SHORT ANTIMICROBIAL PEPTIDES

Abstract

The present invention relates to novel short antimicrobial peptides derived from SHf, to pharmaceutical compositions comprising said peptides and to the uses thereof, in particular as medicament, disinfectant, preservative, agent preventing biofilm formation or pesticide.

Claims

1. An antimicrobial peptide or a pharmaceutically acceptable salt thereof, wherein the antimicrobial peptide comprises the sequence: TABLE-US-00003 (SEQIDNO:1) X1-X2-X3-X4-X5-X6-X7-X8, wherein: X1 is an amino acid selected from the group consisting of: F, hF, 4a-F, (C.sub.1-C.sub.4 alkyl)F, W, R, and K, X2 is an amino acid selected from the group consisting of: F, I, W, hF, K, (C.sub.1-C.sub.4 alkyl)F, L, and R, X3 is an amino acid selected from the group consisting of: F, K, hF, R, (C.sub.1-C.sub.4 alkyl)F, and W, X4 is an amino acid selected from the group consisting of: L, F, R, hF, and W, X5 is an amino acid selected from the group consisting of: S, HmS, R, K, and hF, X6 is an amino acid selected from the group consisting of: R and K, X7 is an amino acid selected from the group consisting of: I, F, R, W, and hF, and X8 is an amino acid selected from the group consisting of: F.sub.amide and R.sub.amide; and wherein peptides of SEQ ID NOs: 2 to 13 are excluded.

2. The antimicrobial peptide according to claim 1, wherein: X1 is an amino acid selected from the group consisting of: F, hF, p-.sup.tBuF, 4a-F, W, R, and K, X2 is an amino acid selected from the group consisting of: F, I, W, hF, K, p-.sup.tBuF, L, and R, X3 is an amino acid selected from the group consisting of: F, K, hF, R, p-.sup.tBuF, and W, X4 is an amino acid selected from the group consisting of: L, F, R, hF, and W, X5 is an amino acid selected from the group consisting of: S, HmS, R, K, and hF, X6 is an amino acid selected from the group consisting of: R, and K, X7 is an amino acid selected from the group consisting of: I, F, R, W, and hF, and X8 is an amino acid selected from the group consisting of: F.sub.amide and R.sub.amide.

3. The antimicrobial peptide according to claim 1, wherein said peptide has a net positive charge.

4. The antimicrobial peptide according to claim 3, wherein said net positive charge is at least +2, or is between +2 to +5.

5. The antimicrobial peptide according to claim 1, wherein the value of hydrophobicity of said peptide is comprised from 50 to 80%.

6. The antimicrobial peptide according to claim 1, wherein said peptide comprises at least three amino acids F.

7. The antimicrobial peptide according to claim 1, wherein said peptide is cyclic.

8. The antimicrobial peptide according to claim 1, wherein said peptide: comprises 3 F and 2 R or 3 K; or comprises 4 F and 1 or 2 or 4 R; or comprises 5 F and 2 or 3 R or 3 K; or comprises 6 F and 1 R or 2 R.

9. The antimicrobial peptide according to claim 8, wherein at least one amino acid F of said peptide is substituted by a hF and/or by a (C.sub.1-C.sub.4 alkyl)F.

10. The antimicrobial peptide according to claim 9, wherein (C.sub.1-C.sub.4 alkyl)F is p-.sup.tBuF.

11. The antimicrobial peptide according to claim 1, wherein said peptide comprises an amino sequence selected from the group consisting of: SEQ ID NO: 14 to SEQ ID NO: 79.

12. The antimicrobial peptide according to claim 1, wherein said peptide comprises an amino sequence selected from the group consisting of: SEQ ID NOs: 19, 25, 29, 30, 32, 33, 34, 38, 39, 41, 42, 43, 49, 50, 51, 53, 55, 56, 65, 66, and 72; or wherein said peptide comprises an amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56.

13. The antimicrobial peptide according to claim 1, wherein: X1 is the amino acid F, X2 is the amino acid p-.sup.tBuF, X3 is an amino acid selected from the group consisting of: K, hF and R, X4 is an amino acid selected from the group consisting of: L and F, X5 is the amino acid R, X6 is an amino acid selected from the group consisting of: R and K, X7 is an amino acid selected from the group consisting of: I and F, and X8 is the amino acid F.sub.amide.

14. A pharmaceutical composition comprising at least one antimicrobial peptide according to claim 1, and a pharmaceutically acceptable support and/or excipient.

