Anti-bacterial 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.5X.sub.6 (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 aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms; X.sub.4 is an uncharged amino acid other than an aromatic amino acid, Glycine (G) and Proline (P); X.sub.5 is any amino acid other than an acidic amino acid or an aromatic amino acid; and X.sub.6 is any amino acid other than an acidic amino acid or an aromatic amino acid, preferably a basic amino acid or Proline (P), wherein when X.sub.3 is not an aromatic amino acid, X.sub.5 is not lysine (K) and X.sub.6 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 composition of the invention may be used as single agents. In other aspects of the invention the agents and composition may be used in conjunction with one or more addition active agents, such as antibiotics, or in combination with UV radiation.

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: 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.5X.sub.6 (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 aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms; X.sub.4 is an uncharged amino acid other than an aromatic amino acid, glycine (G) and proline (P); X.sub.5 is a hydrophobic amino acid, a basic amino acid, a polar amino acid, a thiol-containing amino acid or proline wherein X.sub.5 is not asparagine (N), an aromatic amino acid, or an acidic amino acid; and X.sub.6 is any amino acid other than an acidic amino acid or an aromatic amino acid, wherein when X.sub.3 is not an aromatic amino acid, X.sub.5 is not lysine (K) and X.sub.6 is a basic amino acid or proline (P).

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 is caused by a biofilm.

5. The method of claim 1, wherein the bacterial infection is caused by a bacterium selected from any of the genera Achromobacter, Acinetobacter, Actinobacillus, Aeromonas, Agrobacterium, Alcaligenes, Alteromonas, Bacteroides, Bartonella, Borrelia, Bordetella, Brucella, Burkholderia, Campylobacter, Cardiobacterium, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Edwardsiella, Eikenella, Enterobacter, Enterococcus, Erwinia, Helicobacter, Kingella, Klebsiella, Lactobacillus, Lactococcus, Legionella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Mobiluncus, Moraxella, Morganella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Nocardiopsis, Pantoea, Parachlamydia, Pasteurella, Peptococcus, Peptostreptococcus, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Ralstonia, Rickettsia, Salmonella, Shewenella, Shigella, Sphingobacterium, Sphingomonas, Staphylococcus, Stenotrophomonas, Streptobacillus, Streptococcus, Streptomyces, Treponem and Yersinia.

6. The method of claim 1, wherein the bacterium is a MDR bacterium.

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

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

9. The method of claim 8, 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, or a Tetracycline.

10. The method of claim 1, wherein (i) X.sub.5 is a hydrophobic amino acid, a basic amino acid, a polar amino acid, or proline and not an aromatic amino acid, an acidic amino acid, or asparagine; and /or (ii) X.sub.3 is an aromatic amino acid; and/or (iii) X.sub.4 is a hydrophobic or polar amino acid.

11. The method of claim 1, wherein: (i) the basic amino acid selected from any one of arginine (R), lysine (K), histidine (H), ornithine (Orn), methyllysine (MeK), diaminobutyric acid (Dbu), citrulline (Cit), acetyllysine (AcK), and any basic amino acid selected from the non-conventional amino acids in Table 2; and/or (ii) X.sub.4 is selected from any one of leucine (L), isoleucine (I), valine (V), alanine (A) methionine (M), norleucine (Nor), serine (S) or threonine (T), glutamine (Q), asparagine (N) or cysteine (C) or any hydrophobic or polar amino acid selected from the non-conventional amino acids in Table 2; and/or (iii) X.sub.5 is selected from any one of from V, L, I, A, M, Nor, S, T, Q, H, K, R, G, P or C or any hydrophobic, polar, basic or thiol-containing amino acid selected from the non-conventional amino acids in Table 2; and/or (iv) the aromatic amino acid is selected from any one of tryptophan (W), tyrosine (Y), phenylalanine (F), tert.-butylglycine, cyclohexylalanine, tert.-butylphenylalanine, biphenylalanine or tri tert.-butyltryptophan or any aromatic amino acid selected from the non-conventional amino acids in Table 2.

12. The method of claim 1, wherein the PCNA interacting motif comprises a sequence selected from SEQ ID NOs: 6-21, 1221-1227, 1233-1257 or 22 to 297, 300, 301, 1207, 1209, 1258 to 1288 and 1310.

13. The method of claim 1, wherein the domain that facilitates the cellular uptake of the oligopeptidic compound is a cell penetrating peptide (CPP).

14. The method of claim 13, 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 of SEQ ID NO:326 and a peptide of SEQ ID NO:328; (v) a peptide exhibiting high a-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 13, wherein the CPP is selected from any one of SEQ ID NOs: 302 to 1162, or 1210 to 1220 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 a PCNA interacting motif as set forth in any one of SEQ ID NOs: 28-30, a linker sequence as set forth in SEQ ID NO: 1176 and a cell penetrating signal sequence as set forth in SEQ ID NO: 337.

