WO2013042093A1 - The use of the antibiotic bacitracin in 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 use of the antibiotic bacitracin in the hydrolytic degradation of RNA

The subject of the present invention is a novel use of the antibiotic bacitracin, based on the use of heretofore unknown nucleolytic properties of this drug in the degradation of ribonucleic acids (RNA). Bacitracin is a polypeptide complex of known antibacterial properties, produced by Bacillus subtilis var. Tracy and Bacillus licheniformis.

This drug was discovered in the 1940's and has since been used frequently in antibacterial therapy (Epperson, J., Ming L. Biochemistry39, 4037-4045 (2000); Ming, L., Epperson, J. J. Inoorg. Biochem.91, 46-58 (2002)).

This drug is not absorbed enterically, and is most often used as a component of antibiotic ointments for counteracting skin infections as well as against bacterial eye infections. It is also administered intramuscularly, including infants with bacterial pneumoniae. Moreover, bacitracin is used as a feed additive for preventing infection in animals.

It is thought that like other antibiotics, bacitracin should be administered exclusively in cases of bacterial infections. The probable mechanisms of bacitracin activity is that it inhibits the biosynthesis of cell walls of Gram-positive bacteria (Ming, L., Epperson, J. J. Inoorg. Biochem. 91, 46-58 (2002)).

Bacitracin is a strongly nephrotoxic antibiotic. Bacitracin cofactors include bivalent metals, and activated bacitracin exhibits antibacterial and antifungal properties.

One of the advantages of this antibiotic is that it hardly disperses throughout the organism, and only generates resistant strains to a relatively small degree.

Bacitracin is a drug that has been permitted for marketing for a long time, and thus any questions relating to the side effects connected with its use, such as its nephrotoxicity or allergenicity have been thoroughly studied (Podlewski, J.K., Chwalibogowska-Podlewska A. Leki wspolczesnej terapii - XX edition, Medical Tribune Polska, Warszawa 2010).

It was also reported recently that bacitracin degrades double stranded DNA (Tay, W. et al., J. Am. Chem. Sci. 132, 5652-5661 (2010)). This reaction occurs solely following the binding of some transition metals and occurs via a free radical mechanism. A similar property, the induction of oxidative nucleic acid degradation, is possessed by a series of other antibiotics (Holmes, C. et al., Bioorg. Medicin. Chem. 5, 1235-1248 (1997); Jezowska-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. Da/ton Trans. 8, 1488-1494 (2003b); Wrzesinski, J. et al., Biochem. Biophys. Res. Commun. 331, 267-271 (2005)). It is thought that they may be partially responsible for a number of the side effects of antibiotic therapy. The determination whether these processes are medically significant requires further study, because the concentrations of metal ions which could participate therein are relatively low.

International publication WO 94/04185 describes the use of bacitracin in the inhibition of protein disulphide isomerase (PDI). In the publication by Lara et al. (Lara, H. et al., Virol. 1. 8, 137 (2011)), it was shown that DTNB and bacitracin can inhibit PDI, whereafter viruses, including HIV and RSV, exhibit a limited ability to reduce disulphide bridges in the envelope, which limits their cell penetrance. The direct destruction of viruses by bacitracin was not proven, however. This effect is only indirect. Bacitracin inhibits PDI, thereby disrupting the viral life cycle.

Publications US 4795740 and US 5066783 disclose a pharmaceutical composition effective against HS V, which consists of a mixture of acyclovir and bacitracin (as a protease inhibitor), wherein it was shown in the control sample that bacitracin alone does not exhibit activity against HSV-1.

The state of the art contains no mention of the effect of bacitracin on RNA degradation.

The subject of the present invention is the use of bacitracin in the manufacture of preparations for the degradation of RNA.

According to the present invention it is possible to use the antibiotic bacitracin in a manner different than heretofore, in the degradation of undesirable RNA. This antibiotic could be used against viruses containing RNA as their genetic material. RNA include, amongst others, hepatitis and polio viruses, as well as HIV. Local use could also include use against rapidly multiplying DNA viruses, such as the herpes simplex virus, through attacking viral mRNA.

