Antimicrobial agents and their use in therapy

Abstract

The present invention provides an agent, or a composition containing an agent, for use in treating or preventing a bacterial infection in a subject, wherein said agent comprises: (i) an oligopeptidic compound comprising a PCNA interacting motif and a domain that facilitates the cellular uptake of said compound,
wherein the PCNA interacting motif is X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO: 1) and
wherein: X.sub.1 is a basic amino acid; X.sub.2 is an aromatic amino acid; X.sub.3 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P); X.sub.4 is any amino acid other than Proline (P), an acidic amino acid or an aromatic amino acid; and X.sub.5 is a basic amino acid or Proline (P); or (ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i). In certain aspects the agent and compositions of the invention may be used as single agents. In other aspects of the invention agents and compositions of the invention may be used in conjunction with one or more additional active agents, such as antibiotics.

Claims

1. A method of treating a bacterial infection, said method comprising administering an agent, or a composition containing an agent, to a subject in need thereof, wherein said agent comprises: (i) an oligopeptidic compound comprising a PCNA interacting motif and a cell penetrating peptide (CPP), wherein the PCNA interacting motif is X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5 (SEQ ID NO: 1) and wherein: X.sub.1 is a basic amino acid; X.sub.2 is an aromatic amino acid; X.sub.3 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P); X.sub.4 is any amino acid other than Proline (P), an acidic amino acid, a basic amino acid or an aromatic amino acid; and X.sub.5 is a basic amino acid or Proline (P); or (ii) a nucleic acid molecule comprising a sequence encoding the oligopeptidic compound of (i), wherein the bacterial infection is caused by a bacterium of a genus selected from the list consisting of Acinetobacter, Enterococcus, Escherichia, Micrococcus, Pseudomonas and Staphylococcus.

2. The method of claim 1, wherein said agent or composition is provided as a combined preparation with one or more additional active agents for separate, simultaneous or sequential use or administration.

3. The method of claim 1, wherein said method further comprises a step of UV radiotherapy, which may be administered simultaneously, sequentially or separately to said agent or composition.

4. The method of claim 1, wherein said bacterial infection comprises a biofilm.

5. The method of claim 1, wherein the bacterium is a multi-drug resistant (MDR) bacterium.

6. The method of claim 5, wherein the MDR bacterium is a Methicillin-resistant Staphylococcus aureus (MRSA) bacterium or an Enterococcus faecium bacterium.

7. The method of claim 1, wherein the subject has bacterial infectious disease selected from bacterial pneumonia; and sepsis.

8. The method of claim 1, wherein the subject has cystic fibrosis.

9. The method of claim 1, wherein the infection is a respiratory infection, stomach infection, gastrointestinal tract (GIT) infection, blood infection, skin infection, bladder infection, kidney infection, ear infection or eye infection.

10. The method of claim 2, wherein said one or more additional active agents is an antibiotic.

11. The method of claim 10, wherein said antibiotic agent is selected from one or more of a Macrolide, an Aminocoumarin, an Aminoglycosid, an Ansamycin, a Carbapenem, a Cephalosporin, a Glycopeptide, a Lincosamide, a Lipopeptide, a Monobactam, a Nitrofuran, an Oxazolidonone, a Penicillin, a Penicillin combination, a Polyether antibiotic, a Polypeptide antibiotic, a Quinolone, a sulfonamide, a Tetracycline or another antibiotic.

12. The method of claim 1, wherein the PCNA interacting motif comprises the sequence selected from: TABLE-US-00031 (SEQIDNO:7) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/ T]-[K/R/H]; (SEQIDNO:8) [R/K]-[W/F/Y]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/T]- [K/R]; (SEQIDNO:9) [R/K]-[W/F/]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G/S/T]- [K/R]; (SEQIDNO:10) [R/K]-[W/F]-[L/I/V/A/M/T]-[V/L/I/A/M/G/S/T]- [K/R]; (SEQIDNO:11) [R/K]-[W/F]-[L/I/V/A/M/T]-[V/L/I/A/M/S/T]-[K/R]; (SEQIDNO:12) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[V/L/I/A/M/G]-[K/R]; (SEQIDNO:13) [R/K]-[W/F]-[L/I/A/V/M/T]-[V/L/I/A/M/S/T]-[K/R]; (SEQIDNO:14) [R/K]-[W/F]-[L/I/V/A/M/S/T]-[V/L/A/I/S/T]-[K/R]; (SEQIDNO:15) [R/K]-[W/F]-[L/V/I/A/T]-[V/L/A/I/S/T]-[K/R]; (SEQIDNO:16) [R]-[W/F/Y]-[L/V/I/A]-[V/L/A/S/T/M]-[K/R]; (SEQIDNO:17) [R]-[W/F/Y]-[L/V/I/A/T]-[V/L/A/S/T/M]-[K]; (SEQIDNO:18) [R/K]-[F/Y]-[L/V/I/A]-[V/L/A/I/M]-[K/R]; (SEQIDNO:19) [R/K]-[W/F/Y]-[L/I/V/A]-[V/L/I/A]-[K/R]; (SEQIDNO:20) [R/K]-[W/Y]-[L/V/I/A/S/T]-[V/L/A/S/T/M]-[K/R]; or (SEQIDNO:21) [K]-[F/Y/W]-[I/L/V/A/T]-[V/L/A/I/S/T/M]-[K].

13. The method of claim 1, wherein the PCNA interacting motif comprises the sequence selected from any one of SEQ ID NOs: 28, 22 to 27, 29 to 93, 106 to 177, 190 to 297, 1206 or 1207.

14. The method of claim 1, wherein the CPP is selected from any one of: (i) an antennapedia class peptide; (ii) a protegrin class peptide; (iii) a HIV-TAT class peptide; (iv) an amphipathic class peptide selected from an amphipathic and net positively charged peptide, a proline-rich amphipathic peptide, a peptide based on a Pep-1 peptide (SEQ ID NO: 326) and a peptide based on a MPG peptide (SEQ ID NO: 328); (v) a peptide exhibiting high -helical content; (vi) a peptide comprising oligomers of basic amino acids; (vii) pVEC; (viii) a calcitonin-derived peptide; and (ix) an amphiphilic cyclic CPP.

