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ANTIMICROBIAL PEPTIDES

20200071357 · 2020-03-05

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to peptides of SEQ ID NO: 27 and peptidomimetics thereof and to their use as antimicrobial and anticancer agents. Said peptides being based on the heavy chain (HC) of a centrocin from the sea urchin Echinus esculentus.

    Claims

    1. A peptide that is 12-16 amino acids in length, wherein said peptide comprises an amino acid (AA) sequence of formula (IA) SEQ ID NO:27 TABLE-US-00012 (IA) (SEQIDNO:27) AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.sub.7-AA.sub.8-AA.sub.9-AA.sub.10-AA.sub.11-AA.sub.12 wherein AA.sub.1 is an amino acid that has a hydrophobicity that is less than or equal to the hydrophobicity of glycine and is not an anionic amino acid; AA.sub.2 and AA.sub.3 are each an amino acid with a hydrophobic R group, said R group having at least 4 non-hydrogen atoms; AA.sub.4, AA.sub.5, AA.sub.9, AA.sub.11 and AA.sub.12 are each a cationic amino acid; AA.sub.6is an uncharged amino acid; AA.sub.8 and AA.sub.10 are each an amino acid that is not an anionic amino acid; and AA.sub.7 is an amino acid with a hydrophobic R group, said R group having at least 3 non-hydrogen atoms; or a peptidomimetic thereof.

    2. A peptide or peptidomimetic of claim 1, wherein AA.sub.1 is a cationic amino acid or an uncharged amino acid, preferably an uncharged amino acid.

    3. A peptide or peptidomimetic of claim 1 or claim 2, wherein AA.sub.1 is selected from the group consisting of G, T, S, N, Q, H, K and R.

    4. A peptide or peptidomimetic of any one of claims 1 to 3, wherein AA.sub.1 is an uncharged amino acid selected from the group consisting of G, T, S, N and Q.

    5. A peptide or peptidomimetic of any one of claims 1 to 4, wherein AA.sub.1 is G.

    6. A peptide or peptidomimetic of any one of claims 1 to 3, wherein AA.sub.1 is a cationic amino, preferably lysine (K) or arginine (R), but optionally histidine (H) or any non-genetically coded or modified amino acid carrying a positive charge at pH 7.0.

    7. A peptide or peptidomimetic of any one of claim 1 to 3 or 6, wherein AA.sub.1 is a cationic amino acid selected from the group consisting of H, K and R, preferably K and R, more preferably K.

    8. A peptide or peptidomimetic of any one of claims 1 to 7, wherein at least one of AA.sub.2 and AA.sub.3 has at least 7, preferably at least 9, non-hydrogen atoms in its R group, more preferably both AA.sub.2 and AA.sub.3 have R groups having at least 7, preferably at least 9, non-hydrogen atoms.

    9. A peptide or peptidomimetic of any one of claims 1 to 7, wherein one or both, preferably both, AA.sub.2 and AA.sub.3 has a hydrophobic R group that has a mass of >90Da.

    10. A peptide or peptidomimetic of any one of claims 1 to 9, wherein the hydrophobic R group of AA.sub.2 and/or AA.sub.3 contains a hetero atom such as O, N or S, preferably there is no more than one heteroatom, preferably it is nitrogen.

    11. A peptide or peptidomimetic of any one of claims 1 to 10, wherein AA.sub.2 and AA.sub.3 are each independently selected from the group consisting of W, F, Y, L and I, preferably, AA.sub.2 and AA.sub.3 are each independently selected from the group consisting of W, F and Y.

    12. A peptide or peptidomimetic of any one of claims 1 to 11, wherein at least one, preferably both, of AA.sub.2 and AA.sub.3 is W, F or Y.

    13. A peptide or peptidomimetic of any one of claims 1 to 12, wherein AA.sub.2 and/or AA.sub.3 is W, preferably both AA.sub.2 and AA.sub.3 is W.

    14. A peptide or peptidomimetic of any one of claims 1 to 13, wherein AA.sub.4, AA.sub.5, AA.sub.9, AA.sub.11 and AA.sub.12 are each a cationic amino acid independently selected from the group consisting of K, R, H and any non-genetically coded or modified amino acid carrying a positive charge at pH 7.0.

    15. A peptide or peptidomimetic of any one of claims 1 to 14, wherein AA.sub.4, AA.sub.5, AA.sub.9, AA.sub.11 and AA.sub.12 are each independently selected from the group consisting of K, R and H, preferably R and K.

    16. A peptide or peptidomimetic of any one of claims 1 to 15, wherein AA.sub.4 is R and/or AA.sub.5 is R and/or AA.sub.9 is K and/or AA.sub.11 is R and/or AA.sub.12 is K.

    17. A peptide or peptidomimetic of any one of claims 1 to 16, wherein AA.sub.4 is R, AA.sub.5 is R, AA.sub.9 is K, AA.sub.11 is R and AA.sub.12 is K.

    18. A peptide or peptidomimetic of any one of claims 1 to 17, wherein AA.sub.7 has a hydrophobic R group that has at least 4 non-hydrogen atoms, or at least 7 non-hydrogen atoms, or at least 9 non-hydrogen atoms.

    19. A peptide or peptidomimetic of any one of claims 1 to 18, wherein AA.sub.7 is an amino acid in accordance with the definitions of AA.sub.2 or AA.sub.3 in any preceding claim.

    20. A peptide or peptidomimetic of any one of claims 1 to 19, wherein AA.sub.7 is selected from the group consisting of W, F, Y, L, I, V, P and M, preferably selected from the group consisting of W, F, Y, L, I and V.

    21. A peptide or peptidomimetic of any one of claim 1 to 17 or 20, wherein AA.sub.7 is V.

    22. A peptide or peptidomimetic of any one of claims 1 to 21, wherein one or both of AA.sub.8 and AA.sub.10 is an amino acid in accordance with the definitions of AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.7, AA.sub.9, AA.sub.11 or AA.sub.12 in any preceding claim.

