original RNA was degraded as a result of the reaction (Figs. 2, 3).

Increasing the concentration of bacitracin increased the degree of degradation and at the concentration of 100 μΜ it reached about 95%.

RNA-fish HDV degradation was more efficient than that of RNA-tRNAPhe (Fig. 4, 5). About 60% of RNA was degraded in the presence of 5 μ R bacitracin. However, at 10 μM bacitracin, the degree of degradation reached 80%, and almost complete degradation of HDV-fish RNA was observed for 50 μM bacitracin. The 20-nucleotide RNA-R20 oligomer after 60 minutes of incubation in the presence of 20 µM bacitracin was almost completely degraded into shorter products (Figures 6, 7).

A comparison of the degradation efficiency of three different RNA molecules, used at concentrations of 2 OD / ml, showed that 80-95% of the original RNA was degraded after 60 minutes of incubation at 37 ° C in the presence of 20 μΜ bacitracin. The kinetics of the degradation of RNA-tRNAPhe and RNA-R20 in the presence of 22 µM bacitracin were also investigated (Figures 8-11). In the case of both RNA molecules, approximately 50% degradation was observed after 1 minute incubation. After 20 minutes, the degradation reaction of RNA-tRNAPhe had plateaued at 90% (Fig. 9), while the disappearance of RNA-R20 was almost complete after this time (Fig. 11).

In order to check whether bacitracin shows phosphatase or pyrophosphatase activity, ³² P-labeled nucleoside triphosphates were used: [y- ³² P] ATP and [a- ³² P] UTP, which were incubated with 25 and 250 μΜ bacitracin for 60 minutes at 37 ° C, also in the presence of Mg (II), Mn (II) and EDTA ions. No degradation products of any of these compounds were observed after polyacrylamide gel electrophoresis. Bacitracin has also been shown to have some degree of single-stranded DNA degradation capacity. However, degradation of DNA-M39 (Figure 14) and DNA-M72 (Figure 16) required much higher concentrations of the antibiotic than for RNA molecules. A comparable effect was obtained with at least 10 times higher concentration of bacitracin. The degradation of DNA-M39 and DNA-M72 was observed with 250 μM bacitracin, but not with 25 μΜ antibiotic concentration.

P r z k ł a d 2

In the mechanism of the hydrolytic cleavage of the phosphodiester bond, it is very important whether the phosphate group at the chain breaking site remains on the 3 'or 5' side of the ribose residue. Therefore, it was decided to determine the location of the phosphate group in RNA and DNA fragments after the degradation reaction in the presence of bacitracin.

The migration of RNA-R20 degradation products was compared with that of the alkaline hydrolysis products and the restricted digestions with S1 nuclease and T1 ribonuclease using high-resolution polyacrylamide gel electrophoresis (Figure 19). It is known that the products of alkaline RNA hydrolysis and digestion with ribonuclease T1 have a phosphate group at the 3 'end of the RNA chain, and the products of digestion with S1 nuclease at the 5' end. The migration of RNA fragments resulting from bacitracin degradation is identical to the migration of RNA fragments after alkaline hydrolysis and digestion with RNase T1 and different from digestion with S1 nuclease. This is convincing evidence that the phosphate groups are at the 3 'end of the resulting fragments.

An analogous analysis of the localization site of the phosphate group was performed for the reaction products of DNA-M39 with bacitracin (Fig. 18). Its result indicates that the migration of the products corresponds to the migration after digestion with S1 nuclease. Thus, in the case of DNA degradation by bacitracin, the phosphate groups are located at the 5 'end of the resulting fragments.

From the point of view of the mechanism of RNA and DNA degradation by bacitracin, it is important whether the reaction requires the presence of divalent metal ions. The degradation of HDV-fish RNA was found to be unaffected by the presence of 2 mM EDTA (Figures 12, 13). A similar observation was made for RNA-tRNAPhe.

PL 227 562 B1

In turn, it is striking that the DNA degradation reaction is completely inhibited by EDTA, both in the case of DNA-M39 (Fig. 14; 0.2 mM EDTA was used) and DNA-M72 (Fig. 16; 2 mM EDTA was used). Thus, the DNA degradation reaction requires the absolute presence of divalent metal ions. It was established that they may be, inter alia Mg (II), Mn (II), or Zn (II) ions (Figures 14-16).

