Antimicrobial peptide nanonets and methods of use thereof

Abstract

Disclosed are synthetic β-hairpin antimicrobial peptides (AMPs) of 13–15 amino acids and compositions thereof comprising a pathogen-recognition hairpin turn (e.g., LTA or VpPA) and β-sheet-forming side strands that self-assemble into nanonets and interwoven nanofibers upon contact with bacterial lipoteichoic acid or lipopolysaccharide, or fungal cell wall / membrane components. The peptides trap, immobilize, and kill bacteria and 0 fungi, including antibiotic-resistant strains, reduce motility, and neutralize endotoxin. Coated substrates and medical devices bearing covalently conjugated AMPs prevent or reduce biofilm formation, sustain antifouling activity, and attenuate inflammatory cytokines. Methods of treating infections and immune-related diseases, and kits comprising AMP-coated substrates or medical devices are also provided.

Description

ANTIMICROBIAL PEPTIDE NANONETS AND METHODS OF USE THEREOFFIELD OF THE INVENTION

[0001] The present invention generally relates to synthetic antimicrobial peptides and methods of use thereof. In particular, the present invention relates to synthetic nanonet-forming peptides and methods of their bacterial / fungal trapping and killing uses; and substrates and medical devices and methods of use thereof comprising the synthetic antimicrobial peptides.BACKGROUND

[0002] Bacterial trapping offers a promising strategy to combat antibiotic resistance by targeting mechanisms that help bacteria evade antibiotics, such as internalization in host cells or escaping areas with high local drug concentrations.11-41Human-defensin 6 peptide forms an effective bacteria-trapping nanonet in human small intestine,[5,6]but recreating such nanonet using short synthetic peptides is challenging due to the limited understanding of the design principles. Self-assembling hybrid peptides can form nanofibers that trap bacteria,[7–9]but these nanofibers lacked antibacterial activity, risking bacterial outgrowth and spread. Furthermore, their spontaneous self-assembly may compromise long-term stability for drug administration. Therefore, improved peptide designs are needed to develop nanonets that can effectively trap-and-kill bacteria.

[0003] p-hairpin peptides, with controllable conformational bias and selfassembly,[lo n]have been shown to form cyto-compatible and antibacterial peptide hydrogels.[12,13]These peptides, featuring (XY)nrepeats of hydrophobic and cationic residues flanking a central turn motif,

[0014] fold into P-hairpin facial amphiphiles with distinct hydrophobic and cationic regions, which is a defining structural characteristic of antimicrobial peptides (AMPs).11’1Thus, there is a need for a novel peptide which can tightly wrap around bacterial surfaces and achieve better trapping and killing of bacteria. There is also a need for a novel peptide that can effectively trap or kill fungi.

[0004] There is a further need for practical applications of such peptide.SUMMARY

[0005] The present disclosure describes a series of synthetic P-hairpin antimicrobial peptides (AMPs) which retain the turn motif (Leu-Thr-Ala) of BTT1-3A but possesssystematically modified side strands to improve bacterial or fungal trapping and killing activities.

[0006] In one aspect, the present disclosure refers to a synthetic P-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising the following functional modules:(a) a recognition module comprising a hairpin turn having an amino acid sequence of LTA (SEQ ID NO: 6), for interacting with pathogen-associated molecular patterns (PAMPs) of a bacterium or PAMPs of cell wall or membrane components of a fungus, to initiate amyloid nucleation; and(b) a structural module comprising a first side strand and a second side strand for P-sheet formation;wherein the synthetic -hairpin AMP comprises a sequence selected from any one or more of the group consisting of:cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO:4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof, a = an anionic amino acid, or an analog or derivative thereof;andwherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of the cell wall or membrane component of the fungi, or on or from a bacterium or fungus.

[0007] In another aspect, the present disclosure refers to a pharmaceutical or adjuvant composition comprising the synthetic p-hairpin AMP as disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0008] In another aspect, the present disclosure refers to a method for treating a condition associated with a bacterial or fungal infection, or an immune-related disease in a subject in need thereof, comprising administering the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to thesubject, wherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial surface in the presence of the PAMPs of the bacterium or the PAMPs of the cell wall or membrane component of a fungus, or on or from a bacterium or fungus to (a) trap and kill the bacterium or fungus; and / or (b) trap and reduce motility of the bacterium or fungus.

[0009] A synthetic P-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising a sequence selected from any one or more of the group consisting of:cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO:4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;a = an anionic amino acid, or an analog or derivative thereof;andwherein the AMP kills or reduces bacteria, fungus, or both; and / oreliminates or reduces bacterial activity, fungal activity or both.

[0010] In another aspect, the present disclosure refers to use of the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein, in the manufacture of a medicament for treating a condition associated with a bacterial infection, or an immune-related disease in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial surface or fungal surface in the presence of the PAMPs of the bacterium or fungus, or on or from a bacterium or fungus to (a) trap and kill the bacterium or fungus; and / or (b) trap and reduce motility of the bacterium or fungus.

[0011] In another aspect, the present disclosure refers to a kit comprising the pharmaceutical or adjuvant composition of as disclosed herein, and a dispenser and / or applicator.

[0012] In another aspect the present disclosure refers to a coated substrate comprising the synthetic p-hairpin AMP as disclosed herein. In one example, the coated substrate comprises a substrate coated with a synthetic P-hairpin antimicrobial peptide (AMP) comprising the following functional modules: (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane com-ponent or a fungal cell wall or membrane component to initiate amyloid nucleation, wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA (‘p’: d-Proline) (SEQ ID NO: 38); and (b) a structural module comprising a first side strand and a second side strand for p-sheet formation and molecular stacking during fibrillation, wherein the synthetic p-hairpin AMP comprises a sequence selected from any one or more of the group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X is an aromatic hydrophobic amino acid, or an analog or derivative thereof; c is a cat-ionic amino acid, or an analog or derivative thereof; h is a non-aromatic hydrophobic amino acid, or an analog or derivative thereof, a is an anionic amino acid, or an analog or derivative thereof; and wherein the synthetic p-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to (i) trap and kill the bacterium or the fungus; and / or (ii) trap and reduce motility of the bacterium or the fungus.

[0013] In another aspect, the present disclosure refers to a medical device comprising the coated substrate as described herein.

[0014] In another aspect, the present disclosure refers to a method for preventing or reducing bacterial biofilm formation on a medical device surface, comprising conjugating a synthetic P-hairpin antimicrobial peptide (AMP) onto the medical device surface, wherein the synthetic P-hairpin AMP comprises the following functional modules: (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation;wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA (‘p’: d-Proline) (SEQ ID NO: 38); and (b) a structural module comprising a first side strand and a second side strand for p-sheet formation and molecular stacking during fibrillation, wherein the synthetic P-hairpin AMP comprises a sequence selected from the any one or more of group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X is an aromatic hydrophobic amino acid, or an analog or derivative thereof; c is a cationic amino acid, or an analog or derivative thereof; h is a non-aromatic hydrophobic amino acid, or an analog or derivative thereof; a is an anionic amino acid, or an analog or derivative thereof; and wherein the synthetic -hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to (i) trap and kill the bacterium or the fungus; and / or (ii) trap and reduce motility of the bacterium or the fungus.In another aspect, the present disclosure refers to a method of preventing or treating a biofilm-related infection in a subject in need thereof, comprising implanting a medical device comprising the coated substrate as described herein, into the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to (i) trap and kill the bacterium or the fungus; and / or (ii) trap and reduce motility of the bacterium or the fungus.