    23. A peptide or peptidomimetic of any one of claims 1 to 22, wherein one or both of AA.sub.8 and AA.sub.10 is an uncharged amino acid, preferably selected from the group consisting of W, F, Y, L, I, V, P, M, C, A, G, T, S, N and Q.

    24. A peptide or peptidomimetic of any one of claims 1 to 23, wherein AA.sub.6 is selected from the group consisting of W, F, Y, L, I, V, P, M, C, A, G, T, S, N and Q.

    25. A peptide or peptidomimetic of any one of claims 1 to 22, wherein one or both of AA.sub.8 and AA.sub.10 is a cationic amino acid, preferably H, K or R, more preferably K or R.

    26. A peptide or peptidomimetic of any one of claims 1 to 25, wherein AA.sub.8 and AA.sub.10 are each independently selected from the group consisting of W, F, Y, L, I, V, P, M, C, A, G, T, S, N, Q, H, K and R, preferably each independently selected from the group consisting of W, F, Y, L, I, V, M, A, G, T, S, N, Q, H, K and R.

    27. A peptide or peptidomimetic of any one of claims 1 to 26, wherein AA.sub.6 is selected from the group consisting of W, F, Y, L, I, V, M, A, G, T, S, N and Q.

    28. A peptide or peptidomimetic of any one of claims 1 to 27, wherein AA.sub.6 is an amino acid in accordance with a definition of AA.sub.1 in claim 4 or claim 5, preferably AA.sub.6 is selected from the group consisting of T, S, N and Q.

    29. A peptide or peptidomimetic of any one of claims 1 to 27, wherein AA.sub.6 is an amino acid that has a hydrophobicity that is greater than or equal to the hydrophobicity of glycine, preferably W or A, more preferably W.

    30. A peptide or peptidomimetic of any one of claim 1 to 27 or 29, wherein AA.sub.6 is an amino acid in accordance with the definition of AA.sub.2, AA.sub.3 or AA.sub.7 in any preceding claim.

    31. A peptide or peptidomimetic of any one of claim 1 to 27, 29 or 30, wherein AA.sub.6 is selected from the group consisting of W, F, Y, L, I, V, P or M, or is selected from the group consisting of W, F, Y, L or I, or is selected from the group consisting of W, F or Y, or is W.

    32. A peptide or peptidomimetic of any one of claims 1 to 27, wherein AA.sub.6 is selected from the group consisting of T, S, N, Q, W or A, preferably selected from the group consisting of T, S, N, Q and W or T, S, N or Q, more preferably T or S.

    33. A peptide or peptidomimetic of any one of claim 1 to 27 or 32, wherein AA.sub.6 is selected from the group consisting of T, A or W.

    34. A peptide or peptidomimetic of any one of claim 1 to 27, 32 or 33, wherein AA.sub.6 is selected from the group consisting of T or W.

    35. A peptide or peptidomimetic of any one of claims 1 to 27 or 32 to 34, wherein AA.sub.6 is T.

    36. A peptide or peptidomimetic of any one of claims 1 to 27 or 32 to 34, wherein AA.sub.6 is W.

    37. A peptide or peptidomimetic of any one of claim 1 to 27 or 32 or 33, wherein AA.sub.6 is A.

    38. A peptide or peptidomimetic of any one of claims 1 to 37, wherein AA.sub.8 is an amino acid in accordance with a definition of AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.7, AA.sub.9, AA.sub.11 or AA.sub.12 in any preceding claim.

    39. A peptide or peptidomimetic of any one of claims 1 to 38, wherein AA.sub.8 is an amino acid in accordance with a definition of AA.sub.1 in any preceding claim, preferably AA.sub.8is selected from the group consisting of T, S, N, Q, H, K and R.

    40. A peptide or peptidomimetic of any one of claims 1 to 38, wherein AA.sub.8 is an amino acid that has a hydrophobicity that is greater than or equal to the hydrophobicity of glycine.

    41. A peptide or peptidomimetic of any one of claims 1 to 39, wherein AA.sub.8 is a cationic amino acid in accordance with the definition of AA.sub.4, AA.sub.5, AA.sub.9, AA.sub.11 and AA.sub.12in any preceding claim.

    42. A peptide or peptidomimetic of any one of claim 1 to 39 or 41, wherein AA.sub.8 is selected from the group consisting of H, K and R, preferably K and R, more preferably AA.sub.8 is K.

    43. A peptide or peptidomimetic of any one of claims 1 to 39, wherein AA.sub.8 is uncharged.

    44. A peptide or peptidomimetic of any one of claim 1 to 39 or 43, wherein AA.sub.8 is selected from the group consisting of T, S, N, Q or A.

    45. A peptide or peptidomimetic of any one of claim 1 to 39, 43 or 44, wherein AA.sub.8 is A.

    46. A peptide or peptidomimetic of any one of claims 1 to 45, wherein AA.sub.10 is an amino acid in accordance with a definition of AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.7, AA.sub.9, AA.sub.11 or AA.sub.12 in any preceding claim.

    47. A peptide or peptidomimetic of any one of claims 1 to 46, wherein AA.sub.10 is an amino acid in accordance with a definition of AA.sub.1 in any preceding claim, preferably AA.sub.10 is selected from the group consisting of T, S, N, Q, H, K and R.

    48. A peptide or peptidomimetic of any one of claims 1 to 46, wherein AA.sub.10 is uncharged.

    49. A peptide or peptidomimetic of any one of claims 1 to 46, wherein AA.sub.10 is an amino acid that has a hydrophobicity that is greater than or equal to the hydrophobicity of glycine.

    50. A peptide or peptidomimetic of any one of claim 1 to 46, 48 or 49, wherein AA.sub.10 is not A.

    51. A peptide or peptidomimetic of any one of claims 1 to 46, wherein AA.sub.10 is an amino acid in accordance with a definition of AA.sub.7 any preceding claim.

    52. A peptide or peptidomimetic of any one of claims 1 to 46, wherein AA.sub.10 is an amino acid in accordance with a definition of AA.sub.2 or AA.sub.3 in any preceding claim.