According to the present invention, bacitracin may be used externally, for topically combating viral skin infections. Another possibility is of using this antibiotic therapy of combined viral and bacterial infections, as well as viral only infections, taking into account its mechanism of activity based on RNA degradation, different than thought heretofore. There is also the possibility of using bacitracin in the manufacture of disinfectants.

This advantage of bacitracin, and thus of its new use is the fact that this compound has been known for many years, and is permitted for use in humans and animals. This is very significant in comparison to newly synthesized therapeutics, whose use as a drug requires long-term studies and gigantic financial outlay.

Antiviral therapies used to date make use of low molecular weight compounds which inhibit enzymes important for the viral life cycle such as RNA polymerase essential for the synthesis of the strands of this acid, the protease involved in the generation of the viral envelope proteins, etc. Practically none of the antibiotics used, effective against bacterial infections, is used in antiviral therapy. Quite the opposite, it is thought that these compounds are ineffective against viral infections.

The ability of bacitracin to degrade RNA molecules was a surprising observation, because a series of antibiotics from a number of therapeutic groups examined previously did not exhibit similar properties. The RNA degradation reaction is of a relatively low specificity and occurs without transition metal ions. It is thus not a process that proceeds along the free radical pathway. It occurs most likely via a hydrolytic mechanism, in a similar fashion to proteinaceous enzymes with a molecular mass several dozen times larger. It is also important that RNA degradation was noted at a very low concentration of the antibiotic, at micromolar concentrations, comparable to the serum concentrations found in patients treated with this drug.

The subject of the present invention in its example embodiments is illustrated in the figure, wherein:

Fig. 1 represents the model RNA and DNA used in the evaluation of the nucleolytic properties of bacitracin: tRNAPhe-RNA, phenylalanine-specific tRNA isolated from yeast; ribHDV-RNA, antigenomic ribozyme HDV; R20-RNA, 20-nucleotide HDV fragment; M39- DNA, 39-nucleotide DNA oligomer; M72-DNA, 72-nucleotide DNA oligomer. The main sites of degradation in the presence of bacitracin are shown;

Fig. 2 represents an autoradiogram of the degradation of tRNAPhe-RNA as a result of varying concentrations of bacitracin (Bac), its complex with Cu(II) ions, as well as complexes in the presence of H₂0₂ (concentrations in uM). Reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37°C; time 60 minutes; total concentration of RNA 2 OD/ml. Kl-control lane; K2- 50 uM Cu(II); K3-100 uM Cu(II)-H₂0₂; L-alkaline hydrolysis; T-digestion with ribonuclease Tl;

Fig. 3 represents the effect of the concentration of bacitracin, its complex with Cu(II) ions as well as the complex in the presence of H₂0₂ on the degree of degradation tRNAPhe-RNA;

Fig. 4 represents an autoradiogram showing the degradation of ribHDV-RNA as an effect of varying concentrations of bacitracin (Bac) as well as its complex with Cu(II) ions (concentrations in μΜ), under the following reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37°C; time 60 minutes; total concentration of RNA 2 OD/ml. Kl-control lane; K2- 50 uM Cu(II); L-alkaline hydrolysis; T-digestion with ribonuclease Tl;

Fig. 5 represents the effect of the concentration of bacitracin as well as its complex with Cu(II) ions on the degree of degradation ribHDV-RNA;

Fig. 6 represents an autoradiogram showing the degradation of R20-RNA as an effect of varying concentrations of bacitracin (Bac) as well as its complex with Cu(II) ions (concentrations in μΜ), under the following reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37°C; time 60 minutes; total concentration of RNA 2 OD/ml. Kl-control lane; K2- 50 uM Cu(II); L-alkaline hydrolysis; T-digestion with ribonuclease Tl;