15. The method of claim 1, wherein the CPP is selected from any one of SEQ ID NOs: 337, 302 to 336, 338 to 1162, or 1213 to 1223 or a fragment and/or derivative thereof.

16. The method of claim 1, wherein the agent further comprises a linker domain.

17. The method of claim 1, wherein the agent comprises the sequence as set forth in any one of SEQ ID NOs: 1198, 1182 to 1197, 1199 to 1204 or 1208 to 1212.

18. The method of claim 1, wherein the agent comprises the sequence as set forth in SEQ ID NO: 1198, 1203, 1204 or 1208 to 1212.

Description

(1) The invention will now be further described with reference to the following non-limiting Examples and Figures in which:

(2) FIG. 1 shows graphs that demonstrate that ATX-101 (SEQ ID NO:1198, which contains the APIM motif, RWLVK (SEQ ID NO: 28)) increases the sensitivity of yeast (Saccharomyces cerevisiae) to a cytostatic agent (cisplatin), wherein: (A) shows the growth of yeast in the presence of various concentrations of cisplatin; (B) shows the growth of yeast in the presence of various concentrations of ATX-101 (APIM); (C) shows the growth of yeast in the presence of various concentrations of ATX-101 in combination with 500 M cisplatin; and (D) shows the growth of yeast in the presence of various concentrations of ATX-101 in combination with 125 M cisplatin.

(3) FIG. 2 shows graphs that demonstrate that Hog1 mutant yeast cells have increased sensitivity to ATX-101 in combination with cisplatin relative to wild-type yeast cells, wherein: (A) shows the growth of wild-type yeast in the presence of various concentrations of ATX-101 in combination with 125 M cisplatin; and (B) shows the growth of Hog1 yeast in the presence of various concentrations of ATX-101 in combination with 125 M cisplatin.

(4) FIG. 3 shows a laser scanning microscope image of yeast cells incubated with ATX-101 labelled with FAM.

(5) FIG. 4 shows a graph that demonstrates that the growth of gram-negative bacteria, Pseudomonas aeruginosa ATCC 15692, Acinetobacter baumanni ATCC 19606, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa TO-5A (clinical isolate) is inhibited by varying concentrations of APIM peptide, ATX-101. Growth of the strains is given relative to growth in cultures without ATX-101.

(6) FIG. 5 shows a graph that demonstrates that the growth of gram-positive bacteria, Enterococcus faecium CCUG 37832, Enterococcus faecium CTC 492 and Micrococcus luteus ATCC 9341, is inhibited by varying concentrations of APIM peptide, ATX-101.

(7) FIG. 6 shows a graph that demonstrates that the growth of the gram-positive bacterium Enterococcus faecium CTC 492 is more sensitive to: (A) Ampicillin; and (B) Novobiocin, (both cytotoxic agents, i.e. antibiotics) when grown in the presence of ATX-101.

(8) FIG. 7 shows a graph that demonstrates that the growth of the gram-negative bacterium Escherichia coli NCIMB 12210 is more sensitive to Novobiocin (a cytotoxic agent, i.e. an antibiotic) when grown in the presence of ATX-101.

(9) FIG. 8 shows a graph showing the results of FRET analysis. Normalised FRET (N.sub.FRET) measurements are shown between EYFP (yellow fluorescent protein)/ECFP (cyan fluorescent protein) (Lane 1, background due to dimerisation of the tags). EYFP-APIM motif/ECFP-PCNA for various motifs are shown in the other lanes.

(10) FIG. 9 shows a graph that demonstrates that the growth of the MDR bacterium Methicillin-resistant Staphylococcus aureus (MRSA 1040) is more sensitive to macrolide antibiotics, azithromycin and erythromycin, when grown in the presence of ATX-101.

(11) FIG. 10 shows a graph that demonstrates that various APIM peptide variants are capable of reducing the MIC of erythromycin required to inhibit the growth of the MDR bacterium Methicillin-resistant Staphylococcus aureus (MRSA 1040), wherein: ATX-101-STD is SEQ ID NO: 1198; ATX-101-Sv is SEQ ID NO: 1203; ATX-101-Av is SEQ ID NO: 1204; ATX-101-Ls is SEQ ID NO: 1211; ATX-101-P is SEQ ID NO: 1212; and ATX-101-LT is SEQ ID NO: 1208.

(12) FIG. 11 shows graphs that demonstrate that the growth of the MDR bacterium E. faecium CCUG 37832 (TO-3) is more sensitive to various DNA gyrase inhibitors, when grown in the presence of ATX-101.

(13) FIG. 12 shows (A) a graph showing the growth of E. coli in which the overexpression of various proteins or peptides is induced or not induced and demonstrates that the induction of an APIM peptide (SEQ ID NO: 28), alone or as part of a fusion protein with EYFP, inhibits bacterial growth. (B) shows a Western blot showing levels of EYFP-proteins in un-induced (left) versus induced (right) cell cultures.

(14) FIG. 13 shows graphs showing the growth of E. coli in which the overexpression of various APIM peptides is induced (I) or not induced (NI). The APIM sequences are: (A) SEQ ID NO: 28; (B) SEQ ID NOs: 1206, 1207 and 76; (C) SEQ ID NOs: 41, 40 and 52; (D) SEQ ID NO: 58.

(15) FIG. 14 shows graphs showing the growth of E. coli in the presence of 30 M of various APIM peptides. The same data is presented in (A) and (B), however the control growth curves are not shown in (B). ATX-101 is SEQ ID NO: 1198; ATX-101-P is SEQ ID NO: 1212; FSL is SEQ ID NO: 1206; FLS is SEQ ID NO: 1210; SV is SEQ ID NO: 1203; AV is SEQ ID NO: 1204; LT is SEQ ID NO: 1208; LS is SEQ ID NO: 1211; R11 is SEQ ID NO: 337; Neg contr is no peptide added.