18. The method of claim 1, wherein the agent comprises a sequence as set forth in any one of SEQ ID NOs: 1182 to 1204, 1208 or 1311.

19. The method of claim 1, wherein the bacterial infection is caused by a bacterium of a genus selected from the group consisting of Acinetobacter, Enterococcus, Escherichia, Micrococcus, Pseudomonas and Staphylococcus.

20. The method of claim 1, wherein X.sub.6 is a basic amino acid or proline (P).

21. A product, material, device or implant which is coated, impregnated or chemically bonded with an agent or composition comprising the agent, wherein the agent comprises: 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.5X.sub.6 (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 aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms; X.sub.4 is an uncharged amino acid other than an aromatic amino acid, glycine (G) and proline (P); X.sub.5 is a hydrophobic amino acid, a basic amino acid, a polar amino acid, a thiol-containing amino acid or proline wherein X.sub.5 is not asparagine (N), an aromatic amino acid, or an acidic amino acid; and X.sub.6 is any amino acid other than an acidic amino acid or an aromatic amino acid, wherein when X.sub.3 is not an aromatic amino acid, X.sub.5 is not lysine (K) and X.sub.6 is a basic amino acid or proline (P).

22. The product, material, device or implant of claim 21, wherein when the product, material, device or implant is impregnated: (i) said product or material is a bandage, gauze, surgical tape, cotton swab, puff, fleece, sponge or supportive matrix, diaper, glove, sock, contact lens or contact lens storage case; or (ii) said device or implant is a stent, an ear tube, an artificial eye lens, an orthopedic implant, an artificial bone, a dental implant, a cardiac device, a cosmetic implant, an intra-uterine device, a catheter or a prosthetic device.

23. The product, material, device or implant of claim 21, wherein X.sub.6 is a basic amino acid or proline (P).

24. 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.5X.sub.6 (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 aromatic amino acid or a hydrophobic amino acid that has an R group comprising at least three carbon atoms; X.sub.4 is an uncharged amino acid other than an aromatic amino acid, glycine (G) and proline (P); X.sub.5 is a hydrophobic amino acid, a basic amino acid, a polar amino acid, a thiol-containing amino acid or proline wherein X.sub.5 is not asparagine (N), an aromatic amino acid, or an acidic amino acid; and X.sub.6 is any amino acid other than an acidic amino acid or an aromatic amino acid, wherein when X.sub.3 is not an aromatic amino acid, X.sub.5 is not lysine (K) and X.sub.6 is a basic amino acid or proline (P).

25. A pharmaceutical composition comprising an oligopeptidic compound as defined in claim 24, a nucleic acid molecule encoding said oligopeptidic compound or a vector comprising said nucleic acid molecule, together with at least one pharmacologically acceptable carrier or excipient.

26. The oligopeptidic compound of claim 24, wherein X.sub.6 is a basic amino acid or proline (P).

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 a graph that demonstrates that the growth of gram-negative bacteria, Pseudomonas aeruginosa ATCC 15692, Acinetobacter baumenni ATCC 19606, Escherichla coli ATCC 25922 and Pseudomonas aeruginosa TO-5A (clinical isolate) is inhibited by varying concentrations of APIM peptide, ATX-101 (SEQ ID NO: 1289). Growth of the strains is given relative to growth in cultures without ATX-101.

(3) FIG. 2 shows 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.

(4) FIG. 3 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.

(5) FIG. 4 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.

(6) FIG. 5 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.

(7) FIG. 6 shows graph that demonstrates that the growth of the MDR bacterium Methicillin-resistant Staphylococcus aureus (MRSA 1040) is more sensitive to macrolide antibiotics, azitmomycin and erythromycin, when grown in the presence of ATX-101.

(8) FIG. 7 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: 1298; ATX-101-Sv is SEQ ID NO: 1298; ATX-101-Av is SEQ ID NO: 1299; ATX-101-Ls is SEQ ID NO: 1302; ATX-101-P is SEQ ID NO: 1303; and ATX-101-LT is SEQ ID NO: 1300.

(9) FIG. 8 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.

(10) FIG. 9 show (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: 1290), 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.

(11) FIG. 10 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: 1290; (B) SEQ ID NOs: 1291, 1292 and 1293; (C) SEQ ID NOs: 1294, 1295 and 1296; (D) SEQ ID NO: 1297.

(12) FIG. 11 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: 1289; ATX-101-P is SEQ ID NO: 1303; FSL, is SEQ ID NO: 1304; FLS is SEQ ID NO: 1301; SV is SEQ ID NO: 1298; AV is SEQ ID NO: 1299; LT is SEQ ID NO: 1300; LS is SEQ ID NO: 1302; R11 is SEQ ID NO: 337; Neg contr is no peptide added.