[0015] In another aspect, the present disclosure refers to a medical device comprising the coated substrate as described herein for use in treating preventing or treating a biofilm-related infection in a subject in need thereof, wherein the medical device is for implantation into the subject, wherein the synthetic β-hairpin AMP coated on the substrate self-assembles into nanonets or interwoven nanofibers on or from bacterial surface or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of the cellwall or membrane component of the fungus, or on or from a bacterium or fungus to (i) trap and kill the bacterium or fungus; and / or (ii) trap and reduce motility of the bacterium or fungus.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0017] Figure 1 illustrates the Rational design of P-hairpin peptide to form bacteria nanonet. (A) Schematic illustration of bacteria nanonet formation. BTT1-3A fold into [3-hairpin after binding to bacterial LPS or LTA. The P-hairpin peptide stacks into nanofibrils to form nanonet and trap bacteria. (B) Schematic illustration of three modification strategies for the P-hairpin side strands, focusing on shortening of the peptide, fine-tuning aromaticity, or reducing overall positive charge. Key residues were highlighted in darker gray.

[0018] Figure 2 illustrates the secondary structures and fibrillation capability of the peptides. (A-C) CD spectra of 50 pM peptides were measured with (A) PBS alone, (B) 0.2 mg / mL LPS, and (C) 0.2 mg / mL LTA. The dashed line indicates β-sheet structure of diF-F15in PBS. (D-E) Percentage increase in K114 fluorescence signal for each 20 pM peptide after incubation for 0.5, 1, and 2 hours in the presence of 5 pg / mL (D) LPS and (E) LTA. The dashed line represents the baseline for non-fibrillating control peptide (BTT4). Experiments were repeated three times, with data expressed as mean ± SE. Statistical significance compared to the non-fibrillating control BTT4 was evaluated using Dunnett’s multiple comparisons (ns: p > 0.05, ****: p < 0.0001).

[0019] Figure 3 illustrates that the residue-specific modifications in P-hairpin side strands resulted in nanonets with different morphologies. (A) SEM images of untreated and treated E. coli ATCC 25922 and S. aureus ATCC 29737 with 40 pM of W-W13or 100 μM of E-E13ASYMfor 2 hrs. The arrows in the row of W-W13represent nanonet densely interweave around bacteria, while the arrows in the row of E-E13ASYMrepresent nanonet extensively interweave in the space between the bacteria. Magnification = 15000×. Scale bar = 1 μm. At least three independent fields were imaged per sample to ensure representativeness. (B) Summary of peptide nanonet surface coverage and extensiveness. (Green: High, Yellow: Moderate, Red: Low).

[0020] Figure 4 illustrates that tight trapping by peptide nanonets reduced the spread of motile bacteria. Images of soft agar plates (A, B) showed bacterial spread, with corresponding graphs (C, D) depicting the percentage inhibition of spread for E. coli ATCC 25922 (A, C) and P. aeruginosa PAO1 (B, D) following treatment with different peptide concentrations (2 pM Polymyxin B, 20 μM W-W13, 40 μM BTT1-3A, 40 μM diF-F13, 100 μM E-E13ASYM). After 30 minutes of incubation for nanonet formation, 2 pL of each mixture were seeded in 0.3% LB soft agar and the spreading of bacteria was investigated after 14 hrs. Experiments were repeated three times, with data expressed as mean ± SE. Statistical significance compared to the untreated control was evaluated using Dunnett’s multiple comparisons (ns: p > 0.05, **: p < 0.01, ****: p < 0.0001).

[0021] Figure 5 illustrates predicted secondary structures of peptide libraries by D-I-TASSER. Ab initio secondary structures prediction of peptide libraries by D-I-TASSER All rationally designed peptide libraries exhibited conformational flexibility, suggesting a high propensity to fold from an extended structure into a p-hairpin.

[0022] Figure 6. Illustrates the analysis of spontaneously self-assembled diF-Fis peptide hydrogel. Spontaneous self-assembly of diF-Fis. (A) K114 fluorescence intensity of 20 μM diF-F15after 30 minutes of incubation in lx PBS, LPS, or LTA. (B) Z-stack confocal imaging showing the 3D structure of 64 pM diF-Fis self-assembled in 1x PBS alone, stained with 20 pM of K114 dye. Scale bar = 50 pm. (C) SEM image showing the ultrastructure of the dense peptide hydrogel nanofiber network formed by 64 pM diF-Fis after 30 minutes of incubation in 1x PBS alone. Magnification = 15,000*. Scale bar = 1 pm.

[0023] Figure 7 shows SEM images of bacteria nanonet trapping E. coli ATCC 25922 with different morphologies. Residue-specific modifications in P-hairpin side strands sequence resulted in nanonets with different morphologies. SEM images of E. coli ATCC 25922 treated with 40 pM of each peptide for 2 hrs. The arrows in the last row represent nanonet densely interweave around bacteria, while the arrows in the second row represent nanonet extensively interweave in the space between the bacteria. Magnification = 15000×. Scale bar = 1 μm. At least three independent fields were imaged per sample to ensure representativeness.

[0024] Figure 8 shows SEM images of bacteria nanonet trapping S. aureus ATCC 29737 with different morphologies. Residue-specific modifications in P-hairpin side strands resulted in nanonets with different morphologies. SEM images of S. aureus ATCC29737 treated with 40 pM of each peptide for 2 hrs. The arrows in the last row represent nanonet densely interweave around bacteria, the arrows in the second row represent nanonet extensively interweave in the space between the bacteria, while the purple arrows represent random aggregated structures. Magnification = 15000×. Scale bar = 1 μm. At least three independent fields were imaged per sample to ensure representativeness.

[0025] Figure 9 shows agglutination curves of E. coli ATCC 25922 treated with peptides. Peptide nanonets agglutinated E. coli ATCC 25922. Agglutination curves of E. coli treated with peptides of (A) 15 amino acids or (B) 13 amino acids length at 40 pM in lx PBS buffer. Experiments were repeated three times, with data expressed as mean ± SE, normalized against the OD600at 0 hours.

[0026] Figure 10 shows agglutination curves of S. aureus ATCC 29737 treated with peptides. Peptide nanonets agglutinated S. aureus ATCC 29737. Agglutination curves of S. aureus treated with peptides of (A) 15 amino acids or (B) 13 amino acids length at 40 pM in lx PBS buffer. Experiments were repeated three times, with data expressed as mean ± SE, normalized against the OD600at 0 hours.