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

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

Fig. 9 represents the progress of the degradation tRNAPhe-RNA in the presence of bacitracin as well as its complex with Cu(II) ions at a concentration of 22 μΜ depending on the time of incubation; Fig. 10 represents an autoradiogram showing the degradation of R20-RNA in the presence of bacitracin (Bac) as well as its complex with Cu(II) ions (Bac-Cu) at a concentration of 22 μΜ depending on time (in minutes), under the following reaction conditions: 50 mM Tris-HCl buffer pH 7.5; temp. 37°C; time 60 minutes; total concentration of RNA 2 OD/ml. K-control lane; L-alkaline hydrolysis; T-digestion with ribonuclease Tl;

Fig. 11 represents the progress of the degradation R20-RNA in the presence of bacitracin as well as its complex with Cu(II) ions at a concentration of 22 μΜ depending on time of incubation;

Fig. 12 represents an autoradiogram showing the degradation of ribHDV-RNA as an effect bacitracin activity at a concentration of 5 and 25 uM in the presence of selected factors potentially affecting the degradation reaction, under the following reaction conditions: 50 mM HEPES-NaOH buffer pH 7.0; temp. 37°C; time 60 minutes; total concentration of RNA 2 OD/ml, K- control lanes;

Fig. 13 represents the progress of the degradation of ribHDV-RNA as a result of bacitracin at a concentration of 5 and 25 μΜ in the presence of selected factors affecting the degradation;

Fig. 14 represents an autoradiogram showing the degradation of M39-DNA as an effect of varying concentrations of bacitracin (2.5, 25, 250 and 2500 uM) in the presence of selected factors affecting the degradation, under the following reaction conditions: temp. 37°C; time 60 minutes; total concentration of DNA 0.7 OD/ml. K- control lanes;

Fig. 15 represents an autoradiogram showing the degradation of M39-DNA as an effect of varying concentrations of bacitracin (Bac) (concentrations in μΜ) in the presence of selected factors affecting the degradation, under the following reaction conditions: temp. 37°C; time 60 minutes; total concentration of DNA 0.7 OD/ml, K-control lanes;

Fig. 16 represents an autoradiogram representing the degradation of M72-DNA as an effect of varying concentrations of bacitracin (25, 250 and 2500 uM) in the presence of selected factors affecting the degradation, under the following reaction conditions: 50 mM HEPES-NaOH buffer pH 7.0; temp. 37°C; time: 60 minutes; total concentration of DNA 0.2 OD/ml. K- control lane; G+A, C+A-sequence lanes;

Fig. 17 represents an autoradiogram showing the degradation of an R20-RNA molecule incubated in the presence of bacitracin in 50 mM HEPES-NaOH buffer pH 7.0, at a temperature of 37°C for 2 minutes, Panel Bac: 25 and 50 μΜ bacitracin, RNA concentration 8 g/ml, Panel tRNA carrier: 25 uM bacitracin, RNA concentration 8, 0.8, 0.08 and 0.01 g/ml (-). K-control lane; L-alkaline hydrolysis; SI, Tl-digestion with nuclease SI and ribonuclease Tl ;

Fig. 18 shows an autoradiogram showing the degradation products of M39-DNA as an effect of varying concentrations of bacitracin (250 and 2500 μΜ), under the following reaction conditions: 50 mM HEPES-NaOH buffer pH 7.0; temp. 37°C; time 60 minutes; total concentration of DNA 0.2 OD/ml. K-control lane; SI -digestion with nuclease SI, C+A- sequence lane; showing the results of a short and long electrophoresis run;

Fig. 19 represents an autoradiogram showing the R20-RNA degradation products as an effect of varying concentrations of bacitracin (concentrations in μΜ), under the following reaction conditions: RNA concentration 0.01 μ^ιηΐ, 50 mM HEPES-NaOH buffer pH 7.0; temp. 37°C; time 2 minutes, K-control lane; L-alkaline hydrolysis; SI, Tl-digestion with nuclease SI and ribonuclease Tl.