(13) FIG. 12 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: 1289; ATX-101-P is SEQ ID NO: 1303; FSL is SEQ ID NO: 1304; FLS is SEQ ID NO: 1301; SV is SEQ ID NO: 1298; AV is SEQ ID NO: 1299; LT is SEQ ID NO: 1300; LS is SEQ ID NO: 1301; R11 is SEQ ID NO: 337; Neg contr is no peptide added.

(14) FIG. 13 shows graphs showing the growth of E. coli in the presence of ATX-101 (SEQ ID NO: 1289) 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.

(15) FIG. 14 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: 1289) (left panels) or no peptide (right panels) over a period of 36 hours and demonstrates that biofilm formation is inhibited by ATX-101.

(16) FIG. 15 shows micrographs of biofilm formation of the MDR bacterium Methicillin-resistant Staphylococcus auraus (MRSA 1040) in the presence of 0.8 g/ml ATX-101 (SEQ ID NO: 1289) (left panels) or no peptide (right panels) over a period of 36 hours and demonstrates that biofilm formation is inhibited by ATX-101

(17) FIG. 16 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 (EY-EC), background due to dimerisation of the tags). EYFP-APIM motif/ECFP-PCNA for various motifs are shown in the other lanes, wherein the annotations on the X-axis refer to the following APIM sequences: WT (RWLVK, SEQ ID NO: 1290); LA (RWLAK, SEQ ID NO: 1305); AV (RWAVK, SEQ ID NO: 1297); LG (RWLGK, SEQ ID NO: 1306); LS (RWLSK, SEQ ID NO: 1294); SV (RWSVK, SEQ ID NO: 1296); WF (RWFLVK, SEQ ID NO: 1258); FF (RFFLW, SEQ ID NO: 1265); FL (RFLLVK, SEQ ID NO: 1267); and XPA (KFIVK, SEQ ID NO: 1307).

(18) FIG. 17 shows confocal microscope images which show that fluorescently-labelled peptides (FAM-tagged peptides) containing extended APIM sequences are taken up by mammalian cells (see Example 13). The two letter annotations refer to peptides containing the following APIM sequences: WY (RWYLVK, SEQ ID NO: 1259); FF (RFFLVK, SEQ ID NO: 1265); FY (RFYLVK, SEQ ID NO: 1266), FV (RFVLVK, SEQ ID NO: 1269); and FI (RFILVK, SEQ ID NO: 1268)

(19) FIG. 18 shows graphs showing the results of cytotoxicity assays with various APIM peptides, as described in Example 14. The annotations in the legend refer to the following APIM sequences; WT-101, MDRWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1289); ATX-A, MDRALVKWKKKRKIRRRRRRRRRR (SEQ ID NO: 1206) (negative control); ATX-FF, MDRFFLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1182); ATX-FL MDRFLLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1187); ATX-WY, MDRWYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1184); ATX-FV, MDRFVLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1185); and ATX-FI MDRFILVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1186).

(20) FIG. 19 shows graphs showing the growth of E. coli in which the overexpression of various APIM peptides is (A) not induced: (B) induced after 1 hour of growth: and (C) induced after 2 hours of growth. The annotations in the legend refer to the following: BL21, no plasmid (negative control); ATX-101 is SEQ ID NO: 1290; ATX-FI is SEQ ID NO: 1268; ATX-FF is SEQ ID NO: 1265; ATX-FV is SEQ ID NO: 1269; ATX-FY is SEQ ID NO. 1266; ATX-WY is SEQ ID NO: 1259; ATX-WW is SEQ ID NO: 1310; and Short is SEQ ID NO: 1312 (negative control).

(21) FIG. 20 shows graphs depicting the growth curves of cultures of E. coli BL21 overexpressing variants of several APIM peptides (101, 101-FV, 101-FY, 101-WY, 101-WW, 101-FF, 101-FI) and a negative control (ATX-A) measured by CFU (A and C) and OD.sub.600 (B and D). Both induced and not induced cultures are included in the figures. The annotations in the legend (mentioned above) refer to the following sequences: 101, MDRWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1289); 101-FV, MDFRFVLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1155); 101-FY, MDRFYLVKWKKKRKIRRRIRRRRRRRR (SEQ ID NO: 1183); 101-WY, MDRWYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1184); 101-WW, MDRWWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1311); 101-FF, MDRFFLVKVVKKKRKIRRRRRRRRRRR (SEQ ID NO: 1182); 101-FI, MDRFILVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1186); and ATX-A, MDRALVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1206).