[0027] Figure 11 shows the antibacterial activity of the peptide libraries against bacteria and their relative binding affinity to bacterial LPS or LTA. Residue-specific modifications in P-hairpin side strands altered antibacterial activity. (A) MIC90screening of peptides from different modification groups (1. Reduced length, 2. Increased aromaticity, 3. Reduce positive charge) against E. coli ATCC 25922 and S. aureus ATCC 29737. The solid black triangle (▼) indicated the P-hairpin peptide that formed bacteria-trapping nanonets. (B, C) Percentage of BODIPY-Cadaverine (BC) dye displaced by different concentration of peptides from (B) LPS or (C) LTA. All experiments were performed at least three times.

[0028] Figure 12 illustrates the cytotoxicity of peptides against HaCaT keratinocytes. (A-C) MTS assay results showed the viability of HaCaT keratinocytes treated with peptides at concentrations up to 1000 pM. Experiments were repeated three times, with data expressed as mean ± SE. (D) Table summarizing the CC50 of each peptide.

[0029] Figure 13 shows the comparison of peptide-induced membrane damage in HaCaT keratinocytes. Membrane disruption in HaCaT keratinocytes by P-hairpin peptide. Propidium iodide staining ofHaCaT keratinocyte nuclei indicated cell membrane damage after treatment with 25 pM of peptides for 1 hour. Scale bar = 100 pm. All experiments were repeated three times.

[0030] Figure 14 shows the SEM images of bacteria nanonet trapping P. aeruginosa PAO1 with different morphologies. Residue-specific modifications in p-hairpin side strands resulted in nanonets with different morphologies. SEM images of P. aeruginosa PAO1 treated with 100 pM of E-E13ASYM, 40 pM W-Wn or 40 pM diF-Fn for 2 hrs. The arrows in the last row represent nanonet densely interweave around bacteria, while the arrows in the second row represent nanonet extensively interweave in the space between the bacteria. Magnification = 15000×. Scale bar = 1 μm. At least three independent fields were imaged per sample to ensure representativeness.

[0031] Figure 15 shows the diameter of spreading zone in soft agar plates. Tight trapping of peptide nanonet reduced spreading of motile bacteria. Diameter of spreading zone in soft agar plates for (A) E. coli ATCC 25922 or (B) P. aeruginosa PAO 1 following treatment with different peptide concentrations (2 pM Polymyxin B, 20 μM W-W13, 40 μM BTT1-3A, 40 μM diF-F13, 100 μM E-E13ASYM. After 30 minutes of incubation for nanonet formation, 2 pL of each mixture were seeded in 0.3% LB soft agar and the spreading of bacteria was investigated after 14 hrs. Experiments were repeated three times, with data expressed as mean ± SE. Statistical significance compared to the untreated control was evaluated using Dunnett’s multiple comparisons (*: p < 0.05, **: p < 0.01, ****: p < 0.0001).

[0032] Figure 16 shows the image of soft-agar plates showing the growth of non-motile bacteria treated with different peptides. Non-motile bacteria were more vulnerable to killing by antibacterial peptides. Images of soft agar plates of E. coli K12 BW25113 (Non-motile E. coli AflhD mutant) following treatment with different peptide concentrations (2 pM Polymyxin B, 20 μM W-W13, 40 μM BTT1-3A, 40 μM diF-F13, 100 μM E-E13ASYM). After 30 minutes of incubation for nanonet formation, 2 pL of each mixture were seeded in 0.3% LB soft agar and the growth of bacteria was observed after 14 hrs. Experiments were repeated three times.

[0033] Figure 17 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide BTT1-3A.

[0034] Figure 18 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide Btt1-3A13.

[0035] Figure 19 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide Btt1-3A11.

[0036] Figure 20 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide F-F15.

[0037] Figure 21 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide diF-F15.

[0038] Figure 22 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide F-F15.

[0039] Figure 23 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide diF-F13.

[0040] Figure 24 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide W-W13.

[0041] Figure 25 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide F-F11.

[0042] Figure 26 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide W-W11.

[0043] Figure 27 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide E-E13.

[0044] Figure 28 shows LC-MS analytical characterization of the peptides. A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide E-E13ASYM.

[0045] Figure 29 shows examples of some (a) non-canonical aromatic amino acids; (b) non-canonical charged (cationic or anionic) amino acids; and (c) non-canonical nonaromatic hydrophobic amino acids.

[0046] Figure 30 shows anti-inflammatory effect of peptide nanonets. (A) Schematic illustration of nanonets of different morphologies and the mechanism of peptide nanonets to entrap free LPS. (B) FITC-LPS trapping index of pre-formed nanonets was determined after 1 h pre-incubation with LPS (2 ug / mL), followed by 1 h incubation with FITC-LPS (2 ug / mL). (C) Table summary of the effective concentration (EC50) and maximal efficacy (Emax) of pre-formed peptide nanonets in trapping free LPS. Experiments were repeated three times and results are presented as mean ± SE.

[0047] Figure 31 shows antibacterial activity of peptide nanonets of different morphology. (A) MIC90screening of different peptide nanonets against laboratory strains and clinical isolates. (B) Outer membrane damage and (C) inner membrane damage of E. coli M2 were quantified using NPN and PI dyes, respectively. Polymyxin B was used asa positive control in both assays. Experiments were repeated three times. Data are expressed as mean ± SE.

[0048] Figure 32 shows peptide nanonets enhance antibiotic potency and prevent resistance development. (A) Chequerboard results show the interaction between rifampicin and different peptide candidates against E. coli M2. Darker blue indicates higher bacterial growth. Solid black boxes highlight synergistic concentrations. (B) Corresponding FICI values from the chequerboard heatmaps (Synergy: <0.5; Additive: >0.5 to <1). All experiments were repeated three times. (C) Changes in rifampicin MICs were monitored over 18 days of serial passage, either alone or in combination with peptides at concentrations based on the initial synergistic MICs against E. coli M2. Three independent experiments (n = 3) were performed, with one representative result shown.

[0049] Figure 33 shows aromatic residues affect the formation of bacteria-specific peptide nanonets. Schematic illustration of peptide motifs that form tight nanonets and their respective modifications with unnatural aromatic acid residues of varying hydrophobicity, except Cha which is non-aromatic.