(16) FIG. 15 shows graphs showing the growth of E. coli in the presence of 30 M of various APIM peptides after the bacteria have been irradiated with UVC. The same data is presented in (A) and (B), however the control growth curves are not shown in (B). ATX-101 is SEQ ID NO: 1198; ATX-101-P is SEQ ID NO: 1212; FSL is SEQ ID NO: 1206; FLS is SEQ ID NO: 1210; SV is SEQ ID NO: 1203; AV is SEQ ID NO: 1204; LT is SEQ ID NO: 1208; LS is SEQ ID NO: 1211; R11 is SEQ ID NO: 337; Neg contr is no peptide added.

(17) FIG. 16 shows graphs showing the growth of E. coli in the presence of ATX-101 (SEQ ID NO: 1198) at various concentrations, without UVC irradiation (left panels) or with UVC irradiation (right panels). R11 is SEQ ID NO: 337 and Neg contr is no peptide added. (A) 15 M, (B) 7.5 M and (C) 3.75 M.

(18) FIG. 17 shows micrographs of biofilm formation of the MDR bacterium Methicillin-resistant Staphylococcus aureus (MRSA 1040) in the presence of 7 g/ml ATX-101 (SEQ ID NO: 1198) (left panels) or no peptide (right panels) over a period of 36 hours and demonstrates that biofilm formation is inhibited by ATX-101.

(19) FIG. 18 shows micrographs of biofilm formation of the MDR bacterium Methicillin-resistant Staphylococcus aureus (MRSA 1040) in the presence of 0.8 g/ml ATX-101 (SEQ ID NO: 1198) (left panels) or no peptide (right panels) over a period of 36 hours and demonstrates that biofilm formation is inhibited by ATX-101.

EXAMPLES

(20) The inventors have surprisingly found that an oligopeptidic compound comprising an APIM motif and a cell-penetrating peptide is imported into microbial cells and is capable of inhibiting the growth of said cells. It is thought that oligopeptidic compounds comprising an APIM motif may compete with PCNA-like proteins in microorganisms for proteins that interact with said PCNA-like proteins, thereby inhibiting various cellular processes, e.g. DNA synthesis, signal transduction etc. The effects of oligopeptidic compounds comprising an APIM motif on microbial cell have been established in both bacteria and fungi using an exemplary cell-penetrating APIM-containing peptide ATX-101 (SEQ ID NO:1198, which contains the APIM motif, RWLVK (SEQ ID NO: 28)).

(21) The data presented below suggest that oligopeptidic compounds comprising an APIM motif are useful as antimicrobial agents, e.g. antibiotics and antimycotics, either alone or in combination with other cytostatic or cytotoxic agents. Accordingly, the data supports the use of oligopeptidic compounds comprising an APIM motif in the treatment or prevention of microbial infections or microbial infectious diseases.

Example 1: Determining the Effect of ATX-101 on Yeast (Saccharomyces cerevisiae

(22) The homozygote diploid mutant library of yeast strains was purchased from EUROSCARF, Institute of Microbiology, University of Frankfurt. Growth studies on wild-type (wt) yeast and the Hog1 mutant were performed. Microplates (Greiner 655163) containing 120 l 2MES and 1.5 N-base medium per well were inoculated with 20 l (per well) of each yeast strain (from frozen stock cultures) and a reference strain (wild type BY4743). The microplate cultures were grown over night (ON) (30 C., 900 rpm at 3 mm amplitude, 85% humidity), ensuring growth well into the stationary growth phase in order to reduce variation caused by differences in growth rates. At day 2, 10 l was transferred from the ON-cultures to 200 l 2MES 1.5 N-base per well in 96-well microplates, mixed (900 rpm at 3 mm amplitude, 30 sec) and grown to OD 0.15-0.2 before addition of different doses of cisplatin and/or ATX-101. The OD (600 nm) in each well was read every 20 minutes using an integrated Beckman Coulter Paradigm microplate reader. The growth of the yeast strains in the microplates was monitored for approximately 25 hours.

(23) FIG. 1 shows that cisplatin at a concentration of 125 M is not sufficient to retard growth of the yeast cells (FIG. 1A). A cisplatin concentration of 500 M is required to retard growth and even at 2000 M, growth is not completely inhibited. Similarly, ATX-101 (labeled as APIM) at a concentration of 10 M does not retard growth and a concentration of 20 M or more is necessary to retard growth of the yeast cells (FIG. 1B). However, the combination of cisplatin at 500 M and ATX-101 at 20 M is capable of inhibiting growth of the yeast cells after approximately 7 hours (FIG. 10). Thus, ATX-101 (an APIM peptide) and cisplatin (a cytostatic agent) have a synergistic effect on yeast, when combined. In fact, the effect is seen when cisplatin is used at a concentration that, alone, has no effect on yeast growth, 125 M (FIG. 1D). It is also evident from this experiment that high concentrations of ATX-101 alone will be capable to inhibiting growth of yeast cells.

(24) FIG. 2 shows that Hog1 yeast mutants (FIG. 2B) show increased sensitivity to ATX-101 in combination with cisplatin. A combination of 10 M ATX-101 and 125 M cisplatin is sufficient to retard growth and higher concentrations of ATX-101 result in complete inhibition of cell growth. Hog1 is a protein kinase in the a mitogen activated protein (MAP) kinase pathway in yeast, which is required for adaptation to osmotic stress. Thus, these data suggest that ATX-101 may be acting by interfering with intracellular signalling in addition to DNA synthesis and repair mechanisms.

(25) Based on these results, APIM peptides are expected to be useful as antimycotic agents for all fungi.