    53. A peptide or peptidomimetic of any one of claim 1 to 46, 51 or 52, wherein AA.sub.10 is selected from the group consisting of W, F, Y, L, I, V, P and M, preferably selected from the group consisting of W, F, Y, L, I and V.

    54. A peptide or peptidomimetic of any one of claim 1 to 46 or 51 or 53, wherein AA.sub.10 is V.

    55. A peptide or peptidomimetic of any one of claims 1 to 46, wherein AA.sub.10 is a cationic amino acid in accordance with the definition of AA.sub.4, AA.sub.5, AA.sub.9, AA.sub.11 and AA.sub.12in any preceding claim.

    56. A peptide or peptidomimetic of any one of claim 1 to 46 or 55, wherein AA.sub.10 is selected from the group consisting of H, K and R, preferably selected from the group consisting of K and R.

    57. A peptide or peptidomimetic of any preceding claim, wherein AA.sub.6 is T, A or W, preferably T or W, and/or AA.sub.8 is A or K or R, preferably A, and/or AA.sub.10 is V.

    58. A peptide or peptidomimetic of any preceding claim, wherein AA.sub.6 is T, AA.sub.8 is A and AA.sub.10 is V.

    59. A peptide of claim 1, wherein said peptide comprises, or consists of, the amino acid sequence GWWRRTVAKVRK (SEQ ID NO:10), or a peptidomimetic thereof.

    60. A peptide of claim 1, wherein said peptide is 12 amino acids in length and consists of the amino acid sequence GWWRRTVAKVRK (SEQ ID NO:10).

    61. A peptide of claim 1, wherein said peptide comprises, or consists of, the amino acid sequence GWWRRWVAKVRK (SEQ ID NO:20), or a peptidomimetic thereof.

    62. A peptide of claim 1, wherein said peptide is 12 amino acids in length and consists of the amino acid sequence GWWRRWVAKVRK (SEQ ID NO:20).

    63. A peptide of claim 1, wherein said peptide comprises, or consists of, the amino acid sequence GWWRRAVAKVRK (SEQ ID NO:8), or a peptidomimetic thereof.

    64. A peptide of claim 1, wherein said peptide is 12 amino acids in length and consists of the amino acid sequence GWWRRAVAKVRK (SEQ ID NO:8).

    65. A peptide of claim 1, wherein said peptide comprises, or consists of, the amino acid sequence GWWRRWVKKVRK (SEQ ID NO:22), or a peptidomimetic thereof.

    66. A peptide of claim 1, wherein said peptide is 12 amino acids in length and consists of the amino acid sequence GWWRRWVKKVRK (SEQ ID NO:22).

    67. A peptide of claim 1, wherein said peptide comprises, or consists of, the amino acid sequence KWWRRWVKKVRK (SEQ ID NO:23), or a peptidomimetic thereof.

    68. A peptide of claim 1, wherein said peptide is 12 amino acids in length and consists of the amino acid sequence KWWRRWVKKVRK (SEQ ID NO:23).

    69. A peptide of claim 1, wherein said peptide comprises, or consists of, the amino acid sequence RWWRRWVRRVRR (SEQ ID NO:25), or a peptidomimetic thereof.

    70. A peptide of claim 1, wherein said peptide is 12 amino acids in length and consists of the amino acid sequence RWWRRWVRRVRR (SEQ ID NO:25).

    71. A peptide or peptidomimetic of any preceding claim, wherein said peptide comprises, or consists of, the amino acid sequence GWWRRTVAKVRK (SEQ ID NO:10), or a sequence substantially homologous thereto, wherein said substantially homologous sequence contains 1, 2 or 3 amino acid substitutions compared to the given amino acid sequence (SEQ ID NO:10), and wherein (i) if the G at position 1 is replaced, the replacement amino acid is in accordance with AA.sub.1 as defined in any preceding claim; (ii) if one or both of the W residues at positions 2 and 3 is replaced, the replacement amino acid is in accordance with AA.sub.2 or AA.sub.3 as defined in any preceding claim; (iii) if one or more of the residues at positions 4, 5, 9, 11 and 12 is replaced, the replacement amino acid is in accordance with AA.sub.4, AA.sub.5, AA.sub.9, AA.sub.11 or AA.sub.12 as defined in any preceding claim; (iv) if one or more of the residues at positions 6, 8 or 10 is replaced, the replacement amino acid is in accordance with AA.sub.6, AA.sub.8or AA.sub.10, as defined in any preceding claim; and (v) if the V at position 7 is replaced, the replacement amino acid is in accordance with AA.sub.7 as defined in any preceding claim.

    72. A peptide or peptidomimetic of any preceding claim, wherein said peptide or peptidomimetic is 12 amino acids in length.

    73. A peptide or peptidomimetic of any preceding claim, wherein the molecule is a peptide.

    74. A peptide or peptidomimetic of any preceding claim, wherein said peptide or peptidomimetic is amidated at the C-terminus.

    75. A formulation comprising a peptide or peptidomimetic as defined in any one of the preceding claims in admixture with a suitable diluent, carrier or excipient.

    76. The formulation of claim 75, which is a pharmaceutical formulation.

    77. A peptide or peptidomimetic of any one of claims 1 to 74 for use in therapy.

    78. A peptide or peptidomimetic of any one of claims 1 to 74 for use in the treatment of a microbial infection, preferably a bacterial or fungal infection.

    79. Use of a peptide or peptidomimetic as defined in any one of claims 1 to 74 as an antimicrobial agent, preferably an antibacterial or antifungal agent.

    80. A peptide or peptidomimetic of any one of claims 1 to 74 for use in the treatment of cancer.

    81. A method of treating a microbial infection, preferably a bacterial or fungal infection, said method comprising administering to a patient in need thereof a therapeutically effective amount of a peptide or peptidomimetic as defined in any one of claims 1 to 74.

    82. A method of treating cancer, said method comprising administering to a patient in need thereof a therapeutically effective amount of a peptide or peptidomimetic as defined in any one of claims 1 to 74.