(22) FIG. 21 shows graphs depicting the growth curves of cultures of E. coli BL21 measured by CFU and data 1, 3 and 5 hours after adding variants of severe APIM peptides (101, 101-FF, 101-WW, 101-Y, 101-pen, 101-WW-pen, 101-FF-pen and 101-7aa) and a negative control (R11). BL21 represents the growth with the addition of a peptide. The annotations in the legend (mentioned above) refer to the following sequences: 101, MDRWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1289); 101-WW, MDRWWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1311); 101-FI, MDRFILVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1186); 101-Y, MDRYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1313); 101-pen MDRWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1314); 101-WW-pen, MDRWWLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1315); 101-FF-pen, MDRFFLVKWKKKRKIRQIKIWFQNRRMKWKK (SEQ ID NO: 1316); and 101-7aa, MDRWLVKGAQPKVLRRRRRRRRRRR (SEQ ID NO: 1317),

(23) FIG. 22 shows a graph depicting the percentage reduction in mutation frequency for E. coli BL21 and E. coli BL21 with overexpression of different variants of several APIM peptides (101-FF, 101-FI, 101-FV, 101-FY. 101-WY and 101-WW) and a negative control peptide (short). Data are shown for parallel (P) 1 to 7 and presented as means+/SEM. The annotations in the legend (mentioned above) refer to the following sequences: 101-FF, MDRFFLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1182); 101-FI MDRFILVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1186); 101-FV, MDRFVLVKWXKKRKIRRRRRRRRRRR (SEQ ID NO: 1185); 101-FY, MDRFYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1183); 101-WY, MDRWYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1184); 101-WW, MDRWWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1311); and short, MDRWIXWKKKRKIRRRRRIRRRRRR (SEQ ID NO: 1318).

(24) FIG. 23 shows a graph depicting the mutation frequency for E. coli BL21 and E. coli BL21 with overexpression of different variants of several APIM peptides (101-FF, 101-FI, 101-FV, 101-FY, 101-WY and 101-WW) and a negative control peptide (short). Data are shown for parallel (P) 1 to 7 and presented as means+/SEM. The annotations in the legend (mentioned above) refer to the following sequences: 101-FF, MDRFFLVKWKKKRKIRRRRRIRRRRRR (SEQ ID NO: 1182); 101-FI, MDRFILVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1186); 101-FV, MDRFVLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1185); 101-FY, MDRFYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1183); 101-WY, MDRWYLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1184); 101-WW, MDRWWLVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1311); and short, MDRWLKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1318).

(25) FIG. 24 shows the mutation spectra of rpoB (1525-1722 SEQ ID NOs: 1319 (1525-1596 bp) and 1320 (1690-1722 bp)) from rifampicin resistant colonies (A) E. coli BL21 induced, (B) E. coli BL21 not induced, and overexpression of variants of the APIM-peptide (C) 101-WY, (D) 101-WW, (E) 101-FI and (F) 101-FF. Data represent mutant colonies from different parallels. Spontaneous mutations are shown above sequence line and mutations found after LN exposure below line. Number of colonies varies due to low mutation frequency in BL21 with overexpression of variants of the APIM peptides. Number of colonies, n=not UV/UV, BL21 n=10/10, BL21 not induced n=10/10, 101-WY n=10/10, 101-WW n=10/8, 101-FI n5/9, 101-FF n=3/12.

(26) FIG. 25 shows graphs showing the results of cytotoxicity assays with various standard and extended APIM peptides. (A)-(C) show the levels cell survival of HEK 293 cells treated with 6 M of peptides alone (A), with 0.5 M cisplatin (b) or with 1 M cisplatin (C) as described in Example 18, (D)-(I) show the levels of cell survival of HEK 293 cells treated with 12 M of peptides alone (D) and (G), with 0.5 M cisplatin (E) and (H) or with 1 M cisplatin (F) and (I) as described in Example 18. The annotations in the legend refer to APIM sequences as described above.

(27) FIG. 26 snows graphs showing the results of cytotoxicity assays with various standard and extended APIM peptides. (A)-(C) show the levels cell survival of U2OS delis treated with 4 M of peptides alone (A), with 0.5 M cisptatin (B) or with 1 M cisplatin (C) as described in Example 18. (D)-(F) show the levels of cell survival of U2OS cells treated with 6 M of peptides alone (D), with 0.5 M cisplatin (E) or with 1 M cisplatin (F) as described in Example 18. (G)-(I) show the levels of cell survival of U2OS cells treated with 12 M of peptides alone (G), with 0.5 M cisplatin (H) or with 1 M cisplatin (I) as described in Example 18. The annotations in the legend refer to APIM sequences as described above.

(28) FIG. 27 shows graphs showing the results of cytotoxicity assays with various standard and extended APIM peptides. (A)-(C) show the levels cell survival of U2OS cells treated with 12 M of peptides alone (A), with 0.5 M cisplatin (B) or with 1 M displatin (C) as described in Example 14. The annotations in the legend refer to APIM sequences as described above.