Example 2: Cellular Import of ATX-101

(26) Yeast cells (Saccharomyces cerevisiae) were grown in LB media. A fluorescently labelled APIM peptide (ATX-101-FAM) was added to the media and incubated for 1-2 minutes before an aliquot of cells was removed and analyzed on a Zeiss LSM 510 Meta laser scanning microscope equipped with a Plan-Apochromate 63/1.4 oil immersion objective.

(27) FIG. 3 shows that ATX-101 is imported into yeast cells, thereby demonstrating that it must be acting intracellularly, i.e. unlike many antimicrobial compounds ATX-101 is not antimicrobial due to its effects on cell walls or membranes, i.e. it does not function by permeabilizing cells.

Example 3: Determining the Effect of ATX-101 on Bacteria

(28) The minimum inhibitory concentration (MIC) of ATX-101 was determined for various gram negative and gram positive bacteria. The bacteria, Pseudomonas aeruginosa ATCC 15692, Acinetobacter baumanni ATCC 19606, Escherichia coli ATCC 25922, Pseudomonas aeruginosa TO-5A (clinical isolate), Enterococcus faecium CCUG 37832, Enterococcus faecium CTC 492, Micrococcus luteus ATCC 9341 and Escherichia coli NCIMB 12210 were grown in the presence of various concentrations of ATX-101 and/or antibiotic.

(29) Robotic MIC Assay:

(30) ATX-101 was dissolved in Mueller-Hinton broth to 1.25 times of the desired assay concentration. Antibiotics were dissolved in Mueller-Hinton broth and Mueller-Hinton broth with ATX-101 at a concentration of 1.25 times the highest desired assay concentrations. Antibiotics were pharmaceutical grade purchased from Sigma-Aldrich.

(31) Two-fold serial dilutions of antibiotics were made in Mueller-Hinton with different concentrations of ATX-101, and the solutions were placed in four parallel wells in Nunc 384-well micro plates (30 l per well in Nunc 242757 microplates). A group of 8 wells with no addition of antibiotics for each ATX-101 concentration was included on each micro plate as a growth reference.

(32) At the day of analysis, overnight TSB cultures inoculated from freeze stocks (6 ml in 50 ml tube tilted to 45-degrees angle, 200 rpm, 2.5 cm amplitude, 37 C.) were diluted in TSB until the OD600 was 0.10, and further diluted 1:40 in Mueller-Hinton broth. Each well in the 384-well assay plates was inoculated with 7.5 l of the diluted culture, giving the same dilution of the culture in the assay cultures. The microplates were placed in plastic bags and incubated without shaking at 37 C. The optical density at 600 nm in the microwells was measured after approximately 19 hours of incubation, and the relative growth yield in each well was calculated based on the growth in the reference groups. The MIC value was set to the highest concentration giving less than 30% growth in all 4 parallel wells within the sample groups. The microplates were further incubated for 6 hours, and optical density in the cultures was measured once more for confirmation of the estimated MIC-values.

(33) FIG. 4 shows that the growth of various gram negative bacteria is inhibited by ATX-101 from about 1 M. FIG. 5 shows that the growth of various gram positive bacteria is inhibited by ATX-101 from about 1 M. The MIC for the gram negative bacteria tested to date was determined to be in the range of 5-10 g/ml. The MIC for gram positive bacteria tested to date was determined to be in the range of 1 g/ml. These data suggest that, in general, gram positive bacteria are more sensitive to APIM peptides than gram negative bacteria. However, based on these results, APIM peptides are expected to be useful as antibiotic agents for all bacteria.

(34) FIG. 6 shows that Enterococcus faecium CTC 492 shows increased sensitivity to ampicillin and novobiocin in combination with ATX-101. A combination of 6.5 g/ml or 13 g/ml ATX-101 is sufficient to inhibit bacterial growth at a concentration of about 0.5 g/ml ampicillin, whereas a concentration of 1 g/ml ampicillin without ATX-101 was required to achieve the same level of inhibition. Similarly, 6.5 g/ml or 13 g/ml ATX-101 enhances the inhibition of bacterial growth at a concentration of about 0.5-2 g/ml novobiocin. FIG. 7 shows that that ATX-101 is capable of reducing the MIC for novobiocin by 16-fold. Thus, these data suggest that ATX-101 has an additive or synergistic inhibitory effect on bacterial growth in combination with an antibiotic. FIG. 6 also demonstrates that higher concentrations of ATX-101, e.g. 20 g/ml, are sufficient to kill bacteria, as no growth was observed at this concentration, with or without ampicillin or novobiocin.

(35) Based on these results, APIM peptides are expected to be useful in combination with other antibiotic agents, i.e. may enable known antibiotics to be used effectively at lower concentrations.

Example 4: Determining the Effect of Other APIM Peptides on Bacteria

(36) The inventors have shown that other APIM peptides, i.e. comprising PCNA binding motifs that are different to the motif in ATX-101, are also effective antibiotics. In this respect, peptides comprising the sequences: MDRWSVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1203) and MDRWAVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1204), i.e. wherein X.sub.3 and X.sub.4 are SV and AV, respectively, are particularly effective and show lower MICs than ATX-101. These data indicate that the antimicrobial effect arises from the PCNA interacting motif, because the other domains in these peptides are identical.

Example 5: In Silico Characterisation of APIM Consensus Motif

(37) The inventors have performed sequence analyses to determine how much variation within the APIM motif occurs naturally, i.e. in native sequences across a number of species. As PCNA is highly conserved across eukaryotic organisms, it is expected that sequence variation of the APIM motif in orthologues of polypeptides that are thought to interact with PCNA is representative of the variation that may be used in the oligopeptidic compounds of the invention, i.e. variation of amino acids within the APIM motif at some positions, particularly X.sub.3 and X.sub.4, may be permitted without losing affinity to PCNA.