    83. Use of a peptide or peptidomimetic as defined in any one of claims 1 to 74 in the manufacture of a medicament for treating a microbial infection, preferably a bacterial or fungal infection.

    84. Use of a peptide or peptidomimetic as defined in any one of claims 1 to 74 in the manufacture of a medicament for treating cancer.

    Description

    EXAMPLE 1

    [0169] Increased microbial resistance to commercial antibiotics has led to an extensive search for novel antimicrobial agents to overcome this challenge. Antimicrobial peptides (AMPs) have the ability to kill bacterial pathogens and have therefore attracted interest as novel antimicrobial lead compounds. EeCentrocin 1 is a potent AMP, originally isolated from the marine sea urchin Echinus esculentus. The AMP has a hetero-dimeric structure with the pharmacophore located in its largest monomer (the heavy chain, HC), containing 30 amino acids. In the present study, the pharmacophore has been located within the HC and structure-activity relationship studies and sequence modification of the identified pharmacophore has been done. A lead peptide identified is superior in antifungal activity compared to the other peptides with minimal inhibitory concentrations (MICs) in the low micromolar range and also retains good antibacterial activity. In addition, the peptide displayed minor haemolytic activity.

    [0170] Materials and Methods

    [0171] Solid Phase Peptide Synthesis (SPPS)

    [0172] The non-brominated heavy chain (HC) of EeCentrocin 1, the truncated peptide HC(1-16), and the modified peptide HC(1-16)A8 were synthesised commercially (GenicBio Ltd., Shanghai, China). The other peptides were synthesized by microwave assisted Fmoc-based solid phase peptide synthesis (Fmoc-SPPS). All Fmoc-amino acids and solvents were purchased from Sigma-Aldrich (MO, USA) whereas Rink amide ChemMatrix resin was obtained from Biotage (Uppsala, Sweden). The most efficient procedure involved using Rink amide ChemMatrix resin (loading 0.47-0.49 mmol/gram), which was swelled in N,N-dimethylformamide (DMF) in a 10 ml fritted reaction vial for 20 min with microwave heating at 70 C. Fmoc-amino acids (4.2 eq.) were dissolved in N-methyl-2-pyrrolidone (NMP) prior to in situ coupling with O-(6-chlorobenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HCTU, 4.12 eq.) and N,N-diisopropylethylamine (DIEA, 8.4 eq.) as base, and coupling for five min with microwave heating at 75 C. Fmoc-Arg(Pbf)-OH was coupled at room temperature for 60 min to avoid -lactamisation of its side-chain and we found it necessary to double couple the N-terminal Gly-residue to avoid Gly-1 deletion peptides. Fmoc-cleavage was performed with a solution of 20% piperidine in DMF (4.5 ml for three min and repeated for 10 min) at room temperature, and the resin washed with DMF (44.5 ml for 0.45 min). After the final coupling and Fmoc-cleavage of the N-terminal Gly-residue, the resin was washed thoroughly with dichloromethane (DCM) and dried overnight in a desiccator. A 10 ml solution of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% H.sub.2O was used as cleavage cocktail and added to the 10 ml fritted reaction vial with gentle stirring every hour for 3-3.5 h. The solution was filtered on a Supelco Visiprep vacuum manifold and the cleavage process was repeated with 5 ml of the cleavage cocktail for 0.5-1 h. The solution was concentrated in vacuo and ice-cold diethyl ether was added for precipitation of the crude peptide. The precipitated crude peptide was washed three times with ice-cold diethyl ether to remove traces of the cleavage cocktail.

    [0173] High-Performance Liquid Chromatography

    [0174] The peptides were purified by RP-HPLC using a Waters 2690 module equipped with a Waters 996 photodiode array detector and an XBridge C.sub.18, 5 m, 10250 mm column (Waters, Mass. USA). The mobile phases consisted of buffer A: H.sub.2O/0.1% TFA and buffer B1: 80% ACN/20% H.sub.2O/0.1% TFA (Sigma-Aldrich). Depending on the individual peptide (hydrophobicity and co-eluting reagents), linear gradients for purification went from 5, 10, 15 or 17% buffer B to 35% buffer B in 24 min and with a flow of 2 ml/min for one min initially, followed by 5 ml/min during the run. The purity of all peptides was estimated to be above 95%.

    [0175] Mass Spectrometry

    [0176] Molecular weight and purity of the peptides were confirmed using a 6540B Q-TOF mass spectrometer with a dual ESI source, coupled to a 1290 Infinity UHPLC system, controlled by the MassHunter software (Agilent, Calif., USA). The peptides were separated using a Zorbax C.sub.18, 2.150 mm, 1.8 m column (Agilent). System details and typical parameters are found in Table C. A specific gradient running from 3-20% buffer B2 (ACN/0.1% formic acid) was applied for the determination of retention times of the 12-mer alanine-scan peptides.

    TABLE-US-00007 TABLE C Typical parameters for HPLC-MS HPLC system 1290 Infinity analytical binary G4220A pump with degasser 1290 Infinity TCC column oven G1316C 1290 Infinity Autosampler with G4226A thermostat Gradient 5% to 60% buffer B2 Flow-rate 0.4 ml/min Column temperature 40 C. Q-TOF Dual ESI Positive ion mode ADC 2 GHz (analog-to-digital) acquisition Gas temperature 300 C. Drying gas 8 L/min Nebulizer gas 35 Psig Capillary voltage 3.5 kV Fragmentor 175 V Skimmer 65 V Drying and nebulizer gas N.sub.2 Max range m/z for sample 100-3200 acquisition Reference mass 121.050873, 922.009798 Software Masshunter Acquisition Vesion B.06.01 Build 6.01.6157 MassHunter Qualitative Vesion B.07.00 Build Analysis 7.0.7024.29 service pack 1

    [0177] Antibacterial Assay

    [0178] The peptides were screened for antibacterial activity against two strains of Gram-positive and two strains of Gram-negative bacteria; Corynebacterium glutamicum (Cg, ATCC 13032), Staphylococcus aureus (Sa, ATCC 9144), Pseudomonas aeruginosa (Pa, ATCC 27853) and Escherichia coli (Ec, ATCC 25922).