EXAMPLES

(29) The inventors have surprisingly found that an oligopeptidic compound comprising a conventional APIM motif and a cell-penetrating peptide is imported into bacterial 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 bacteria 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 bacterial cells have been established in both gram positive and gram negative bacteria using an exemplary cell-penetrating APIM-containing peptide ATX-101 (SEQ ID NO:1289, which contains the APIM motif, RWLVK (SEQ ID NO: 1290)).

(30) The data presented below suggest that oligopeptidic compounds comprising an APIM motif, including an extended or longer APIM motif as defined herein, are useful as anti-bacterial agents, e.g. antibiotics, either alone or in combination with other cytostatic or cytotoxic agents. Accordingly, the data supports the use of oligopeptidic compounds comprising an APIM motif, including an extended or longer APIM motif as defined herein, in the treatment or prevention of bacterial infections or bacterial infectious diseases.

(31) The inventors have also unexpectedly found that conventional APIM sequence may be substantially modified as described herein to produce extended or longer APIM peptides that are capable of interacting with PCNA. As shown by the experimental results discussed below (Examples 12-14), the extended or longer APIM peptides interact with PCNA with a similar or improved affinity relative to the conventional APIM peptides, but are not cytotoxic to normal, healthy animal cells. Accordingly, it can be inferred from the data, provided herein that peptides containing the extended or longer APIM sequence as defined herein would also find utility in the treatment or prevention of bacterial infections or bacterial infectious diseases.

Example 1

Determining the Effect of ATX-101 on Bacteria

(32) The minimum inhibitory concentration (MIC) of ATX-101 (SEQ ID NO: 1289) was determined for various gram negative and gram positive bacteria. The bacteria, Pseudomonas aeruginosa ATCC 15692, Acinetobacter baumenni ATCC 19600, Escherichia coli ATCC 25922, Pseudomonas aeruginosa TO-5A (clinical isolate), Enterococous faecium CCUG 37832, Enterococcus faecium CTC 492, Micrococcus luteus ATCC 9341 and Escherichie coli NCIMB 12210 were grown in the presence of various concentrations of ATX-101 and/or antibiotic.

(33) Robotic MIC Assay:

(34) 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.

(35) 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 is 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.

(36) 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 OD800 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 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.

(37) FIG. 1 shows that the growth of various gram negative bacteria is inhibited by ATX-101 from about 1 M. FIG. 2 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.

(38) FIG. 3 shows that Enterococcus faecium CTC 492 shows increased sensitivity to ampicillin and novobiocin 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. 4 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. 3 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.

(39) 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 2

Determining the Effect of Other APIM Peptides on Bacteria

(40) 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: 1298) and MDRWAVKWKKKRKIRRRRRRRRRRR (SEQ ID NO: 1299) 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 3

In Silica Characterisation of APIM Consensus Motif

(41) 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 may be permitted without losing affinity to PCNA.

(42) 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: 1308) 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 gellus (236 orthologues) Xenopus tropicalis (200 orthologues); Dania 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:

(43) TABLE-US-00060 (SEQIDNO:1309) [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],

(44) wherein specific combinations of amino acids at positions 3 and 4 that were identified in the orthologues include:

(45) 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 or 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 being LL, LA, LV, AL, VL, VI, LI, VV, VA, IV, II, AV, IA and AI.

(46) 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 4

In Vivo Characterisation of APIM Consensus Motif

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

(48) 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.

(49) 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.

(50) 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.

(51) 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.

(52) FIG. 5 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 5

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

(53) 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.

(54) 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.

(55) 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.

(56) At the day of analysis, overnight TSB cultures inoculated 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.

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

(58) TABLE-US-00061 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 6

The Effect of APIM Peptides on MDR Bacteria

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

(60) 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.

(61) TABLE-US-00062 TABLE 4 Starting concentration of Factor of MIC of ATX-101 Strain ATX-101 (g/ml) inhibition (g/ml) Staphylococcus aureus 150 1 150 NCTC 6571 Staphylococcus aureus 150 0.25 37.5 MRSA 1040s Staphylococcus aureus 150 0.0625 9.375 MRSA 1096

(62) 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 in Example 3, 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 anti-bacterial activity, its combination with the APIM sequence greatly improves its activity, thereby indicating that the APIM sequence is responsible for the antibacterial effect.