(38) The inventors used identified 657 human polypeptide sequences that comprise the motif [K/R]-[F/W/Y]-[A/L/V/I]-[A/L/V/I]-[K/R] (SEQ ID NO:19) from a possible 21,673 polypeptide sequences. Of the 657 sequences identified, 291 were excluded because insignificant information about the function of the polypeptides was available. The remaining 366 were considered to be polypeptides that are likely to interact with PCNA and these sequences were used to identify orthologues in: Bos taurus (288 orthologues); Rattus norvegicus (286 orthologues); Mus musculus (312 orthologues); Gallus gallus (236 orthologues); Xenopus tropicalis (200 orthologues); Danio rerio (189 orthologues); Caenrhabditis elegans (102 orthologues); Drosophila melanogaster (136 orthologues); and Saccharomyces cerevisiae (65 orthologues). Alignment of the domains of the orthologues that comprise the APIM motif suggested that the motif may defined as:

(39) [R/K/H]-[W/F/Y]-[L/I/V/A/M/S/T/N/Q/C]-[L/I/V/A/M/G/S/T/N/Q/R/H/K/C]-[K/R/H/P] (SEQ ID NO: 2), wherein specific combinations of amino acids at positions 3 and 4 that were identified in the orthologues include:

(40) LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS, LT, IS, MV, TV, AA, IM, LN, LQ, VM, TL, SL, IT, VT, LG, MA, ML, NL, QL, QI, TI, SI, AS, VS, SV, CA, IG, LR, VR, TK and IR. Particularly common combinations are LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA, AI, AM, LM, LS and LT, the most common being LL, LA, LV, AL, VL, VI, LI, IL, VV, VA, IV, II, AV, IA and AI.

(41) Thus, the broadest definition of the APIM motif was derived from this analysis, and all polypeptides comprising an APIM motif according to this definition could reasonably be expected to interact with, i.e. bind to, PCNA.

Example 6: In Vivo Characterisation of APIM Consensus Motif

(42) This work described in this Example investigates interaction between APIM peptides and PCNA.

(43) In living S-phase cells, PCNA tagged with green fluorescent protein (EGFP) forms distinct foci representing sites of replication and thus can be used as a S-phase marker.

(44) PCNA tagged with cyan fluorescent protein (ECFP) was co-expressed with various APIM peptide constructs fused with yellow fluorescent protein (EYFP). To examine the degree of proximity of APIM peptides and PCNA, fluorescence resonance energy transfer (FRET) was measured.

(45) Live HeLa cells were examined 16-24 hours after transient transfection (by Fugene 6 (Roche Inc.) according to the manufacturer's recommendations) of ECFP and EYFP fusion constructs. Fluorescent images were acquired using a Zeiss LSM 510 Meta laser scanning microscope equipped with a Plan-Apochromate 63/1.4 oil immersion objective. Enhanced cyan fluorescent protein (ECFP) was excited at =458 nm and detected at =470-500 nm and enhanced yellow fluorescent protein (EYFP) was excited at =514 nm and detected at =530-600 nm, using consecutive scans. The thickness of the slice was 1 m.

(46) Fluorescent resonance energy transfer (FRET) occurs if the tags (EYFP and ECFP) are less than 100 (10 nm) apart. We detected FRET using the sensitised emission method, measuring acceptor (EYFP) emission upon donor (ECFP) excitation. We had FRET when the intensity of emitted light from EYFP after excitation of the ECFP fluorochrome was stronger than the light emitted by ECFP or EYFP-tagged proteins alone, after excitation with the EYFP and ECFP lasers respectively (bleed through), given by the equation: FRET=I.sub.2I.sub.1 (I.sub.D2/I.sub.D1)I.sub.3 (I.sub.A2/I.sub.A3) is >0. FRET was normalised for expression levels using the equation: N.sub.FRET=FRET/(I.sub.1I.sub.3).sup.1/2. N.sub.FRET was calculated from mean intensities (I) within a region of interest (ROI) containing more than 25 pixels where all pixels had intensities below 250 and the average intensities were between 100 and 200 for both the donor and the acceptor constructs. Channel 1 (ECFP) and 3 (EYFP) were measured as described above for imaging, and channel 2 (FRET) was excited with =458 nm and detected at =530-600 nm. I.sub.D1, D2, D3 and I.sub.A1, A2, A3 were determined for cells transfected with ECFP and EYFP constructs only, with same settings and same fluorescence intensities as co-transfected cells (I.sub.1 and I.sub.3). ECFP-PCNA and EYFP-PCNA were included as positive controls, and due to dimerisation of co-expressed tags, ECFP and EYFP proteins expressed from empty vectors were included as negative controls in all experiments.

(47) FIG. 8 shows that a significant FRET signal could be detected for all of the variants tested, which verifies that a variety of peptides within the APIM motif definition described herein (and that occur in polypeptides that are expected to interact with PCNA) are capable of interacting with PCNA and would therefore be expected to find utility in the method and uses described herein, i.e. as anti-microbial peptides.

Example 7: Determining the Effect of APIM Peptides on the Minimum Inhibitory Concentration of Various Antibiotics on Bacteria

(48) The APIM peptide ATX-101 was used to determine the effect of APIM peptides on the MIC for various antibiotics on a range of bacteria.

(49) The APIM peptide was dissolved in Mueller-Hinton broth to 1.25 times of the desired assay concentration. Antibiotics were dissolved in Mueller-Hinton broth and Mueller-Hinton broth with APIM at a concentration of 1.25 times the highest desired assay concentrations. Antibiotics were pharmaceutical grade purchased from Sigma-Aldrich.

(50) Two-fold serial dilutions of antibiotics were made in Mueller-Hinton with different concentrations of APIM, and the solutions were placed in four parallel wells in Nunc 384-well micro plates (30 l per well in Nunc 242757 microplates). A group of 8 wells with no addition of antibiotics for each APIM concentration was included on each micro plate as growth reference.