    [0179] Cultures stored at 80 C. in glycerol were transferred to Mller-Hinton plates (MH, Difco, Lawrence, Kans., USA) and incubated for 24 h at 35 C. A few colonies of each bacterial strain were transferred to 5 ml liquid MH medium and left shaking at room temperature overnight at 600 rpm. Cultures of actively growing bacteria (20 l) were inoculated in 5 ml MH medium and left shaking for 2 h at room temperature. The antibacterial assays were performed as previously described by Sperstad, S. V., et al. ((2009) Mol. Immunol. 46, 2604-2612) with the following exception: bacterial cultures were diluted with medium to 2.5-310.sup.4 bacteria/ml concentrations. An aliquot of 50 l (1250-1500 bacterial cells) was added to each well in 96-well Nunclon microtiter plates (Nagle Nunc Int., Denmark) preloaded with peptide solution (50 l).

    [0180] The microtiter plates were incubated for 24 h at 35 C. with optical density recorded every hour using an Envision 2103 multilabel reader, controlled by a Wallac Envision manager (PerkinElmer, Conn., USA). Minimum inhibitory concentration (MIC) was defined as a sample showing complete inhibition (as measured by optical density at 595 nm) compared to the negative (growth) controls, consisting of bacteria and water. Oxytetracycline (20 M) served as a positive (inhibition) control.

    [0181] The synthetic peptides were tested for antibacterial activity in concentrations ranging from 200 to 0.1 M in two-fold dilutions. All tests were performed in triplicates.

    [0182] Antifungal Assay

    [0183] The synthetic peptides were also screened for antifungal activity against Candida albicans (ATCC 10231), Aureobasidium pullulans and Rhodotorula sp. (the last two were obtained from Professor Arne Tronsmo, The Norwegian University of Life Sciences, As, Norway). The antifungal assay was performed as previously described (Sperstad, S. V., et al. (2009) Dev. Comp. Immunol. 33, 583-591). Briefly, fungal spores were dissolved in potato dextrose broth (Difco, Lawrence, Kans., USA) to a concentration of 410.sup.5 spores/ml. The spores (50 l) were inoculated on 96-well Nunclon microtiter plates containing the synthetic peptides (50 l) dissolved in MQ-H.sub.2O. Fungal growth and MIC (defined as the lowest concentration of peptide giving no visible growth) were determined visually after incubation for 24 h at room temperature. The negative (growth) control consisted of medium and fungal solution. The peptides were tested for activity in concentrations ranging from 100 to 0.1 M in two-fold serial dilutions. All tests were performed in triplicates.

    [0184] Haemolytic Assay

    [0185] Selected synthesised peptide analogues were also screened for haemolytic activity using human red blood cells as described previously (Sperstad, S. V., et al. (2009) Dev. Comp. Immunol. 33, 583-591). The assay was performed on 96-well U-shaped microtiter plates (Nagle Nunc) with 50 l peptide sample, 40 l phosphate-buffered saline (PBS) and 10 l red blood cells. After one hour of incubation at 37 C. in a shaker, the plate was centrifuged at 200 g for 5 min and the supernatants (60 l) were carefully transferred to a new flat-bottomed polycarbonate microtiter plate (Nagle Nunc) and the release of haemoglobin (absorbance at 550 nm) was measured on a Synergy H1 multimode reader (BioTek, Vt., USA). Cell suspension added 0.05% Triton X-100 (Sigma-Aldrich, Mo., USA) in PBS served as positive (100% haemolysis) control and cell suspension added PBS served as negative (0% haemolysis, blank) control. The percent haemolysis was calculated using the formula [(Asample-Abaseline)/(Atriton-Abaseline)]100. The cytotoxic peptide melittin (Sigma-Aldrich) was used as a positive control peptide and for comparison. The experiment was performed in triplicates with peptide concentrations ranging from 200 M to 1.56 M in two-fold dilutions.

    [0186] Bioinformatics

    [0187] Peptide properties were calculated with PEPCALC (http://pepcalc.com) from Innovagen, and helical wheel projections made with Pepwheel at the EMBOSS suite http://www.bioinformatics.nl/cgi-bin/emboss/pepwheel. Secondary structures were predicted using PEP-FOLD3 (Plamiable, A., et al. (2016) Nucleic Acids Res. 44, W449-W454) at the Mobyle portal and resulting figures were made with BIOVIA Discovery Studio Visualizer v4.5.0.15071. Homology search was performed with BLAST searching for non-redundant protein sequences at the National Centre for Biotechnological Information (NCBI) homepage (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Zhang, Z., et al. (2000) J. Comput. Biol. 7, 203-214).

    [0188] Results and Discussion

    [0189] Truncation of Non-Brominated HC

    [0190] In silico modelling of the EeCentrocin 1 HC revealed that the N-terminal part of the sequence most likely forms an -helix. In addition, the N-terminal part has an abundance of hydrophobic and positively charged residues. The last 14 C-terminal amino acids were removed from the HC of EeCentrocin 1, resulting in the peptide HC(1-16). The peptide contains two Trp and six positively charged (Arg/Lys) residues. Truncation led to reduced antibacterial activity, especially against S. aureus (the truncation experiments are presented in Table 1). However, the Gram-positive C. glutamicum was still sensitive to HC(1-16) albeit at slightly higher concentrations. An 8-fold decrease in activity was also observed against the Gram-negative test bacteria (Escherichia coli and Pseudomonas aeruginosa).