Examples

According to the invention, we performed studies on the nucleolytic properties of bacitracin, whose goal was to obtain answers to the following questions:

1) What is the specificity of bacitracin towards various types of phosphodiester bonds, meaning whether it degrades RNA or DNA molecules, and does it possibly exhibit phosphatase or pyrophosphatase activity,

2) Via which mechanism does the degradation of nucleic acids proceed,

3) Does the degradation of RNA DNA occur preferentially at particular sites of the nucleotide or fragments of the nucleotide sequence, and is the reaction affected by the secondary structure of the molecules,

4) Taking into account the possibility of the practical use of bacitracin, how do the degradation conditions affect its activity, meaning the minimum active concentration of bacitracin, dependence on RNA/DNA concentration and the effect of bivalent and monovalent metal ions,

5) Does the inclusion of ribonuclease inhibitors to a reaction using a commercial bacitracin preparation, or preincubation at a high temperature affect the properties bacitracin .

In these experiments we used a commercial bacitracin preparation from Sigma-Aldrich Co. As the model molecules we used: tRNA specific for phenylalanine isolated from yeast 76 nucleotide long (tRNAPhe-RNA) as well as two HDV RNA fragments, antigenomic ribozyme HDV 72 nucleotides long (ribHDV-RNA) and a 20-nucleotide virus fragment (R20- RNA). The model DNA molecules used were: a 39-nucleotide oligomer DNA (M39-DNA) as well as a 72-nucleotide oligomer DNA (M72-DNA) (Fig. 1).

RNA and DNA molecules were tagged with a ³²P isotope at the 5' end using T4 polynucleotide kinase and [γ- P]ATP according to a standard procedure, bacitracin reaction products were separated electrophoretically in high-resolution polyacrylamide gels, and visualised using imaging screens and a computerised radioactivity scanner: FLA-5100 (Fujifilm), and a quantitative analysis using the Multi Gauge program.

Example 1

We conducted a reaction between bacitracin and tRNAPhe-RNA at a concentration of 2 OD/ml, under the following conditions: a 60 minute incubation at a temperature of 37°C in the presence of 5 uM of the antibiotic.

As a result of the reaction about 40% of the initial RNA was degraded (Fig. 2, 3). An increase in the concentration of bacitracin caused an increase in the degree of degradation, and at 100 uM it reached about 95%.

The degradation of ribHDV-RNA was much more effective than of tRNAPhe-RNA (Fig. 4, 5). In the presence of 5 uM bacitracin 60% of the initial RNA was degraded. At 10 μΜ bacitracin, the degree of degradation reached 80%, and an almost complete degradation of ribHDV-RNA was observed at 50 uM bacitracin. The 20-nucleotide oligomer R20-RNA following 60 minutes of incubation in the presence of 20 uM bacitracin underwent almost complete degradation to shorter products (Fig. 6, 7).

A comparison of the effectiveness of the degradation of three different RNA molecules used at concentrations of 2 OD/ml showed that following 60 minutes of incubation at a temperature of 37°C in the presence of 20 μΜ bacitracin, 80-95% of the initial RNA underwent degradation. The kinetics of the degradation of tRNAPhe-RNA as well as of R20-RNA in the presence of 22 uM bacitracin have been examined (Fig. 8-11). For both RNA molecules, about 50% degradation was observed already following 1 minute of incubation. Following 20 minutes the degradation reaction of tRNAPhe-RNA reached a plateau at 90% (Fig. 9), whereas the degradation of R20-RNA following this time was almost complete (Fig. 11).

In order to demonstrate whether bacitracin exhibits phosphatase or pyrophosphatase activity, P nucleoside triphosphates: [γ- P]ATP as well as [a- P]UTP, which were incubated with 25 and 250 μΜ bacitracin for 60 minutes at a temperature of 37°C, including in the presence of Mg(II) and Mn(II) ions and EDTA, were used. No degradation products of any of these compounds following polyacrylamide gel electrophoresis have been observed.