(63) TABLE-US-00063 TABLE5 Peptidesequence APIMsequence MIC(g/ml) MDRWSVKWKKKRKIRRRRRRRRRRR RWSVK 32 (SEQIDNO:1298) (SEQIDNO:1296) MDRWAVKWKKKRKIRRRRRRRRRRR RWAVK 32 (SEQIDNO:1299) (SEQIDNO:1297) MDRWLSKWKKKRKIRRRRRRRRRRR RWLSK 32 (SEQIDNO:1302) (SEQIDNO:1294) MDRWLTKWKKKRKIRRRRRRRRRRR RWLTK 32 (SEQIDNO:1300) (SEQIDNO:1293) MDRWLVPWKKKRKIRRRRRRRRRRR RWLVP 32 (SEQIDNO:1303) (SEQIDNO:1292) MDRWLVKWKKKRKIRRRRRRRRRRR RWLVK 32 (SEQIDNO:1289) (SEQIDNO:1290) RRRRRRRRRRR(SEQIDNO:337) 118

(64) Next, the effect of APIM peptides on the MIC of antibiotics was determined using Staphylococcus aureus MRSA 1040s. Table 6 and FIG. 6 show that ATX-101 reduces the MIC for erythromycin and azithromycin significantly. Table 7 and FIG. 7 demonstrate that similar effects are observed for other APIM variants. These data indicate that APIM peptides may be particularly effective in treating MRSA infections, either alone or in combination with antibiotics, particularly macrolide antibiotics.

(65) TABLE-US-00064 TABLE 6 MIC (g/ml) when combined with 10 g/ml MIC g/ml of ATX-101 ATX-101 16 Erythromycin >1034 2 Azithromycin >1034 8

(66) TABLE-US-00065 TABLE7 Concentration MICof ofAPIM Erythromycin APIM peptide (Relative Peptidesequence sequence (g/ml) values) MDRWLVKWKKKRKIRRRRRRRRRRR RWLVK 7.5 1000 (SEQIDNO:1289) (SEQID NO:1290) MDRWLVKWKKKRKIRRRRRRRRRRR RWLVK 15 125 (SEQIDNO:1289) (SEQID NO:1290) MDRWSVKWKKKRKIRRRRRRRRRRR RWSVK 15 100 (SEQIDNO:1298) (SEQID NO:1296) MDRWAVKWKKKRKIRRRRRRRRRRR RWAVK 15 200 (SEQIDNO:1299) (SEQID NO:1297) MDRWLSKWKKKRKSRRRRRRRRRRR RWLSK 15 200 (SEQIDNO:1302) (SEQID NO:1294) MDRWLTKWKKKRKIRRRRRRRRRRR RWLTK 15 100 (SEQIDNO:1300) (SEQID NO:1293) MDRWLVPWKKKRKIRRRRRRRRRRR RWLVP 15 100 (SEQIDNO:1303) (SEQID NO:1292)

(67) 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.

(68) 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, Surfaceta, Trimeth, Flumeq, Levoflox, Pruliflox, Metronid and Nitrofur. FIG. 8 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 7

Overexpression of APIM Peptides in E. coli

(69) In order to verify further that the anti-microbial effect of the APIM peptides arise 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.

(70) 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.

(71) FIG. 9A 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: 1290) has antibacterial properties even in the absence of a cell-penetrating peptide.

(72) FIG. 10 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: 1293), RFSLK (SEQ ID NO: 1291) and RWLVP (SEQ ID NO: 1292) are particularly effective. However, all of the variants tested show a significant inhibitory effect on bacterial growth.

Example 8

Determination of APIM Peptide Interaction with the -clamp Protein from E. coli

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

(74) 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.

(75) 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.

(76) TABLE-US-00066 TABLE8 APIM Peptidesequence sequence Kd MDRWLVKWKKKRKIRRRRRRRRRRR RWLVK 101 (SEQIDNO:1289) (SEQID NO:1290) MDRWSVKWKKKRKIRRRRRRRRRRR RWSVK 408 (SEQIDNO:1298) (SEQID NO:1296) MDRWAVKWKKKRKIRRRRRRRRRRR RWAVK 57 (SEQIDNO:1299) (SEQID NO:1297) MDRWLSKWKKKRKIRRRRRRRRRRR RWLSK 115 (SEQIDNO:1302) (SEQID NO:1294) MDRWLTKWKKKRKIRRRRRRRRRRR RWLTK 22 (SEQIDNO:1300) (SEQID NO:1293) MDRWLVPWKKKRKIRRRRRRRRRRR RWLVP 366 (SEQIDNO:1303) (SEQID NO:1292) MDRFLSKWKKKRKIRRRRRRRRRRR RFLSK 511 (SEQIDNO:1301) (SEQID NO:1295) MDRFSLKWKKKRKIRRRRRRRRRRR RFSLK 20 (SEQIDNO:1304) (SEQID NO:1291) R11(SEQIDNO:337) Nd

Example 9

Determination of the Effect of UV Radiation on the Anti-microbial Properties of APIM Peptides

(77) 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.550 was measured every hour. Values were normalised and average was plotted.

(78) FIGS. 11A 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. 11B shows the same growth curves as FIG. 14A without the controls (no peptide was added), which are the two highest grow curves in FIG. 11A. Thus, FIG. 11B 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.