[0050] Figure 34 shows aromatic residues affect the secondary structure and selfassembly of the peptides. CD spectra of 50 pM peptides containing (A) Trp analogs and (B) Phe analogs were measured in PBS alone, or in the presence of 0.2 mg / mL LPS or 0.2 mg / mL LTA. The red solid lines indicate the [3-sheet structure of Fl and F2 in PBS Black solid triangles indicate the negative peak for random coil, while black open triangles indicate the characteristic positive and negative peaks of |3-sheet structures. Percentage increase in K114 fluorescence for each 20 μM peptide with (C-D) Trp analogs and (E-F) Phe analogs was measured after 0.5, 1, and 2 hours of incubation with 5 pg / mL (C, E) LPS or (D, F) LTA. The blue dashed line represents the baseline signal of the non-fibrillating control peptide (BTT4). The double-gradient scale indicates peptide hydrophobicity, with darker red representing higher hydrophobicity and darker blue representing higher hydrophilicity. Experiments were repeated three times, with data expressed as mean ± SE. Statistical significance compared to the non-fibrillating control BTT4 was evaluated using Dunnett’s multiple comparisons test (ns: p > 0.05, *: p < 0.05, **: p < 0.005, ***: p < 0.0005, ****: p < 0.0001).

[0051] Figure 35 shows (A) K114 fluorescence was measured after 0.5 hours of incubation with or without 20 μM Phe analogs in 1x PBS. Data represent mean ± SE from at least three independent experiments. Statistical significance was assessed usingDunnett’s multiple comparisons test (ns: p > 0.05, ****: p < 0.0001). (B-C) SEM images show dense peptide hydrogel nanofiber networks formed by (B) 64 pM Cha and (C) 64 pM Phe(3,4,5-F) after 30 minutes of incubation in 1x PBS alone. Magnification: 15,000x; scale bar: 1 μm.

[0052] Figure 36 shows aromatic residues influence the formation and morphology of peptide nanonets. Schematic representation of nanonets with distinct morphologies. SEM images of E. coli ATCC 25922, either untreated or treated for 2 hours with 40 pM of W-WB, Wl, W2, W3, W4, Fl, F3, or F4; 80 pM of W5 or F5; or 100 pM of F7 or F8. Red arrows indicate nanonets densely interwoven around bacterial surfaces; yellow arrows indicate nanonets extensively interwoven in the space between bacteria; and purple arrows indicate exposed bacterial surfaces. Images were captured at 15,000x or 50,000× magnification. Scale bars represent 1 pm or 200 nm, respectively. At least three independent fields were imaged per sample to ensure representativeness.

[0053] Figure 37 shows aromatic residues influence the antibacterial activity and cytotoxicity of P-hairpin peptides. (A) MIC90 screening of different peptide nanonets against a panel of pathogenic microorganisms. (B) Table summarizing the CC50 values of each peptide against HaCaT keratinocytes. All experiments were repeated three times. (C-D) 3D scatter plot showing the correlation between peptide hydrophobicity (logP), antibacterial activity (MIC), and biocompatibility (CC50) for E. coli ATCC 25922 and S. aureus ATCC 29737, respectively. The grey dashed line serves as a visual guide highlighting the region where bacteria-specific self-assembly tends to occur, forming distinct morphologies such as tight nanonets, semi-tight nanonets or loose-entangled nanonets.

[0054] Figure 38 shows changing cationic residues (c) and non-aromatic hydrophobic amino acids (h) have minimal impact on nanonet properties. (A) Chemical structure of the parent peptide E-E13ASYMand its modified analog E-E13ASYM(ch). Blue font indicates hydrophobic amino acids, while red font indicates cationic amino acids. (B-C) CD spectra of 50 pM peptides containing E-E13ASYManalogs measured in (B) PBS alone and (C) in the presence of 0.2 mg / mL LPS or 0.2 mg / mL LTA. (D-E) Percentage increase in K114 fluorescence of 20 μM peptides incubated with 5 pg / mL (D) LPS or (E) LTA for 0.5, 1, and 2 hours. The blue dashed line indicates the baseline signal of the non-fibrillating control peptide (BTT4). Experiments were repeated three times, with data expressed as mean ± SE. Statistical significance compared to BTT4 was assessed usingDunnett’s multiple comparisons test (***: p < 0.0005, ****: p < 0.0001). (F-G) SEM images of E. coli ATCC 25922 either untreated or treated with 100 pM of (F) E-E13ASYMor (G) E-E13ASYM(ch) for 2 hours. Yellow arrows indicate nanonets extensively interwoven in the space between bacteria; purple arrows indicate exposed bacterial surfaces. Images were acquired at 15,000× magnification. Scale bars = 1 μm. At least three independent fields were imaged per sample to ensure representativeness. (H) Table summarizing MIC90 values of peptides against E. coli ATCC 25922 and their cytotoxicity (CC50) against HaCaT keratinocytes. All experiments were repeated three times.

[0055] Figure 39 shows the summary of tryptophan analogs (X) with modified side chains considered as potential substitutes in peptide nanonet formation.

[0056] Figure 40 shows the summary of phenyalanine (X) analogs with modified side chains considered as potential substitutes in peptide nanonet formation

[0057] Figure 41 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide Wl.

[0058] Figure 42 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide W2.

[0059] Figure 43 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide W3.

[0060] Figure 44 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide W4.

[0061] Figure 45 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide Fl.

[0062] Figure 46 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide F2.

[0063] Figure 47 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide F3.

[0064] Figure 48 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide F4.

[0065] Figure 49 shows A) Analytical HPLC and B) ESI (+) mass spectrum of the peptide E-EisASYM(ch).

[0066] Figure 50 show the schematic of peptide coating onto the device surface B) Illustration of the surface functionalization strategy for immobilizing fibrillating peptides onto a polymer substrate. The process involves initial silanization of the polymer surfaceto introduce reactive amine or hydroxyl groups, followed by treatment with glutaraldehyde to introduce aldehyde functionalities. Subsequent Schiff base formation between the aldehyde groups and the primary amines of the peptide enables stable covalent attachment. C) ATR spectrograph of peptide, PU and Peptide-coated PU.

[0067] Figure 51 shows the evaluation of PC-PU antimicrobial activity a) Quantitative results for bacterial biofilm formation, b) Qualitative comparison of bacterial biofilm development on polymer substrates, c) Diameter of bacterial colonies incubated in soft agar, d) Time-kill curves of UPEC incubated with PC-PU, Colistin and in a Drug-Free environment. Colistin was used as positive control. Yellow dotted lines indicate the limit of detection (100 CFU / mL).

[0068] Figure 52 shows comparative analysis of the impact of incubation of UPEC with PU and PC-PU substrates on cellular surfaces through confocal microscopy. UPEC cells incubated with A) PU substrate or B) PC-PU substrate and stained with SYTOX Green. UPEC cells incubated with PC-PU substrates were also stained with C) PI. D) Double labelling of SYTOX Green with PI revealed an overlapping fluorescence signal (yellow pixels). Scale bars = 10pm.

[0069] Figure 53 shows the comparative analysis of the impact of incubation of UPEC with PU and PC-PU substrates on cellular surfaces through SEM microscopy. SEM images of unmodified (A) PU substrate and UPEC incubated with (B) PU and (C) PC-PU substrate. Magnification = 10000x Scale bars = 1 μm.