    TABLE-US-00008 TABLE 1 Sequences and in vitro antibacterial (MIC, M) activities of EeCentrocin 1 HC and truncated analogues. Amino acid sequence and position Peptide Mw (Da) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 HC 3255.7 G W W R R T V D K V R N A G R K V A G HC(1-16) 1985.3 G W W R R T V D K V R N A G R K HC(1-16)A8 1940.3 G W W R R T V A K V R N A G R K HC(2-16)A7 1883.2 W W R R T V A K V R N A G R K HC(1-12)A8 1527.8 G W W R R T V A K V R N HC(1-12)A8K12 1541.9 G W W R R T V A K V R K HC(1-9)R8 1243.5 G W W R R T V R K Amino acid sequence and position MIC (M) Peptide 20 21 22 23 24 25 26 27 28 29 30 C-term Charge Cg Sa Pa Ec HC F A S K A C G A L G H OH +6 0.4 3.1 1.6 0.8 HC(1-16) OH +5 0.8 200 12.5 6.3 HC(1-16)A8 NH.sub.2 +7 0.8 12.5 3.1 3.1 HC(2-16)A7 NH.sub.2 +7 0.4 12.5 25 6.3 HC(1-12)A8 NH.sub.2 +5 1.6 50 6.3 6.3 HC(1-12)A8K12 NH.sub.2 +6 0.4 12.5 1.6 3.1 HC(1-9)R8 NH.sub.2 +5 3.1 100 25 50 MIC: Minimal Inhibitory Concentration

    [0191] The negatively charged Asp8 residue was replaced by an Ala-residue and the C-terminal residue amidated in HC(1-16)A8. The peptide HC(1-16)A8 was antibacterial at low concentrations and clearly pointed to the importance of eliminating negatively charged groups. Thus, all subsequent synthesised AMPs were prepared with a C-terminal amide.

    [0192] Early in the process, it was found that eliminating the N-terminal Gly1 was not beneficial for antibacterial activity as shown for the resulting peptide HC(2-16)A7. This peptide displayed a remarkable drop in antibacterial activity against the Gram-negative P. aeruginosa compared to the HC(1-16)A8 analogue. However, the activity towards the other test strains remained similar.

    [0193] As the N-terminal truncation proved unsuccessful against P. aeruginosa, the focus was directed towards further truncation of the C-terminal end, producing the peptide HC(1-12)A8. The antibacterial activity of HC(1-12)A8 was somewhat reduced compared to the larger HC(1-16)A8 against all strains. In an attempt to improve the antibacterial activity of this 12-residue peptide, the C-terminal Arg-Lys-motif, which was recognised in the 16-residue peptides, was reinstated. This also made it possible to replace the original Asn12-residue, which could compromise peptide integrity by forming aspartimide side-products in SPPS involving chain-elongation through its side-chain and not the peptide-backbone. The resulting peptide HC(1-12)A8K12 (FIG. 1) was the most potent shortened AMP tested with antibacterial activities towards C. glutamicum and P. aeruginosa identical to the original HC peptide.

    [0194] An analogue HC(1-9)R8 was synthesised to further shorten the peptide sequence and also reinstate the C-terminal Arg-Lys-motif. However, the potency of this 9-residue peptide was much lower than the previous peptides.

    [0195] Accordingly, HC(1-12)A8K12 peptide was chosen as lead peptide, and represented an AMP two-fifths (40%) of the sequence-size (length) of the original HC (full-length HC).

    [0196] Alanine-Scan of the Lead Peptide HC(1-12)A8K12

    [0197] In order to investigate the importance of individual residues of the lead peptide, each amino acid was substituted by Ala and antibacterial activity was recorded for each peptide. The peptides were named (apart from the lead peptide, HC(1-12)A8K12) according to the original amino acid, position and substitution, i.e. the peptide where Gly was substituted with Ala in position 1, was named G1A (Table 2). Table 2 shows the antibacterial results from the Ala-scan on the lead peptide HC(1-12)A8K12.

    TABLE-US-00009 TABLE 2 Antibacterial activity (MIC, M) of alanine scan peptides derived from the lead peptide HC(1-12)A8K12. The nomenclature indicates which amino acid has been exchanged with alanine (e.g. G1A: Gly1 is exchanged with Ala). The table also shows sequences, molecular weights (Mw), retention time (RT, min) on a RP-HPLC column, and peptide net charge. Amino acid sequence and position MIC (M) Peptide Mw (Da) 1 2 3 4 5 6 7 8 9 10 11 12 C-term Charge RT Cg Sa Pa Ec G1A 1555.9 A W W R R T V A K V R K NH.sub.2 +6 5.3 0.8 25 6.3 12.5 W2A 1426.7 G A W R R T V A K V R K NH.sub.2 +6 2.1 3.1 100 200 50 W3A 1426.7 G W A R R T V A K V R K NH.sub.2 +6 2.2 0.8 25 100 100 R4A 1456.7 G W W A R T V A K V R K NH.sub.2 +5 7.9 1.6 50 12.5 6.3 R5A 1456.7 G W W R A T V A K V R K NH.sub.2 +5 7.3 0.8 100 25 6.3 T6A 1511.8 G W W R R A V A K V R K NH.sub.2 +6 5.9 1.6 12.5 3.1 3.1 V7A 1513.8 G W W R R T A A K V R K NH.sub.2 +6 4.0 1.6 100 12.5 12.5 HC(1-12)A8K12 1541.9 G W W R R T V A K V R K NH.sub.2 +6 5.2 0.4 12.5 1.6 3.1 K9A 1484.8 G W W R R T V A A V R K NH.sub.2 +5 7.1 1.6 100 6.3 6.3 V10A 1513.8 G W W R R T V A K A R K NH.sub.2 +6 4.1 1.6 100 6.3 12.5 R11A 1456.7 G W W R R T V A K V A K NH.sub.2 +5 6.8 1.6 100 12.5 12.5 K12A 1484.8 G W W R R T V A K V R A NH.sub.2 +5 6.9 0.8 50 6.3 6.3 MIC: Minimal Inhibitory Concentration.

    [0198] All Ala-scan peptides displayed antibacterial activity, but to a different degree against the different strains. Overall, C. glutamicum was the most sensitive strain to the peptides. The other Gram-positive strain, S. aureus, was the least sensitive in the experiment, resisting all the AMPs in concentrations below 12.5 M. Four AMPs were antibacterial towards S. aureus in concentrations <50 M; the lead peptide (HC(1-12)A8K12), G1A, W3A and T6A, and an additional two AMPs were antibacterial towards S. aureus at 50 M concentrations; R4A and K12A. Against the two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa) the lead peptide, T6A, K9A and K12A were antibacterial at concentrations 6.3 M, whereas W2A and W3A were the least antibacterial with MICs 50 M against the Gram-negative bacteria. The stand-out (i.e. best) performer in terms of antibacterial profile (antibacterial activity across the strains tested) was the lead peptide (HC(1-12)A8K12).