(51) At the day of analysis, overnight TSB cultures inoculated from freeze stocks (6 ml in 50 ml tube tilted to 45-degrees angle, 200 rpm, 2.5 cm amplitude, 37 C.) were diluted in TSB until the OD.sub.600 was 0.10, and further diluted 1:40 in Mueller-Hinton broth. Each well in the 384-well assay plates was inoculated with 7.5 l of the diluted culture. The microplates were placed in plastic bags and incubated without shaking at 37 C. The optical density at 600 nm in the microwells was measured after approximately 19 hours of incubation, and the relative growth yield in each well was calculated based on the growth in the reference groups. The MIC value was set to the highest concentration giving less than 30% growth in all 4 parallel wells within the sample groups. The microplates were further incubated for 6 hours, and optical density in the cultures was measured once more for confirmation of the estimated MIC-values.

(52) Table 3 shows that APIM peptides are capable of reducing the MIC for numerous antibiotics by at least 50% in various bacteria.

(53) TABLE-US-00024 TABLE 3 APIM conc. Est MIC Strain Antibiotic g/ml (g/ml) E. faecium CTC 492 Ampicillin 0 1 E. faecium CTC 492 Ampicillin 6.5 0.5 E. faecium CTC 492 Ampicillin 13 0.5 E. faecium CTC 492 Apramycin 0 64 E. faecium CTC 492 Apramycin 6.5 64 E. faecium CTC 492 Apramycin 13 32 E. faecium CTC 492 Rifampicin 0 16 E. faecium CTC 492 Rifampicin 6.5 32 E. faecium CTC 492 Rifampicin 13 4 E. faecium CTC 492 Rifampicin 20 8 E. faecium CTC 492 Erythromycin 0 4 E. faecium CTC 492 Erythromycin 6.5 4 E. faecium CTC 492 Erythromycin 13 1 E. faecium CTC 492 Chloramphenicol 0 2 E. faecium CTC 492 Chloramphenicol 6.5 2 E. faecium CTC 492 Chloramphenicol 13 1 E. faecium CTC 492 Imipenem 0 4 E. faecium CTC 492 Imipenem 6.5 2 E. faecium CTC 492 Imipenem 13 2 E. faecium CTC 492 Tobramycin 0 64 E. faecium CTC 492 Tobramycin 6.5 64 E. faecium CTC 492 Tobramycin 13 32 E. faecium CTC 492 Monensin 0 1 E. faecium CTC 492 Monensin 6.5 0.5 E. faecium CTC 492 Monensin 13 0.5 E. coli NCIMB 12210 Chloramphenicol 0 2 E. coli NCIMB 12210 Chloramphenicol 2 2 E. coli NCIMB 12210 Chloramphenicol 4 2 E. coli NCIMB 12210 Chloramphenicol 8 1 E. coli NCIMB 12210 Novobiocin 0 64 E. coli NCIMB 12210 Novobiocin 2 32 E. coli NCIMB 12210 Novobiocin 4 8 E. coli NCIMB 12210 Novobiocin 8 4 A. baumanii ATCC 19606 Rifampicin 0 2 A. baumanii ATCC 19606 Rifampicin 2 2 A. baumanii ATCC 19606 Rifampicin 4 1 A. baumanii ATCC 19606 Rifampicin 8 1 A. baumanii ATCC 19606 Imipenem 0 0.25 A. baumanii ATCC 19606 Imipenem 2 0.125 A. baumanii ATCC 19606 Imipenem 4 0.125 A. baumanii ATCC 19606 Gentamicin 0 16 A. baumanii ATCC 19606 Gentamicin 2 16 A. baumanii ATCC 19606 Gentamicin 4 8 A. baumanii ATCC 19606 Monensin 0 64 A. baumanii ATCC 19606 Monensin 2 64 A. baumanii ATCC 19606 Monensin 4 64 A. baumanii ATCC 19606 Monensin 8 32 A. baumanii ATCC 19606 Vancomycin 0 32 A. baumanii ATCC 19606 Vancomycin 2 32 A. baumanii ATCC 19606 Vancomycin 4 16 A. baumanii ATCC 19606 Novobiocin 0 8 A. baumanii ATCC 19606 Novobiocin 2 8 A. baumanii ATCC 19606 Novobiocin 4 8 A. baumanii ATCC 19606 Novobiocin 8 2

Example 8: The Effect of APIM Peptides on MDR Bacteria

(54) The efficacy of APIM peptides as antibiotics against MDR bacteria was tested using strains of MRSA and MDR E. faecium.

(55) Table 4 demonstrates that APIM peptides, exemplified using ATX-101, are particularly effective against MRSA, as the MIC of APIM peptide needed to inhibit growth of two strains of MRSA was greatly reduced in comparison to a control strain of S. aureus.

(56) TABLE-US-00025 TABLE 4 Starting concentration of Factor of MIC of ATX-101 Strain ATX-101 (g/ml) inhibition (g/ml) Staphylococcus 150 1 150 aureus NCTC 6571 Staphylococcus 150 0.25 37.5 aureus MRSA 1040s Staphylococcus 150 0.0625 9.375 aureus MRSA 1096

(57) In view of the anti-bacterial effect of ATX-101 on strains of MRSA, the MIC for a variety of APIM variants was determined using Staphylococcus aureus MRSA 1040s. The results in Table 5 show that all APIM peptides have similar antibacterial activity, i.e. variation of the APIM sequence within the parameters defined herein, particularly at positions 3 and 4, does not reduce activity. A cell penetrating peptide (SEQ ID NO: 337) present in all of the APIM peptides, was used as a negative control. Whilst the CPP alone demonstrates some antibacterial activity, its combination with the APIM sequence greatly improves its activity, thereby indicating that the APIM sequence is responsible for the antibacterial effect.