It has been proved that bacitracin possesses a the ability to degrade single stranded DNA to some degree. However, the degradation of M39-DNA (Fig. 14) and M72-DNA (Fig. 16) required much higher antibiotic concentrations than for RNA. A comparable effect was achieved using an at least 10-fold concentration of bacitracin. M39-DNA and M72-DNA degradation was observed using 250 uM bacitracin, but not at 25 μΜ antibiotic concentration.

Example 2

In the hydrolytic cleavage mechanism of phosphodiester bonds, it is important to note whether the phosphate group at the chain cleavage site remains at the 3' or 5' end of the ribose residue. We thus decided to determine the site of localization of the phosphate group in the RNA and DNA fragments following the degradation reaction in the presence of bacitracin .

The migration of the degradation products of R20-RNA with the migration of alkaline hydrolysis products as well as limited digestion products with nuclease S 1 and ribonuclease Tl using high resolution PAGE has been compared (Fig. 19). It is known that RNA alkaline hydrolysis products as well as ribonuclease Tl digestion products possess a phosphate group at the 3' end of the RNA chain, whereas nuclease SI digestion products possess it at the 5' terminus. The migration of RNA fragments following degradation with bacitracin is identical with the migration of RNA fragments following alkaline hydrolysis as well as RNase Tl digestion, and different than following digestion with the SI nuclease. This is convincing evidence that phosphate groups are found at the 3' end of the arising fragments.

An analogous localization of the phosphate group was performed for the reaction products of M39-DNA with bacitracin (Fig. 18). This result shows that the product migration corresponds to the migration following digestion with nuclease S 1. Thus, in the case of DNA degradation, the phosphate groups are found at the 5' end of the arising fragments.

From the point of view of the RNA and DNA degradation mechanisms by bacitracin, it is significant whether the reaction requires bivalent metal ions. It turned out that the degradation of ribHDV-RNA is not affected by the presence of 2 mM EDTA (Fig. 12, 13). A similar observation was made for tRNAPhe-RNA. It is striking, however, that the degradation of DNA is completely inhibited by EDTA, both in the case of M39-DNA (Fig. 14; we used 0.2 mM EDTA) and M72-DNA (Fig. 16; we used 2 mM EDTA). Thus, the degradation of DNA requires the absolute presence of bivalent metal ions. It was determined that these may be, amongst others, Mg(II), Mn(II) or Zn(II) (Fig. 14-16).

Example 3

According to the above description, an RNA degradation reaction has been conducted. It has been determined that bacitracin degrades RNA beside guanosine residues (Fig. 2,4,6). The degradation occurs in single-stranded RNA regions, for tRNAPhe-RNA primarily in the D loop (G15, G18, G19) and at T C (G57), for ribHDV-RNA in the looped regions L3/P3 (G28, G30, G31) and L4 (G58), and in the single stranded R20-RNA at the G12, G13, G14, G17 and G18 residues (Fig. 2,4,6). It is interesting that a similar specific degradation of RNA is exhibited by the Tl ribonuclease. Furthermore, it has been observed the removal by bacitracin of the phosphate group from the 5' end (or from the 5'-terminal nucleotide) of RNA molecules. This effect was observed both for tRNAPhe-RNA as well as ribHDV-RNA (Fig. 2, 4). We did not observe it in the case of R20-RNA (Fig. 6). This may stem from the fact that in the first two model RNAs, the 5'-terminal nucleoside is guanosine, in the third it is cytidine.

It turned out that the specific degradation of DNA by bacitracin is significantly different than the RNA degradation. The degradation sites in M72-DNA as well as M39-DNA are very infrequent, and preferentially occur in the sequence near some cytidine residues (Fig. 16,18). In conjunction with the information that the degradation of DNA requires the presence of some bivalent metal ions, it may be suggested that the sites of degradation of DNA are localised near the sites of binding of these ions.