    [0199] In general, all Ala-substitutions of the lead peptide resulted in reduced antibacterial activity. The peptide T6A was generally the most potent AMP after the lead peptide. However, while T6A was only marginally less potent than the lead peptide against P. aeruginosa, a four-fold dilution separated T6A and the lead peptide towards C. glutamicum. This observation indicated a selective drop in the antibacterial activity of T6A towards C. glutamicum. The lead peptide and T6A were different in that Thr is a polar residue without charge whereas Ala is smaller and more hydrophobic.

    [0200] Replacement of the positively charged residues (R4A, R5A, K9A, R11A and K12A) with Alanine resulted in 2-8 fold reduction of antibacterial activity against all strains tested. Of notice, Lys12, which is positioned at the C-terminal end of the peptide, seemed to be the least important positively charged residue.

    [0201] An interesting pair when considering individual amino acid substitutions were the Tryptophan substitutions represented by the peptides W2A and W3A. In the current Ala-scan, Ala-substitutions of W2 or W3 resulted in a dramatic loss of antibacterial activity. W3A and G1A have similar potency towards the Gram-positive bacteria, but W3A is noticeably less potent than G1A towards the Gram-negative bacteria. The one AMP that was consistently the least potent towards all strains was W2A, which indicates that Trp2 may be a more important residue for antibacterial activity than Trp3. The exception was the data against E. coli where W2A was somewhat more potent than W3A. As shown in Table 2, the retention times (hydrophobicity) for W2A and W3A on a C18 RP-HPLC column was highly reduced compared to the lead peptide HC(1-12)A8K12. This illustrated the importance of the hydrophobic character contributed by the two Tryptophan-residues (both located in the hydrophobic face of the -helix) to the antibacterial activity.

    [0202] Replacement of the polar uncharged Gly1 with the small hydrophobic Ala (G1A) resulted in 2-4 fold increase in MIC against all bacterial strains compared to the lead peptide. In the helical wheel projection (FIG. 2B) Gly1 is positioned in the polar and cationic face of the -helix. In addition, glycine does not have a side chain and may therefore provide increased flexibility to this region of the peptide.

    [0203] Ala-substitution of the hydrophobic Val7 (V7A) or Val10 (V10A), both positioned in the hydrophobic region of the -helix (FIG. 2B), resulted in 4-8 fold decrease in activity against all bacterial strains tested. Valine contains an isopropyl side chain, in contrast to the methyl side chain displayed by Alanine. Replacement of Val with Ala would therefore slightly decrease the size of the hydrophobic sector and thereby the amphipacity of the peptide. As shown in Table 2, the retention times are reduced for V7A and V10A, indicating reduced hydrophobicity for these two peptides compared to the lead peptide.

    [0204] In a further experiment, the peptide GWWRRWVAKVRK (amidated at C-terminus) was also tested for antibacterial activity. This peptide differs from the lead peptide HC(1-12)A8K12 in that the T at position 6 has been replaced by a W. This peptide showed good antibacterial activity, with a MIC against C. glutamicum of 0.8 M, a MIC against S. aureus of 3.1 M, a MIC against P. aeruginosa of 1.6 M and a MIC against Escherichia coli of 1.6 M.

    [0205] Antifungal Activity

    [0206] The synthesised peptides were subjected to antifungal screening against the moulds A. pullulans and Rhodotorula sp., and the yeast C. albicans. Surprisingly, the lead peptide HC(1-12)A8K12 was superior in activity compared to the other peptides, including the full HC peptide (Table 3). The lead peptide was antifungal at concentrations (MICs) ranging from 1.6-6.3 M, whereas the other peptides had MICs from 12.5 M and upwards. Thus, the lead peptide HC(1-12)A8K12 was the stand-out (i.e. best) performer in terms of antifungal activity. In addition, whereas the full HC is more potent against bacteria than fungi, the lead peptide HC(1-12)A8K12 is equally active against both types of microorganisms, i.e. showing broad-spectrum antimicrobial activity.

    [0207] In a further experiment, the peptide GWWRRWVAKVRK (amidated at C-terminus) was also tested for antifungal activity. This peptide differs from the lead peptide HC(1-12)A8K12 in that the T at position 6 has been replaced by a W. This peptide showed good antifungal activity, with a MIC against C. albicans of 12.5 M and a MIC against Rhodotorula sp. of 1.6 M.

    [0208] Haemolytic Activity

    [0209] To test whether the lead peptide or the other peptides were cytotoxic, their haemolytic activity on human red blood cells was determined. The data obtained indicated a correlation between the antibacterial and the haemolytic activities. The peptides showing highest haemolytic activities were the lead peptide and the full HC, with 25% and 75% haemolysis at 200 M respectively (Table 3 and FIG. 3). At concentrations closer to the MIC-values, the haemolytic activity of the lead peptide is negligible. All the other peptides displayed minor (5%) or no haemolytic activity at 200 M. By contrast, the bee venom peptide melittin displayed 100% haemolysis at concentrations as low as 6.3 M (FIG. 3).