(58) TABLE-US-00026 TABLE5 MIC Peptidesequence APIMsequence (g/ml) MDRWSVKWKKKRKIRRR RWSVK 32 RRRRRRRR (SEQIDNO:52) (SEQIDNO:1203) MDRWAVKWKKKRKIRRR RWAVK 32 RRRRRRRR (SEQIDNO:58) (SEQIDNO:1204) MDRWLSKWKKKRKIRRR RWLSK 32 RRRRRRRR (SEQIDNO:40) (SEQIDNO:1211) MDRWLTKWKKKRKIRRR RWLTK 32 RRRRRRRR (SEQIDNO:76) (SEQIDNO:1208) MDRWLVPWKKKRKIRRR RWLVP 32 RRRRRRRR (SEQIDNO:1207) (SEQIDNO:1212) MDRWLVKWKKKRKIRRR RWLVK 32 RRRRRRRR (SEQIDNO:28) (SEQIDNO:1198) RRRRRRRRRRR 118 (SEQIDNO:337)

(59) Next, the effect of APIM peptides on the MIC of antibiotics was determined using Staphylococcus aureus MRSA 1040s. Table 6 and FIG. 9 show that ATX-101 reduces the MIC for erythromycin and azithromycin significantly. Table 7 and FIG. 10 demonstrate that similar effects are observed for other APIM variants.

(60) These data indicate that APIM peptides may be particularly effective in treating MRSA infections, either alone or in combination with antibiotics, particularly macrolide antibiotics.

(61) TABLE-US-00027 TABLE 6 MIC (g/ml) when combined with 10 g/ml of MIC g/ml ATX-101 ATX-101 16 Erythromycin >1034 2 Azithromycin >1034 8

(62) TABLE-US-00028 TABLE7 Concen- MICof tration Eryth- ofAPIM romycin APIM peptide (Relative Peptidesequence sequence (g/ml) values) MDRWLVKWKKKRKIRRR RWLVK 7.5 1000 RRRRRRRR (SEQID (SEQIDNO:1198) NO:28) MDRWLVKWKKKRKIRRR RWLVK 15 125 RRRRRRRR (SEQID (SEQIDNO:1198) NO:28) MDRWSVKWKKKRKIRRR RWSVK 15 100 RRRRRRRR (SEQID (SEQIDNO:1203) NO:52) MDRWAVKWKKKRKIRRR RWAVK 15 200 RRRRRRRR (SEQID (SEQIDNO:1204) NO:58) MDRWLSKWKKKRKIRRR RWLSK 15 200 RRRRRRRR (SEQID (SEQIDNO:1211) NO:40) MDRWLTKWKKKRKIRRR RWLTK 15 100 RRRRRRRR (SEQID (SEQIDNO:1208) NO:76) MDRWLVPWKKKRKIRRR RWLVP 15 100 RRRRRRRR (SEQID (SEQIDNO:1212) NO:1207)

(63) Additive effects were also observed when APIM peptides were combined with various antibiotics to treat a MDR strain of E. faecium (E. faecium CCUG 37832 (TO-3)), which is commonly associated with endocarditis, urinary tract infections and infections in wounds.

(64) The MIC for APIM peptide ATX-101 on E. faecium CCUG 37832 (TO-3) was determined to be 7.5 g/ml. Accordingly, concentrations of 8 g/ml and 16 g/ml of ATX-101 were combined with various antibiotics selected from: 2,4-Diamin, S. methizol, S. methoxa, S. dimetho, Sulfaceta, Trimeth, Flumeq, Levoflox, Pruliflox, Metronid and Nitrofur. FIG. 11 shows that the MIC of each antibiotic could be reduced by combining it with an APIM peptide. Thus, these data indicate that APIM peptides may be particularly effective in treating E. faecium infections (particularly MDR E. faecium infections), either alone or in combination with antibiotics, particularly DNA gyrase inhibitors.

Example 9: Overexpression of APIM Peptides in E. coli

(65) In order to verify further that the anti-microbial effect of the APIM peptides arises from the APIM sequence, peptides containing only the APIM sequence (i.e. without a cell-penetrating peptide) were over-expressed in E. coli using the expression vector pET28. The APIM peptide was expressed alone or as part of a fusion protein with EYFP. Expression of EYFP alone was used as a control.

(66) The expression vectors containing the respective peptides were transfected into the bacterial strain E. coli BL21 (ripl). Single colonies, 4-6 of each strain, were inoculated in 150 ml LB media (+Km/Clm) in 96 wells plates, and incubated at 37 C. Overnight cultures were diluted 1:100 and grown for 1 h before induction with 1 mM IPTG (initiating peptide expression). OD was measured every hour.

(67) FIG. 12A shows that expression of the APIM peptide, either alone or as part of a fusion protein with EYFP, inhibits bacterial growth. This result demonstrates that the APIM sequence RWLVK (SEQ ID NO: 28) has antibacterial properties even in the absence of a cell-penetrating peptide.

(68) FIG. 13 shows that APIM variant sequences are similarly effective at inhibiting bacterial growth when overexpressed in E. coli. FIG. 13B indicates that the APIM variants RWLTK (SEQ ID NO: 76), RFSLK (SEQ ID NO: 1206) and RWLVP (SEQ ID NO: 1207) are particularly effective. However, all of the variants tested show a significant inhibitory effect on bacterial growth.

Example 10: Determination of APIM Peptide Interaction with the -Clamp Protein from E. coli

(69) Microscale thermophoresis (MST) was used to determine the dissociation constant for various APIM containing peptides.

(70) The -clamp protein from E. coli was labeled with a fluorescent molecule. Concentration of PCNA was kept constant, whereas dilutions of each APIM containing peptide were prepared (1:1). In a mix of protein and peptide, the signal was recorded in all capillaries with varying concentrations of the unlabeled peptide, and any change of thermophoretic properties was observed as a change in fluorescence intensity.