15. The antimicrobial peptide according to claim 1, wherein the antimicrobial peptide is formulated as a pharmaceutical composition as a medicament.

16. The antimicrobial peptide according to claim 1, wherein the antimicrobial peptide is formulated as a pharmaceutical composition for preventing and/or treating an infection due to a bacterium, a virus, a fungus, or a parasitic.

17. The antimicrobial peptide according to claim 1, wherein said peptide is a disinfectant, preservative or pesticide.

Description

EXAMPLES

Materials and Methods

Peptide Synthesis

[0139] All SHf analogs were synthesized using solid-phase standard Fmoc chemistry protocols, as previously described (Raja et al., Structure, antimicrobial activities and mode of interaction with membranes of novel phylloseptins from the painted-belly leaf frog, Phyllomedusa sauvagii, PLoS One, 2013, 8:e70782) but with the following modifications. Synthesis was carried out on a CEM Liberty Blue automated microwave peptide synthesizer (CEM Corporation, Peptide Synthesis Platform, IBPS, Sorbonne University, Paris, France) using Protide Rink Amide LL resin (CEM Corporation, USA, 0.19 mmol/g substitution). Post-deprotection washing with N,N-dimethylformamide (DMF) was followed by coupling using a diisopropyl carbodiimide (DIC)/Oxyma activation method. The peptidyl resin was cleaved and deprotected by incubation (3 h at room temperature) with an acidic mixture containing 94% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS), 2.5% H.sub.2O and 2.5% 1,2-ethanedithiol (EDT). Resin was removed by filtration and the peptide was precipitated in cold ether. The crude material was then subjected to semi-preparative RP-HPLC on a Phenomenex Luna? C18(2) semi-preparative column (10 ?m, 250?10 mm) eluted at a flow rate of 5 mL/min by a 20-70% linear gradient of acetonitrile (0.07% TFA) in 0.1% TFA/water (1% acetonitrile/min). Peptide purity was assessed by analytical RP-HPLC, followed by MALDI-TOF analysis (Mass Spectrometry and Proteomics Platform, IBPS, Sorbonne University, Paris, France).

Bacterial and Yeast Strains

[0140] The following strains were used: [0141] Gram-negative bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 13883, [0142] Gram-positive bacteria: Staphylococcus aureus ATCC 25923, multi-drug resistant Staphylococcus aureus ATCC BAA-44, Streptococcus pyogenes ATCC 19615, Listeria ivanovii Li4pVS2, Enterococcus faecalis ATCC 29212,

Tests of Antibacterial Activity

[0143] For each strain, a standard inoculum of approximately 10.sup.6 bacteria/mL (exponential growth phase) was prepared. To this end, a colony isolated on LB agar previously inoculated with one of the strains was cultured in 4 ml of LB broth medium, except for S. pyogenes and L. ivanovii which were grown in BHI (Brain Heart Infusion) from a colony isolated on BHI agar. Liquid cultures were then incubated for 2 to 3 hrs at 37? C. with shaking for the bacteria to reach exponential growth phase. After centrifugation, most of the bacterial suspensions were diluted in Mueller-Hinton (MH) broth medium to an OD.sub.630nm of 0.01, which corresponds to a concentration of approximately 10.sup.6 cfu/mL (cfu: colony forming unit). A different medium was used for E. faecalis (LB) and for S. pyogenes and L. ivanovii (BHI).

[0144] The minimum inhibitory concentration (MIC) of each peptide was determined by a test of growth inhibition in broth medium. MIC is defined as the lowest concentration of peptide able to inhibit the growth of the bacterial strain tested after 18-24 hrs of incubation at 37? C. The test was performed in a sterile 96-well microtiter plate. A series of increasing concentrations of peptide (2 to 400 ?M) was first prepared in sterile MilliQ water. 50 ?L of each peptide concentration were mixed into the well with 50 ?L of bacterial suspension (10.sup.6 cfu/ml). The microtiter plate was then incubated for 18-24 hrs at 37? C. with shaking. Bacterial growth was determined by measuring OD at 630 nm (turbidity) on a plate reader. Tests were carried out in triplicate for each peptide concentration and at least three independent experiments were performed to determine the MIC value.

[0145] The growth inhibition negative control was obtained by replacing the solution containing the peptide with 50 ?L of sterile MilliQ water. The positive control allowing the complete inhibition of bacterial growth was obtained by replacing the solution containing the peptide with 50 ?L, of 0.7% formaldehyde.