P r z k ł a d 3

An RNA degradation reaction was performed as described above. Bacitracin was found to degrade RNA at guanosine residues (Figures 2, 4, 6). The degradation takes place in the RNA single-stranded regions, for RNA-tRNAPhe mainly in the D loop (G15, G18, G19) and TΨC (G57), for RNA-fish HDV in the L3 / P3 loop regions (G28, G30, G31) and L4 (G58 ), for single-stranded RNA-R20 at residues G12, G13, G14, G17 and G18 (Figures 2, 4, 6). Interestingly, ribonuclease T1 exhibits similar RNA degradation specificity. In addition, bacitracin has been observed to remove the phosphate group from the 5 '(or 5' terminal nucleotide) end of RNA molecules by bacitracin. Such an effect was observed for RNA-tRNAPhe and RNA-fishHDV (Figures 2, 4). However, it was not observed for RNA-R20 (Fig. 6). This may be due to the fact that guanosine is the 5 'nucleoside in the first two model RNAs, and cytidine in the third.

It turned out that the specificity of DNA degradation by bacitracin is clearly different from that observed in the case of RNA degradation. The degradation sites of DNA-M72 and DNA-M39 are very few and occur preferentially in sequence around some cytidine residues (Figures 16, 18). In combination with the information that DNA degradation requires the presence of some divalent metal ions, it can be suggested that the DNA degradation sites are located near the sites of strong binding of these ions.

P r z k ł a d 4

The RNA degradation reaction was carried out to investigate the dependence of this process on the RNA concentration value. The observed degree of RNA degradation depended on its total concentration. The degradation efficiency of three different RNA molecules, used at a concentration of 2 OD / ml, ie 80pg / ml, incubated with 20 µΜ bacitracin for 60 minutes at 37 ° C, was 80-95% of the original amount of RNA. Lowering the concentration of RNA-R20 to 8, 0.8, 0.08 and approx. 0.01 pg / ml, with the use of 25 pM bacitracin and the two-minute incubation time, led to an enhanced degradation effect (Fig. 17). When using the ³² P-labeled RNA R20 having a concentration of approx. 0.01 pg / ml, and 2 minutes of incubation in the presence of 10 μΜ bacitracin observed in the total degradation of the RNA starting material and 15% degradation of bacitracin 1 pM (FIG. 19).

The effect of selected chemical factors that could potentially alter the efficiency of RNA degradation was also tested (Figures 12, 13). Comparing the RNA-fish HDV reaction with 5 and 25 pM bacitracin, without and after the addition of 5 mM Mg (II) ions, some differences in product degradation were observed, with the overall degree of RNA degradation slightly altered (Fig. 12). The presence of 100 mM NaCl had a similar little effect on degradation. In the reaction with 25 μΜ bacitracin, after 60 minutes, 15% of the original RNA remained instead of approx. 5%.

The reactions with bacitracin were carried out in two different buffers: 50 mM Tris-HCl pH 7.5 and 50 mM HEPES-NaOH pH 7.0, without any significant differences in the course of the reaction with RNA and DNA.

P r z k ł a d 5

The following experiments were performed to reduce the likelihood that the nucleolytic properties of bacitracin, isolated from bacterial cultures, are the result of contamination of the commercial preparation with other nuclease.

The RNA-fish HDV degradation reaction in the presence of 5 and 25 µM bacitracin was carried out with the addition of the protein ribonuclease inhibitor RNasin (Promega), without any significant differences in the effectiveness of the antibiotic (Fig. 12, 13). Pre-incubation of bacitracin at 100 ° C for 5 minutes, before the reaction of 5 µM bacitracin with RNA-fishHDV for 60 minutes, caused only a slight reduction in the ability of the antibiotic to degrade; the amount of non-degraded RNA increased from about 70 to 80%.

Pre-incubation of the bacitracin solution for 5 minutes at 100 ° C, under the conditions in which most protein DNases are deactivated, did not significantly affect the degradation of DNA-M72

Claims (3)

1. The use of bacitracin for the hydrolytic degradation of RNA in vitro. 2. The use according to claim 1 The method of claim 1, wherein the degraded RNA is viral RNA. 3. Use according to claim 1 The method of claim 1 or 2, comprising the preparation of a disinfectant preparation.