(71) Table 8 shows that various APIM peptides show specific interactions with the -clamp protein (a low Kd value indicates a strong interaction, whereas a high Kd value indicates a weak interaction). The R11 peptide (SEQ ID NO: 337) was used as a control and no data could be obtained by MST for this peptide, indicating that this peptide does not interact with the -clamp protein. This data further verifies that the APIM sequence contributes to the antibacterial effects of the APIM peptides.

(72) TABLE-US-00029 TABLE8 APIM Peptidesequence sequence Kd MDRWLVKWKKKRKIRRRRRRRRRRR RWLVK 101 (SEQIDNO:1198) (SEQID NO:28) MDRWSVKWKKKRKIRRRRRRRRRRR RWSVK 408 (SEQIDNO:1203) (SEQID NO:52) MDRWAVKWKKKRKIRRRRRRRRRRR RWAVK 57 (SEQIDNO:1204) (SEQID NO:58) MDRWLSKWKKKRKIRRRRRRRRRRR RWLSK 115 (SEQIDNO:1211) (SEQID NO:40) MDRWLTKWKKKRKIRRRRRRRRRRR RWLTK 22 (SEQIDNO:1208) (SEQID NO:76) MDRWLVPWKKKRKIRRRRRRRRRRR RWLVP 366 (SEQIDNO:1212) (SEQID NO:1207) MDRFLSKWKKKRKIRRRRRRRRRRR RFLSK 511 (SEQIDNO:1210) (SEQID NO:41) MDRFSLKWKKKRKIRRRRRRRRRRR RFSLK 20 (SEQIDNO:1209) (SEQID NO:1206) R11(SEQIDNO:337) Nd

Example 11: Determination of the Effect of UV Radiation on the Anti-Microbial Properties of APIM Peptides

(73) Pre-cultures of E. coli BL21 (ripl) were grown over-night in LB at 37 C. The cultures were then diluted 1:100 and 150 ml/well was added/well in 96 wells plates. The cultures were further incubated for 1 h and each plate was exposed to UVC, 2 J/cm.sup.2, with a Stratalinker. The plates were incubated for 30 minutes following UV treatment and various APIM peptides, 15 M or 30 M of each peptide, were added to 6 parallel wells. OD.sub.660 was measured every hour. Values were normalized and average was plotted.

(74) FIGS. 14A and B show the effect of various APIM peptides (30 M) on bacteria that have not been exposed to UV radiation. The graph in FIG. 14B shows the same growth curves as FIG. 14A without the controls (no peptide was added), which are the two highest grow curves in FIG. 14A. Thus, FIG. 14B shows that there is some re-growth 5 hours after the APIM peptides were added. However, this assay shows that all of the APIM peptides tested have a significant inhibitory effect on bacterial growth.

(75) FIGS. 15A and B are equivalent to FIGS. 14A and B when the bacteria have been exposed to UV radiation. These Figures demonstrate that there is no re-growth when the APIM peptides are combined with UV radiation. Furthermore, no re-growth was observed in samples even when they were incubated over-night (data not shown). This suggests that UV radiation sensitizes the cells to APIM peptides or vice versa. Thus, these data demonstrate that a combination of APIM peptides and UV radiation, particularly UVC radiation, may be useful in treating bacterial infections.

(76) The treatments using APIM peptides at 15 M (data not shown) showed similar effects to the 30 M treatment, which is shown in FIGS. 14 and 15.

(77) FIG. 16 shows that treatment with UV radiation is effective even when using lower concentrations of APIM peptides. APIM peptide concentrations of 15 M (FIG. 16A), 7.5 M (FIG. 16B) and 3.75 M (FIG. 16C) were all effective at inhibiting bacterial growth following treatment with UV radiation. Overall, these data show that bacteria are more 2-5 fold more sensitive to APIM peptides after UV-irradiation.

Example 12: Effect of ATX-101 on Methicillin Resistant Staphylococcus Aureus (MRSA) Biofilm Under Flow

(78) APIM peptides were tested to determine whether they have an effect on biofilm formation.

(79) The IBIDI flow-system coupled with EVOS Auto Imaging system was optimized and used for testing the effect of ATX-101 on MRSA biofilm under flow. MRSA 1040 (u50) was used as model organism; it normally produces a dense biofilm in the growth channel during 36 hours of flow. The effect of 3 different concentrations of ATX-101 was tested (7 g/ml, 3.5 g/ml and 0.8 g/ml). The flow system was programmed with share stress similar to those found in capillary networks (3.49 dyne/cm.sup.2), 2% Luria Bertani (LB) was used for dilution and flow medium. Good effect of ATX-101 was observed for all tested concentrations, the highest and lowest concentrations are presented in FIGS. 17 and 18, respectively.

Example 13: Determining the Minimum Inhibitory Concentration (MIC) of Various APIM Peptides on Bacteria

(80) The MICs for various APIM peptides were determined for various bacteria, as described above. The results are shown in Table 9 and show that all of the tested variants have anti-bacterial properties, when used alone, across a variety of bacteria. The results also demonstrate that the APIM peptides are particularly effective against MDR bacteria, e.g. E. faecium TO-3 and MRSA 1040.

(81) TABLE-US-00030 TABLE9 MIC(M) E.faecium E.faecium MRSA S.aureus Peptidesequence P.aeruginosa E.coli A.baumanii TO-3 TO-12 1040 NCTC6571 MDRWLVKWKKKRKIRRRRRRRRRRR 37.9 nd nd 7.5 16.8 7.5 37.9 (SEQIDNO:1198) MDRWSVKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 37.9 7.5 37.9 (SEQIDNO:1203) MDRWAVKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQIDNO:1204) MDRWLSKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 16.8 7.5 37.9 (SEQIDNO:1211) MDRWLTKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQIDNO:1208) MDRWLVPWKKKRKIRRRRRRRRRRR nd 37.9 25.3 7.5 25.3 7.5 37.9 (SEQIDNO:1212) MDRFLSKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 25.3 7.5 37.9 (SEQIDNO:1210) MDRFSLKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQIDNO:1209)