(79) FIGS. 15A and B are equivalent to FIGS. 11A 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.

(80) The treatments using APIM peptides at 15 M (data not shown) showed effects to the 30 M treatment, which is shown in FIGS. 11 and 12.

(81) FIG. 13 shows that treatment with UV radiation is effective even when using lower concentrations of APIM peptides. APIM peptide concentrations of 15 M (FIG. 13A), 7.5 M (FIG. 13B) and 3.75 M (FIG. 13C) 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 10

Effect of ATX-101 n Methicillin Resistant Staphyllococcus aureus (MRSA) Biofilm Under Flow

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

(83) 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. 14 and 15, respectively.

Example 11

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

(84) 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.

(85) TABLE-US-00067 TABLE9 MIC(M) P. E.faecium E.faecium MRSA S.aureus Peptidesequence 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:1289) MDRWSVKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 37.9 7.5 37.9 (SEQIDNO:1298) MDRWAVKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQIDNO:1299) MDRWLSKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 16.8 7.5 37.9 (SEQIDNO:1302) MDRWLTKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQIDNO:1300) MDRWLVPWKKKRKIRRRRRRRRRRR nd 37.9 25.3 7.5 25.3 7.5 37.9 (SEQIDNO:1303) MDRFLSKWKKKRKIRRRRRRRRRRR nd 37.9 nd 7.5 25.3 7.5 37.9 (SEQIDNO:1301) MDRFSLKWKKKRKIRRRRRRRRRRR nd nd nd 7.5 25.3 7.5 37.9 (SEQIDNO:1304)

Example 12

In Vivo Characterisation of an Extended APIM Consensus Motif

(86) The work described in this Example investigates the interaction between extended APIM peptides and PCNA.

(87) The inventors have determined unexpectedly that the conventional APIM sequence may be substantially modified without reducing the affinity interaction of the peptide with PCNA. In particular, the inventors have determined that an aromatic or hydrophobic amino acid may be inserted into the APIM sequence to generate a new consensus sequence, as defined by SEQ ID NO: 1. The inventors have also determined that the insertion of an aromatic or hydrophobic amino acid, particularly an aromatic amino acid, allows an increase in flexibility at the C-terminal end of the APIM sequence. Whilst not wishing to be bound by theory, it is thought that the insertion of an aromatic or hydrophobic amino acid, particularly an aromatic amino acid, within the APIM sequence improves the affinity of the peptide, such that it is not essential to include a basic amino acid at the C-terminal end of the APIM sequence to maintain the capacity of the peptide to bind to PCNA.

(88) FRET assays were used to determine the capacity of the extended APIM peptides (peptides containing an extended or longer APIM sequence) to bind to PCNA, as described in Example 4.

(89) FIG. 16 shows the FRET signal for a variety of APIM peptides containing a pentamer motif and three peptides containing an extended hexamer APIM sequence. A significant FRET signal could be detected for all of the extended APIM variants tested. Furthermore, all of the extended APIM peptides generate a signal that is equivalent to, or higher than, the APIM peptides containing a pentamer motif. These results verify that a variety of peptides within the APIM motif definition described herein are capable of interacting with PCNA. Accordingly, peptides containing the extended APIM sequence would therefore be expected to find utility in the method and uses described herein akin to peptides containing the pentamer APIM sequence, i.e., as anti-bacterial peptides, e.g. for treating or preventing a bacterial infection or a bacterial infectious disease as evidenced by the data in the Examples above.

(90) It is particularly surprising that the extended APIM sequence is capable of facilitating the interaction between peptides containing the sequence and PCNA because the extended sequence does not typically occur in proteins that are known to interact with PCNA. Moreover, it was completely unexpected that this extended sequence would facilitate the interaction with PCNA with a similar or improved affinity relative to various pentamer sequences.

Example 13

Import of Extended APIM-containing Peptides

(91) Fluorescently-labelled (FAM-tagged) extended APIM peptide constructs (oligopeptidic compounds as defined herein) were incubated with HeLa cells.

(92) It was found that all of the extended APIM peptides were imported into said cells, showing that the cell penetrating peptide coupled to the extended APIM sequence as defined herein is sufficient to mediate the cellular uptake of the peptides (FIG. 17). This demonstrates that the oligopeptidic compounds of the invention are readily imported into cells and are available to interact with PCNA.

Example 14

Peptides Containing the Extended APIM Sequence are not Cytotoxic to Animal Cells

(93) Cytotoxicity of the peptides containing the extended APIM sequence was investigated by an MTT assay as described below. The ATX-101 peptide, which contains a standard pentamer APIM sequence was used as a control.