[0070] Figure 54 shows SEM analysis of PU and PC-PU against UPEC over 24 hours to understand the nanonet morphology on the polymer surface B) Confocal laser scanning microscopy images of peptide coated on various polymer surfaces (100x magnification) following live / dead staining against UPEC over 4 weeks. All bacteria incoolum was standardized at 106 CFU / ml.

[0071] Figure 55 MIC Screening of Fibrillating and non-Fibrillation peptides against Uropathogenic strains, Control (Colistin against UPEC and KpCP and Cirpofloxacin againt p. mirabilis).

[0072] Figure 56 shows the evaluation of the antifouling efficacy of the peptide-coated surface against Uropathogenic Escherichia coli (UPEC) over a period of 4 weeks, using Confocal laser scanning microscopy images (100x magnification) following live / dead staining (B) SEM analysis showing surface morphology and extent of bacterialfouling at 2 and 4 weeks. (C) Crystal violet staining quantifying total biofilm biomass on coated and uncoated surfaces. Bacterial inoculum was standardized to 106CFU / ml.

[0073] Figure 57 shows the motility assay of bacteria trapped in biofilms after week 2 and week 4.

[0074] Figure 58 shows the evaluation of the antifouling efficacy of the peptide-coated surface against clinical isolates over 1 week using confocal laser scanning microscopy images (100x magnification) following live / dead staining. Bacterial inoculum was standardized to 106CFU / ml.

[0075] Figure 59 shows the evaluation of the anti-inflammatory activity of the peptide-coated surface using ELISA, results showing the reduction in the concentration of pro-inflammatory cytokine TNF-a produced by (A) HEK293 (B) RAW 264.7 (C) Evaluation of the antioxidant activity of the peptide-coated surface using HEK293 cells Confocal microscopy images of DCFDA-stained HEK293 cells showing decreased ROS fluorescence intensity.

[0076] Figure 60 shows ex vivo evaluation of the antimicrobial efficacy of the peptide-coated surface using a porcine urinary bladder model. (A) Schematic representation of the experimental setup illustrating placement of control and test tissues in conjunction with uncoated (control) and peptide-coated catheters. (B) Quantification of bacterial colonization CFU analysis. (C) Biofilm biomass assessment using crystal violet staining (D) SEM images of tissue surfaces depicting morphological differences and extent of biofilm formation between control and peptide-coated groups.

[0077] Figure 61 shows A) ATR-FTIR spectra of polymer surfaces coated with fibrillating peptides W-Wn and F-F15. B) SEM of peptide-coated polymer surfaces illustrating distinct fibrillation morphologies and antifouling activity.DETAILED DESCRIPTION

[0001] Inspired by spiders that weave extensive nets to initially trap prey and then tightly interweave them to confine prey and prevent escape, the inventors' design goal is to achieve better control of peptide nanofibers interweaving on or from bacterial surfaces to enhance trapping and limit bacteria spread. By strategic substituting residues on the side strands with either aromatic or negatively charged residues, nanonets with distinct morphologies were created: either tightly interwoven nanonets on or from bacterial surfaces or extensive hydrogel-like nanonets with minimal bacterial coverage.Furthermore, it was demonstrated that these tightly interwoven nanonets effectively encage motile bacteria, significantly reducing their spread compared to loosely trapping nanonets. Furthermore, it was demonstrated that these nanonets are effective at killing fungi.

[0002] Tn the inventors' previous work, a series of [3-hairpin peptides with novel turn motifs that exhibited potent antibacterial activity was designed.

[0016] Introducing the novel turn motif (Leu-Thr-Ala) in the 15-mer BTT1-3A enabled it to self-assemble into nanonets specifically in the presence of bacteria, rather than forming non-specific peptide hydrogels.

[0017] Initially, BTT1-3A was a highly water-soluble random coil. Upon binding to pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide (LPS) or lipoteichoic acid (LTA), it exhibited conformational bias by folding into [3-hairpin and self-assembling into nanofibers, leading to the formation of bacteri -trapping nanonets (Figure 1A). Furthermore, BTT 1-3 A demonstrated broad-spectrum antibacterial activity, forming extensive nanonets that trap-and-kill various bacterial species while also sequestering endotoxins to reduce inflammation.[17,18]However, while BTT1-3A nanofibers form extensive nanonets, they tend to entangle with themselves rather than tightly wrapping around bacterial surfaces. To this end, a library of BTT1-3A analogues was designed, which retained the turn motif (Leu-Thr-Ala) but were systematically modified at side strands via three major approaches to control bacteria nanonet morphology while preserving bacterial specificity: 1) shortening the peptides from both termini, 2) fine-tuning aromaticity or 3) reducing the overall positive charge (Figure IB).

[0003] Precise control over peptide nanonet architecture is instrumental in advancing the development of antibacterial nanonets. Here, there is presented a novel design strategy to control bacteria nanonet morphology through rational modification of the [3-hairpin side strands, leveraging the unique chemical properties of amino acid side chains. By finetuning both the terminal ends and aromaticity of the hydrophobic residue, diF-F13 and W-W13 were identified as the key candidates for bacterial trapping and killing. These two peptides, with similar physicochemical properties, interweave tightly and effectively trap motile Gram-negative bacteria such as E. coli and P. aeruginosa. Moreover, the W-Wn peptide emerged as the lead candidate, with increased nanofibers interweaving on or from bacterial surfaces, forming a tightly interwoven nanonet that effectively trapped and killed both Gram-positive bacteria such as S. aureus and Gram-negative bacteria such as E. coli. The key candidates also had good compatibility with skin cells, posting minimumrisk for skin irritation or inflammation. In contrast, asymmetric glutamic acid substitutions on the cationic residues of the E-E13ASYMpeptide redirected the nanofibers to self-interweave, forming extensive nanonets with minimal bacterial coverage and no antibacterial activity. Using these nanonets with distinct morphologies and function, it was demonstrated that the formation of tightly interwoven nanonets on or from bacterial surfaces significantly reduced the spread of motile E. coli and P. aeruginosa, outperforming both loosely trapping nanonets and conventional potent antibiotic. The present findings pave the way for the development of novel peptide-based nanonets, offering a promising strategy to target bacterial motility and prevent spreading of bacteria.

[0004] synthetic P-hairpin AMP

[0005] In one aspect, the present disclosure refers to a synthetic P-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising the following functional modules:(a) a recognition module comprising a hairpin turn having an amino acid sequence of LTA (SEQ ID NO: 6), for interacting with pathogen-associated molecular patterns (PAMPs) of a bacterium or PAMPs of cell wall or membrane components of fungus to initiate amyloid nucleation; and(b) a structural module comprising a first side strand and a second side strand for P-sheet formation;wherein the synthetic P-hairpin AMP comprises a sequence selected from any one or more of the group consisting of:cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO:4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;a = an anionic amino acid, or an analog or derivative thereof;andwherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or thePAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus.