    TABLE-US-00010 TABLE 3 Antifungal (MIC, M) and haemolytic activities (% haemolysis at 200 M and 25 M) of EeCentrocin 1 HC, truncated analogues and alanine scan peptides. MIC (M) C. A. Rhodotorula Haemolysis (%) Peptide albicans pullulans sp. 200 M 25 M HC 100 12.5 12.5 74.6 13.5 HC(1-16) 100 50 25 0.0 0.0 HC(1-16)A8 Nt Nt Nt 5.0 0.0 G1A 25 50 12.5 1.6 0.0 W2A 50 50 25 0.0 0.0 W3A 50 50 25 0.0 0.0 R4A 25 50 50 3.6 0.0 R5A 25 25 12.5 0.0 0.0 T6A 25 25 12.5 4.1 0.9 V7A 25 25 12.5 0.0 0.0 HC(1-12)A8K12 3.1 6.3 1.6 25.1 2.4 K9A 25 25 12.5 5.1 0.0 V10A 25 50 12.5 1.0 0.0 R11A 25 50 12.5 0.0 0.0 K12A 25 50 12.5 0.0 0.0 MIC: Minimal Inhibitory Concentration Nt: Not tested

    CONCLUSION

    [0210] Natural AMPs can be challenging to synthesise due to both large size and post-translational modifications. However, those properties are not always necessary for the antimicrobial activity as the pharmacophore may be located in only a minor sequence-motif and post-translational modifications can have a variety of purposes. In the present study, the pharmacophore of the antimicrobial peptide EeCentrocin 1 HC was located to the N-terminal part of the sequence. Truncation of EeCentrocin 1 HC, and selected amino acid substitutions, combined with C-terminal amidation, led to the production of a 12-residue lead peptide with potent antibacterial and antifungal activities. The lead peptide HC(1-12)A8K12 possibly forms an -helical structure. This is supported by helical wheel projection and secondary structure predictions. The peptide also displays low haemolytic activity at the MIC, making it a promising lead peptide for further drug development.

    EXAMPLE 2

    [0211] Further peptides were synthesised and tested for their antimicrobial activity. The methodology used is as per the description in Example 1.

    [0212] These peptides are named HC(1-12)W6A8K12, HC(1-12)A3W6A8K12, HC(1-12)K6A8K12, HC(1-12)W6K8K12, HC(1-12)K1W6K8K12 and HC(1-12)R1W6R8,9,12-NH2 and their amino acid sequences are set forth in Table 4 below.

    [0213] The antifungal and antibacterial MIC data (M) for these peptides is presented in Table 4 below. A new batch (new synthesis) of the peptide HC(1-12)A8K12 was also prepared and tested.

    [0214] Data from Example 1 is also included in Table 4 below.

    TABLE-US-00011 TABLE4 # MIC(M) PeptideName aa Sequence Cg Sa Pa Ec Ca Ap Rh G1A 12 AWWRRTVAKVRK-NH.sub.2 0.8 25 6.3 12.5 25 50 12.5 W2A 12 GAWRRTVAKVRK-NH.sub.2 3.1 100 200 50 50 50 25 W3A 12 GWARRTVAKVRK-NH.sub.2 0.8 25 100 100 50 50 25 R4A 12 GWWARTVAKVRK-NH.sub.2 1.6 50 12.5 6.3 25 50 50 R5A 12 GWWRATVAKVRK-NH.sub.2 0.8 100 25 6.3 25 25 12.5 T6A 12 GWWRRAVAKVRK-NH.sub.2 1.6 12.5 3.1 3.1 25 25 12.5 V7A 12 GWWRRTAAKVRK-NH.sub.2 1.6 100 12.5 12.5 25 25 12.5 HC(1-12)A8K12 12 GWWRRTVAKVRK-NH.sub.2 0.4 12.5 1.6 3.1 3.1 6.3 1.6 K9A 12 GWWRRTVAAVRK-NH.sub.2 1.6 100 6.3 6.3 25 25 12.5 V10A 12 GWWRRTVAKARK-NH.sub.2 1.6 100 6.3 12.5 25 50 12.5 R11A 12 GWWRRTVAKVAK-NH.sub.2 1.6 100 12.5 12.5 25 50 12.5 K12A 12 GWWRRTVAKVRA-NH.sub.2 0.8 50 6.3 6.3 25 50 12.5 HC(1-12)A8K12+ 0.9%NaCl 12 GWWRRTVAKVRK-NH.sub.2 0.4 6.3 6.3 6.3 Newpeptides HC(1-12)A8K12 12 GWWRRTVAKVRK-NH.sub.2 0.8 25 3.1 6.3 12.5 25 3.1 (newsynthesis) HC(1-12)W6A8K12 12 GWWRRWVAKVRK-NH.sub.2 0.8 3.1 1.6 1.6 12.5 50 3.1 HC(1-12)A3W6A8K12 12 GWARRWVAKVRK-NH.sub.2 0.8 50 12.5 6.3 6.3 25 6.3 HC(1-12)K6A8K12 12 GWWRRKVAKVRK-NH.sub.2 1.6 12.5 50 12.5 50 25 25 HC(1-12)W6K8K12 12 GWWRRWVKKVRK-NH.sub.2 1.6 3.1 3.1 6.3 25 12.5 6.3 HC(1-12)K1W6K8K12 12 KWWRRWVKKVRK-NH.sub.2 0.4 6.3 1.6 3.1 6.3 25 3.1 HC(1-12)R1W6R8,9,12-NH2 12 RWWRRWVRRVRR-NH.sub.2 0.8 3.1 3.1 3.1 12.5 25 3.1

    [0215] The data under the heading New peptides in Table 4 demonstrate that peptides HC(1-12)A8K12, HC(1-12)W6A8K12, HC(1-12)W6K8K12, HC(1-12)K1W6K8K12 and HC(1-12)R1W6R8,9,12-NH2 show good antimicrobial activity. Peptides HC(1-12)W6A8K12, HC(1-12)W6K8K12, HC(1-12)K1W6K8K12 and HC(1-12)R1W6R8,9,12-NH2 show particularly good activity against S. aureus (Sa) relative to other peptides.

    [0216] The peptide HC(1-12)A8K12 (initial batch as reported in Example 1) was also tested for antibacterial activity in growth medium with 0.9% NaCI (which corresponds to 154 mM NaCl, physiological saline). The results show that activity against Gram positive bacteria (Cg and Sa) was retained and the activity against Gram-negative bacteria (Pa and Ec) was only slightly reduced. Many antimicrobial peptides lose their activity when exposed to salt-rich environments, like mucus (a problem for cystic fibrosis patients). Thus, the good activity observed in this experiment in the presence of 0.9% NaCI is beneficial.