Example 4

An RNA degradation reaction to evaluate the dependence of this process on the RNA concentration has been conducted. The observed degradation level of RNA was dependent on its total concentration. The degradation effectiveness of three different RNA molecules, used at concentrations of 2 OD/ml, i.e. 80 μg/ml, incubated with 20 μΜ bacitracin for 60 minutes at a temperature of 37°C was 80-95% of the initial amount of RNA. Decreasing the concentration of R20-RNA to 8, 0.8, 0.08 and about 0.01 μg/ml, using 25 uM bacitracin and a 2 minute incubation led to an increased degradation effect (Fig. 17). In the case of isotope ³²P tagged R20-RNA at a concentration of about 0.01 μg/ml and 2 minutes of incubation, in the presence of 10 μΜ bacitracin, we observed a complete degradation of the initial RNA, and a 15% degradation with 1 μΜ bacitracin (Fig. 19).

We also tested the effect of selected chemical factors which may potentially alter the effectiveness of RNA degradation (Fig. 12,13). Comparing the reaction of ribHDV-RNA with 5 and 25 μΜ bacitracin, with and without an addition of 5 mM Mg(II) ions, we observed some differences in the distribution of the products, with little changes in the total level of RNA degradation (Fig. 12). Likewise, the degradation was little affected by the presence of 100 mM NaCl. In a reaction with 25 μΜ bacitracin, following 60 minutes, instead of about 5%, about 15% of the initial RNA underwent degradation.

The bacitracin reactions were performed in two different buffers, 50 mM Tris-HCl pH 7.5 and 50 mM HEPES-NaOH pH 7.0, without any observed significant differences in the RNA and DNA reactions.

Example 5

The experiment below was performed to decrease the probability that the nucleolytic properties of bacitracin, isolated from bacterial cultures are the result of the contamination of the commercial preparation with other nucleases.

The ribF£DV-RNA degradation reaction in the presence of 5 and 25 μΜ bacitracin was performed with the addition of protein ribonuclease inhibitor, RNasin (Promega), without noting any significant differences in the effectiveness of the antibiotic (Fig. 12,13). Preincubation with bacitracin at a temperature of 100°C for 5 minutes, prior to the reaction with 5 uM bacitracin and ribHDV-RNA for 60 minutes, caused only a slight inhibition of the antibiotic's ability to degrade the RNA. The amount of undegraded RNA increased to about 70 to 80%.

Preincubation of the bacitracin solution for 5 minutes at a temperature of 100°C, conditions under which most proteinaceous DNases are deactivated, did not significantly alter the degradation of M72-DNA (Fig. 16).

Patent documents

4795740 US 1/1989 Cohen et al.

5066783 US 1 1/1991 Cohen et al.

WO 94/04185 3/1994 Ryser et. al.

Non-patent documents

Epperson, J., Ming L. Biochemistry 39, 4037-4045 (2000). Holmes, C. et al., Bioorg. Medicin. Chem. 5, 1235-1248 (1997). Jezowska-Bojczuk, M. et al, Eur. J. Biochem. 269, 5547-5556 (2002). Lara, H. et al., Virol. J. 8, 137 (201 1). Ming, L., Epperson, J. J. Inoorg. Biochem. 91, 46-58 (2002).

Podlewski, J.K., Chwalibogowska-Podlewska A. Leki wspolczesnej terapii - wydanie XX, Medical Tribune Polska Warszawa 2010.

Szczepanik, W. et al., J. Inorg. Biochem. 94, 355-364 (2003a).

Szczepanik, W. et al., J. Chem. Soc. Dalton Trans. 8, 1488-1494 (2003b).

Tay, W. et al., J. Am. Chem. Sci. 132, 5652-5661 (2010).

Wrzesmski, J. et al., Biochem. Biophys. Res. Commun. 331, 267-271 (2005).

Claims

Claims

1. The use of bacitracin in the manufacture of a preparation for the degradation of RNA.

2. The use according to Claim 1, characterised in that the degraded RNA is viral RNA.