[0006] As used herein, the term “peptide” includes a polymer of amino acids linked through peptide bonds or modified peptide bonds, produced synthetically. The term "amino acid” can include L-amino acids, D-amino acids, natural or non-natural amino acids, homologs of naturally occurring amino acids, amino acids with modified side chains, or any analog of an amino acid known to those skilled in the art.

[0007] In one example, the synthetic 0-hairpin AMP as disclosed herein comprises a sequence of cXchcLTAchcXc (SEQ ID NO: 1). In another example, the synthetic -hairpin AMP as disclosed herein comprises a sequence of cXcXcLTAcXcXc (SEQ ID NO: 2). In another example, the synthetic P-hairpin AMP as disclosed herein comprises a sequence of hcXchcLT AchcXch (SEQ ID NO: 3). In another example, the synthetic -hairpin AMP as disclosed herein comprises a sequence of ahchcLTAchahc (SEQ ID NO:4).

[0008] As used herein, X is an aromatic hydrophobic amino acid, or an analog or derivative thereof. An aromatic hydrophobic amino acid is selected from the group consisting of phenylalanine (F), tryptophan (W) and tyrosine (Y). Each X can independently be any natural or non- natural amino acid (L or D stereochemistry) or any analog or derivative of such an amino acid known to those skilled in the art.

[0009] As used herein, c is a cationic amino acid, or an analog or derivative thereof. A cationic amino acid is selected from the group consisting of arginine (R), lysine (K), and histidine (H). Each c can independently be any natural or non- natural amino acid (L or D stereochemistry) or any analog or derivative of such an amino acid known to those skilled in the art.

[0010] As used herein, h is a non-aromatic hydrophobic amino acid, or an analog or derivative thereof. A non-aromatic hydrophobic amino acid is selected from the group consisting of alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), and proline (P). Each h can independently be any natural or non- natural amino acid (L or D stereochemistry) or any analog or derivative of such an amino acid known to those skilled in the art.

[0011] As used herein, a is an anionic amino acid, or an analog or derivative thereof An anionic amino acid is selected from the group consisting of aspartic acid (D) and glutamic acid (E). Each a can independently be any natural or non- natural amino acid (Lor D stereochemistry) or any analog or derivative of such an amino acid known to those skilled in the art.

[0012] The term "analogue" or “analog” as used herein refers to an amino acid residue of the peptide as disclosed herein that is substituted by another amino acid residue and / or wherein one amino acid residue has been deleted from the peptide in the C-terminal of the peptide or wherein one amino acid residue has been added to the C-terminal of the peptide. An analog could be canonical or non-canonical amino acids.

[0013] The term "derivative" as used herein refers to an amino acid residue or analog thereof of the peptide as disclosed herein that is chemically modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters and the like.

[0014] X (an aromatic hydrophobic amino acid), c (a cationic amino acid), h (a nonaromatic hydrophobic amino acid), and a (an anionic amino acid) can be a canonical amino acid, selected from among the 20 standard amino acids that are directly encoded by the universal genetic code and are used to build proteins in almost all organisms. These amino acids are incorporated into proteins during translation, the process by which ribosomes read mRNA sequences and assemble corresponding amino acids into a polypeptide chain. The canonical amino acids include: 1. Alanine; 2. Arginine; 3. Asparagine; 4. Aspartic acid; 5. Cysteine; 6. Glutamic acid; 7. Glutamine, 8. Glycine; 9. Histidine; 10. Isoleucine; 11. Leucine; 12. Lysine; 13. Methionine; 14. Phenylalanine; 15. Proline; 16. Serine; 17. Threonine; 18. Tryptophan; 19. Tyrosine; 20. Valine. These 20 amino acids are considered “canonical” because they are universally used in the genetic code across most forms of life and form the basic building blocks of proteins. In another example, X (an aromatic hydrophobic amino acid), c (a cationic amino acid), h (a nonaromatic hydrophobic amino acid), and a (an anionic amino acid) can be an Non-Canonical Amino Acid (ncAA) which is an amino acid that is not among the 20 standard amino acids encoded by the genetic code but can be found in proteins either through post-translational modification or through specialized biosynthetic pathways or chemical synthesis. Non-canonical amino acids may include Post-Translationally Modified Amino Acids: Some non-canonical amino acids arise when canonical amino acids are chemically modified after they are incorporated into a protein. Examples include: hydroxyproline and hydroxylysine: formed by the hydroxylation of proline and lysine; phosphoserine: formed by the phosphorylation of serine; and methyllysine: formed by the methylation of lysine. Another type of ncAA is Genetically Encoded Non-Canonical Amino Acids. Certainorganisms (especially bacteria) or engineered systems can incorporate non-canonical amino acids into proteins directly during translation by modifying the genetic code or adding specialized tRNAs and enzymes. Examples include: selenocysteine (Sec, U); and pyrrolysine (Pyl, O). Non-canonical amino acids expand the functional diversity of proteins. They enable specialized functions that go beyond what the 20 canonical amino acids can provide, including enhanced chemical reactivity, structural stability, or regulatory roles. Examples of some non-canonical aromatic amino acids can be found in Figure 29a. Examples of some non-canonical charged (cationic or anionic) amino acids can be found in Figure 29b. Examples of some non-canonical non-aromatic hydrophobic amino acids can be found in Figure 29c. These examples are adapted from

[0032] . Non-canonical amino acids could also include unnatural amino acids.

[0015] The above-described canonical amino acids in SEQ ID NO: 1-4 substitutable by their analog or derivative thereof resulting in functionally-equivalent peptides, which according to the invention are intended to include peptides wherein at one or more positions there have been amino acid substitutions, either conservative or nonconservative, provided that such changes result in a peptide which retains and / or enhances the activity of the parent peptide. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. Such variants may for example be made using the methods of protein engineering and site-directed mutagenesis.

[0016] A "conservative" change is wherein a substituted amino acid has similar structural or chemical properties whilst a "non-conservative" change is wherein the substituted amino acid is structurally or chemically different. The terms “conservative substitution”, “non-conservative substitutions”, “aromatic hydrophobic amino acid”, “cationic amino acid”, "non-aromatic hydrophobic amino acid" and “anionic amino acid” are all used consistently with the prior art terminology. Each of these terms is well-known in the art and has been extensively described in numerous publications, including standard biochemistry text books, such as “Biochemistry” by Geoffrey Zubay, Addison-Wesley Publishing Co., 1986 edition, which describes conservative and non-conservative substitutions, and properties of amino acids which lead to their definition. In general, the non-polar amino acids Gly, Ala, Vai, He and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr, the neutral polar amino acids Ser, Thr, Cys, Gin, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Aspand Glu, represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups. Amino acids which are conserved can usually be substituted by other conserved amino acids without significantly affecting the protein's function. Finally, amino acids which are not conserved within a family can usually be freely substituted. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may also be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

[0017] In the present invention, wherein analogs contain chemical structures or side chain functional groups similar to natural amino acids, their properties can be predicted to resemble those of their natural counterparts For example, analogs with similar numbers of aromatic rings should exhibit comparable hydrophobicity, π-π interactions, and overall chemical structure, leading to similar physiochemical properties. This paper

[0032] demonstrates that a skilled person can select analogs with structures similar to natural amino acids, which exhibit such comparable properties that they can be recognized and charged onto tRNA by ribosomal synthetase for protein synthesis.