Cytotoxicity Assay

[0146] Hemolytic experiments were performed using human erythrocytes obtained from healthy adult donors (Etablissement Fran?ais du sang, Paris, France) according to a previously described protocol (Abbassi et al., Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica, Peptides, 2008, 29: 1526-33).

[0147] Briefly, synthetic peptides (1-200 ?M, final concentrations) were incubated (100 ?L, final volume) with erythrocytes (2?10.sup.7 cells) in Dulbecco's phosphate-buffered saline (pH 7.4) for 1 h at 37? C. After centrifugation (12,000?g, 15 s), the absorbance of the supernatant was measured at 450 nm. The LC50 value, which is the average concentration of peptide producing 50% hemolysis, was determined from three independent experiments carried out in triplicate with positive control (100% hemolysis) corresponding to 0.1% triton (v/v).

Results

[0148] Biological activities (antimicrobial activities and cytotoxicity) of SHf, known SHf analogs and of AMPs of the invention are provided in the Table 2.

TABLE-US-00002 TABLE 2 MIC (?M) LC.sub.50 (?M) SEQ ID Gram-negative bacteria Gram-positive bacteria Erythrocytes No Sequence EC PA AB KP SA SP LI EF Human 2 FFFLSRIF.sub.amide 50 >100 >100 >100 25 25 50 100 >200 11 F(p-.sup.tBuF)FLRRIF.sub.amide 6.25 12.5 6.25 100 3 3 6.25 25 >200 19 FF(hF)LRRIF.sub.amide 12.5 25 25 25/50 6.25 12.5 5/6.25 15/25 170 25 F(p-.sup.tBuF)(hF)LRKIF.sub.amide 6.25 6.25 3 6.25/12. 3 NE NE 6.25/12.5 <100 29 F(p-.sup.tBuF)RFRRFF.sub.amide 6.25 3/6.25 12.5 25 3 NE NE >25 <100 30 F(p-.sup.tBuF)KFRRFF.sub.amide 6.25 3/6.25 12.5 25 3 NE NE 25 ~100 32 F(p-.sup.tBuF)KFKKFF.sub.amide 6.25 3 6.25 25 3/6.25 NE NE 12.5 ~100 33 FK(p-.sup.tBuF)LKKIF.sub.amide 6.25 3 25 25 6.25 NE NE 12.5 ~200 34 FFFFSRFF.sub.amide >100 >100 >100 >100 12.5 6.25 25 100 >200 38 FFFRRRFF.sub.amide 25 25 100 >100 6.25/12.5 12.5 15/25 >100 >200 39 FFFFRRFF.sub.amide 12.5 12.5 25 50 6.25 3 12.5 50 >200 41 FFRRFFFF.sub.amide 12.5 12.5 12.5 25 6.25 6.25 10/12.5 50 >200 42 FFFFKRFF.sub.amide 12.5/15 12.5 12.5/15 50 6.25 12.5 12.5/15 >100 >200 43 FFFFRKFF.sub.amide 12.5/15 12.5 12.5 50 6/12.5 12.5 10/12.5 100 >200 49 FRFFRRFF.sub.amide 20/25 6.25 25/50 100 3/6.25 25 10/12.5 >100 >200 50 FFRFRRFF.sub.amide 12.5/15 12.5 100 100 6.25 6.25 10/12.5 100 >200 51 F(hF)FFRRFF.sub.amide 12.5/20 12.5/15 12.5 25 6/6.25 12.5 12.5 >100 159 53 FF(hF)FRRFF.sub.amide 12.5 12.5 12.5 50 3 6.25 5/6.25 50 140 55 FRFRFRFF.sub.amide 5/6.25 5/6.25 12.5/15 12.5 3 12.5 5/6.25 >100 >200 56 FRFRFRFR.sub.amide 3 3/6 25 12.5 6/6.25 6.25 5/6.25 30 >200 65 (p-.sup.tBuF)FKLRRIF.sub.amide 12.5 NE NE NE 1.56/3 NE NE NE >200 66 (p-.sup.tBuF)KFLRRIF.sub.amide 6.25 3 25 6.25 6.25 NE NE 12.5 100-200 72 RFFFRRFF.sub.amide 20/25 12.5 100 100 6.25 25 12.5/15 >100 >200 MIC: Minimum Inhibitory Concentration LC50: Lytic Concentration 50% EC: Escherichia coli; PA: Pseudomonas aeruginosa; AB: Acinetobacter baumannii; KP: Klebsiella pneumoniae; SA: Staphylococcus aureus; SP: Streptococcus pyogenes; LI: Listeria ivanovii; EF: Enterococcus faecalis. NE: Not evaluated.