(94) HEK293 cells (Human embryonic kidney cells) were seeded into 96 well plates (6000 cells/well) and incubated for 3 hours. After 24 hours peptides were added to the cells in serum free media and incubated for 1 h. Fresh media was added and the cells were harvested after additional 24, 72 and 96 hours. MTT was added to the cells (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and OD was measured at 565 nm, and the average from at least 6 wells was used to calculate cell survival. Data is presented in FIG. 18 as growth from one representative experiment and has been reproduced at least 2 times.

(95) The results show that the peptides containing the extended APIM sequence do not affect the growth of normal animal cells, i.e., the peptides are not cytotoxic to animal cells, even though they may bind to PCNA with a greater affinity than some peptides containing the standard pentamer APIM sequence. The absence of cytotoxic activity in normal animal cells also indicates that peptides containing the extended APIM motif would be useful in the methods and uses defined herein as the peptides have similar functional activity to peptides containing the standard pentamer sequence, which have been shown to function as anti-bacterial peptides, as discussed in the Examples above.

Example 16

Overexpression of extended APIM Peptides in E. coli

(96) In order to verify that the extended APIM peptides have an anti-bacterial effect, peptides containing only the extended APIM sequence (i.e. without a cell-penetrating peptide) were over-expressed in E. coli using the expression vector pET28, using the method described in Example 7. Expression of a short version of the APIM sequence, which does not fall within the consensus sequence, was used as a control. The bacterial strain without a plasmid was used as a further control.

(97) FIG. 19 shows that expression of various peptides containing an extended APIM peptide inhibits bacterial growth (measured by OD). This result demonstrates that the extended APIM sequences have anti-bacterial properties even in the absence of a cell-penetrating peptide. FIG. 20 shows the number of colony forming units (CFU) or bacterial growth as measured by OD.sub.600 of E. coli BL21 overexpressing various standard and extended APIM peptides. The results demonstrate that the extended APIM peptides are antibacterial.

Example 16

Antibacterial Activity of API Peptides by Addition to E. coli BL21

(98) To verify that peptides containing an extended APIM sequence are capable of inhibiting bacterial growth, several APIM peptides (without a FAM-tag) were added to cultures of E. coli BL21 at various concentrations. The growth of the bacteria was assessed by counting the number of CFUs.

(99) FIG. 21 shows that all tested APIM peptides were effective at reducing bacterial growth in comparison to control peptides, e.g. R11 (a cell penetrating peptide) or no peptide. Furthermore, the peptides were effective irrespective of the cell-penetrating peptide attached to the APIM sequence.

Example 17

Effect of APIM Peptides on Mutation Frequency in E. coli BL21

(100) The frequency of rifR (mutations in the rpoB gene) was determined by calculating number of rifR per CFU. Cultures of E. coli BL21(ripl) expressing various extended APIM peptides were grown on LB and the cultures were induced with (IPTG) (1 mM) at OD.sub.600=0.3-0.4 inducing protein expression. Diluted aliquots from the culture were mixed with 3 ml soft agar (LB agar plates with 0.5% agar) and plated on LB agar plates (37 C., 16 h) and LB agar plates with rifampicin (100 g/ml) (37 C., 48 h). Some cultures were treated with UV (1.5 J/cm.sup.3). Controls included untransfected E. coli BL21 cells and cells transfected with a vector encoding a short sequence that does not fall within the APIM consensus. FIG. 22 shows the percentage reduction in mutation frequency and demonstrates that expression of APIM peptides significantly reduces mutation frequency. Whilst not wishing to be bound by theory, it is thought that the APIM peptides may impair the interaction between the -clamp and translesion (TLS) polymerases, thereby inhibiting the activity of the TLS polymerases and their effects on mutation frequency.

(101) FIG. 23 shows the mutation frequency in the E. coli cultures and again confirms that expression of APIM peptides significantly reduces mutation frequency.

(102) FIG. 24 shows the mutation spectra of rpoB (1525-1722 bp) from rifampicin resistant colonies in various E. coli cultures. The figure demonstrates that the expression of APIM peptides results in a change in mutation hotspots, which further supports the hypothesis that the APIM peptides may impair the interaction between the -clamp and translesion (TLS) polymerases.

Example 18

Cytotoxicity of Extended APIM-containing Peptides

(103) Cytotoxicity of the peptides containing the extended APIM sequence (without a FAM-tag) was investigated by an MTT assay as described in Example 14, using both HEK 293 and U2OS cells. The ATX-101 peptide (101), which contains a standard pentamer APIM sequence was used as a control. Furthermore, the peptides were tested in combination with various concentrations of a cytostatic agent, cisplatin.

(104) FIGS. 25-27 show that the peptides containing extended APIM sequence do not affect considerably the growth of normal healthy cells, i.e. the peptides are not cytotoxic to healthy cells, and do not significantly potentiate the effects of cytostatic agents.

(105) The data in FIGS. 25 and 27 also demonstrates that APIM peptides may be coupled to various cell penetrating peptides without affecting the toxicity of the peptides.