[0018] Therefore, by comparing the chemical structure of the analog's side chain with that of natural amino acids, a skilled person can predict and identify suitable analogs for potential substitution. Table 1 and 2 and Figure 29, 39 and 40 also shows non exhaustive examples of potential analogs that could be substituted at each amino acid residue position to maintain similar properties as the parent peptide, as well as examples of analogs that should be avoided.

[0019] Table 1. non exhaustive examples of potential amino acid analogsPeptide Motif Natural amino Similar analogs Comments acids (X) (X)W-W13 cXchcLT AchcXc The analogs have similar two-ring aromatic structures dxTrp (W) p-Me-Trp as the natural amino acids, so they are expected to exhibit comparable OH hydrophobicity and contribute similarly to π-π interactions.Trp analog -X >*< V (A23)(AAvoid such Trp analog with more than 2 aromatic ring. L-2- The analogs should napththylalanine maintain similar charge. (Eg: The analogs remain neutrally charged)Vv Avoid Trp analog with negatively charged side chain. diF-Fi3 cXcXcLTAcXcXc The analogs have F-FB cXchcLT AchcXc A- / one aromatic ring hcXchcL 1 fF-F15 T AchcXch structure similar to Phe (F) B-2- that of natural amino thienylalanine acids, so they are expected to have OH f comparable hydrophobicity and contribute similarly to π-π interactions.Avoid analogs with more than one aromatic ring or nonneutral charge for excess pi-pi interaction.Jx 1H, N "'"'''GOOHE- ah ch cLT Ach ah c The analogs have a g^ASYM j ' negatively charged, aliphatic side chain of similar length tothat of Glu.Glu (E) PhosphonatederivativeOH ■» OHH2NGlu analog (C2)

[0020] Table 2. non exhaustive examples of potential amino acid analogs Motif Natural amino acids Similar analogs Comments (X) (X)Cationic residue S3 The analogs have (c) similar positive I s?;ss-" Saw charges and aliphatic side chains of Arg (R) 5-keto-L-argininecomparable length to 0 p natural amino acids. H- ‘N 'xz YC 'OHTherefore, they are NHZexpected to exhibit Lys (K) Ornithine similar physicochemical pH0=^ properties and can be;... S...... 'SH; used interchangeably H2NLys analog (C7)OHO~<HjNiLys analog (C8)pHHjNt HArg analog (CIO)Hydrophobic OH The analogs of Vai, residue (h) Leu, and He exhibit H2N A T A °’US,HO2N very similarHVal / Ile a hydrophobicity and Vai (V) nalog(N16) depend on their aliphatic side chains I OH for van der Waals forces of attraction HzrJy3°:lv.6H HZN \ Different analogs may He (I) Vai analog (N13) be substituted at the h position.Avoid hydrophobicanalogs with aromaticOH. rings, as they can increase hydrophobicity H2N and introduce Leu (L) Leu analog (N17) additional π-πinteractions.

[0021] Tn one example, there are provided functional equivalents of the synthetic [3-hairpin AMP as disclosed herein in which one or more amino acids in the two [3-strands as disclosed herein (e.g. between 1 and 2, 1 and 3, 1 and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, 1 and 10, 1 and 11, 1 and 12, 2 and 4, 3 and 5, 4 and 6, 5 and 7, 6 and 8, 7 and 9, 8 and 10, 9 and 11, 10 and 12, or just 1 amino acid) are independently substituted in any combination

[0022] The functional equivalents of the invention retain the biological activity of the parent peptide. As used herein, biological activity refers to the in vitro and / or in vivo activities of a synthetic P-hairpin AMP as disclosed herein or physiological responses that result upon in vitro contact or in vivo administration of said AMP, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such AMP, compositions and mixtures. Biological activities may be observed in in vitro systems designed to test or use such activities. In the case of a synthetic P-hairpin AMP of the present invention, the AMPs as wells as substitutes by amino acid analogs / derivatives which are functional equivalents suitably retains the ability to form nanonets to trap and kill bacteria or fungi, trap and reduce motility of bacteria, trap a pro-inflammatory cytokine and / or delay the development of antibiotic resistance. Activity may be retained at the same level or at a higher level as the parent AMP. In other words, the fragment or functional equivalent may of similar or identical, or enhanced activity as the parent polypeptide.

[0023] The beta hairpin is a small protein structure motif in which two P-strands, linked by a turn or a short loop, fold to form hydrogen bonds with each other. The two P-strands that are adjacent in primary structure, oriented in an antiparallel direction (the N-terminus of one sheet is adjacent to the C-terminus of the next), and linked by a short loop of two to five amino acids. Beta hairpins can occur in isolation or as part of a series of hydrogen bonded strands that collectively comprise a beta sheet.

[0024] The hairpin turn of the synthetic P-hairpin AMP as disclosed herein acts as the "recognition module" for interacting with bacterial membrane components to initiate peptide conformational change and amyloid nucleation. The hairpin turn of the syntheticP-hairpin AMP as disclosed herein comprises a sequence which is the same as the hairpin turn of the synthetic P-hairpin AMP " BTT1-3A" disclosed in a previous invention of the inventors (PCT / SG / 2023 / 050786) - the hairpin turn has a sequence of (SEQ ID NO: 6): Leu-Thr-Ala, or LTA. The sequence of " BTT1-3A" is LKLKLKLTAKLKLKLISEO ID NO: 5).

[0025] The two side strands of the synthetic P-hairpin AMP as disclosed herein act as the ‘structural module’ for p-sheet formation and molecular stacking during the fibrillation process. The side strands of the synthetic P-hairpin AMP as disclosed herein are rationally modified based on the side strands of BTT1-3A. In one example, the synthetic P-hairpin AMP is diF-Fi3, which comprise the first side strand at the N-terminal of the AMP of the sequence of SEQ ID NO: 9, and the second side strand at the C terminal of the AMP of the sequence of SEQ ID NO: 9• Sequence of the first side strand at the N-terminal of the synthetic P-hairpin AMP diF-Fi3, and the second side strand at the C terminal of the synthetic P-hairpin AMP diF-Fu are the same (SEQ ID NO: 9): KFRFK

[0026] Comparing with the first side strand (LKLKLK) and the second side strand of BTT1-3A (KLKLKL), the first side strand at the N-terminal of the synthetic P-hairpin AMP diF-F 13, and the second side strand at the C terminal of the synthetic P-hairpin AMP diF-Fu have been modified to remove the terminal leucine of the first side strand and second side strand of BTT1-3A, and been introduced with two phenylalanine residues (F) at each side strand at symmetric positions.

[0027] In another example, the synthetic P-hairpin AMP is W-W13, which comprise the first side strand at the N-terminal of the AMP of the sequence of SEQ ID NO: 10, and the second side strand at the C terminal of the AMP of the sequence of SEQ ID NO: 11• Sequence of the first side strand at the N-terminal of the synthetic p-hairpin AMP W-W13 (SEQ ID NO: 10) KWRVK• Sequence of the second side strand at the C terminal of the synthetic P-hairpin AMP W-W13 (SEQ ID NO: 11): KVRWK

[0028] Comparing with the first side strand (LKLKLK) and the second side strand of BTT1-3A (KLKLKL), the first side strand at the N-terminal of the synthetic P-hairpin AMP W-W 13, and the second side strand at the C terminal of the synthetic p-hairpin AMPW-W have been modified to remove the terminal leucine of the first side strand and second side strand of BTT1-3A, and been introduced with one tryptophan residue (W) at each side strand at symmetric positions.

[0029] The above fine-tuning of the side strands of diF-Fi? and W-W13 aims to carefully modulate the overall hydrophobicity of the peptide to stabilize the (3-hairpin and promote nanofiber fibrillation through intramolecular and intermolecular π-π stacking, while preventing spontaneous self-assembly into peptide hydrogels.

[0030] In one example, the synthetic [3-hairpin AMP as disclosed herein is diF-Fu, which comprises the sequence of SEQ ID NO: 7 (KFRFKLIAKFRFK) (hairpin turn is underlined). In another example, the synthetic (3-hairpin AMP as disclosed herein is W-W13, which comprises the sequence of SEQ ID NO: 8 (KWRVKLTAKVRWK) (hairpin turn is underlined).

[0031] For diF-Fu and W-W13, hydrophobic residues at both cross ends of the [3-hairpin peptides were symmetrically substituted with two phenylalanine or tryptophan, known to enhance bacterial membrane interaction and bactericidal activity and antifungal activity of typical AMPs.

[0032] In another example, the synthetic (3-hairpin AMP as disclosed herein is F-F15, which comprises the sequence of SEQ ID NO: 14 (LKFRVKLTAKVRFKL) (hairpin turn is underlined). For F-F15, both side strands of BTT1- A are substituted with one phenylalanine at symmetric positions. This peptide forms extensive nanonets with better potency against both E. coll and S. aureus, while maintaining similar CC50 as BTT1-3A.

[0033] In another example, the synthetic (3-hairpin AMP as disclosed herein is F-F13, which comprises the sequence of SEQ ID NO: 16 (KFRVKLT AK VRFK) (hairpin turn is underlined). For F-F13, terminal leucine of both side strands of BTT1-3A are removed, and both side strands of BTT1-3A are substituted with one phenylalanine at symmetric positions. This peptide demonstrated improved peptide fibrillation for 13-mer and formed extensive nanonet trapping E. coh, although with lesser trapping against S. aureus. It also showed a better potency than BTT1-3A against E. coll, and showed higher cytocompatibility on skin cells than both diF-Fu and W-W13.

[0034] In another example, the synthetic P-hairpin AMP as disclosed herein is E-P^ASYM y jc comprises the sequence of SEQ ID NO: 20 (EVRIKLTAKIEVR) (hairpin turn is underlined). For E-EHAS', M, terminal leucine of both side strands of BTT1-3A are removed, and both side strands of BTT1-3A are substituted with one glutamic acidat asymmetric positions. This peptide forms extensive bacteria responsive nanonets which efficiently agglutinate both Gram-positive and negative bacteria with the best CC50 value (about 477.6 pM).

[0035] Only when the synthetic P-hairpin AMP as disclosed herein in solution encounter a bacterial membrane component or fungal cell wall or membrane componentpathogen-associated molecular patterns (PAMPs) of a bacterium or a fungus, amyloid nucleation is initiated. Amyloid fibrils undergo elongation as hairpin peptides stack at the tail-end of the fibril. Once the nanofibrils grow sufficiently long, they start to form physical cross-links. Eventually, a mature interlocking network of fibrils not only entraps other bacteria in the surroundings, but also forms a tightly interwoven nanonet on or from bacterial surfaces with high surface coverage. The high bacterial surface coverage entraps, kills, and prevents the escape and spread of both non-motile bacteria and motile bacteria. The mature interlocking network of fibrils is termed as "nanonets" to trap and promote bacterial agglutination, and to prevent bacterial post-trapping escape for reducing bacterial spread. In one example, the PAMPs is lipoteichoic acid (LTA) on or from the bacterial surface of a Gram-positive bacterium. In another example, the PAMPs is lipopolysaccharide (LPS) on or from the bacterial surface of a Gram-positive bacterium. In one example, the bacterium is a Gram-positive bacterium. In another example, the bacterium is a Gram-negative bacterium

[0036] In one example, the synthetic P-hairpin AMP as disclosed herein forms a tightly interwoven nanonet on or from bacterial surfaces to trap, kill, and to prevent spread of a Gram-positive bacterium. In another example, the synthetic P-hairpin AMP as disclosed herein forms a tightly interwoven nanonet on or from bacterial surfaces to trap, kill, and to prevent spread of a Gram-negative bacterium. In another example, the synthetic P-hairpin AMP as disclosed herein forms a tightly interwoven nanonet on or from bacterial surfaces to trap, kill, and to prevent spread of both a Gram-positive bacterium and a Gram-negative bacterium. In one example, diF-Fi3 and W-W13 are key candidates of the present invention, that form tightly interwoven nanonet to prevent posttrapping escape of motile bacteria from the nanonets and demonstrate good cytocompatibility.

[0037] Advantageously, formation of nanonets by the synthetic P-hairpin AMP as disclosed herein is selectively responsive to the presence of bacteria cells, bacterial membrane components, or fungal cell wall or membrane components. For example,pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS) and lipotechoic acids (LTA). Self-assembling hybrid peptides often face solubility and stability issues that limit their clinical application. In the literature, incorporating aromatic residues is a common strategy to enhance the activity of antimicrobial peptides (AMPs). However, this approach often reduces solubility and promotes unwanted aggregation. No current method successfully balances these factors to achieve both high antimicrobial efficacy and precise control over nanonet formation. This challenge is addressed by finetuning the peptide side-strands to control nanonet morphology and antimicrobial function while maintaining bacterial specificity and enhancing killing activity. This design provides greater spatiotemporal control, as the peptides remain highly soluble and stable in water without spontaneous self-assembly, forming nanonets only at specific infection sites This makes them suitable for long-term drug formulation and targeted administration.

[0038] In another example, the synthetic p-hairpin AMP as disclosed herein forms an interwoven nanonet on or from fungal surfaces to trap, kill, and to prevent spread of a fungus.

[0039] Besides diF-Fn, W-Wn, F-F15, F-F13, and E-Ei3ASYM, other synthetic P-hairpin AMPs which are analogues of BTT1-3A are also synthesized:• Bttl-3Ais: terminal leucine of both side strands of BTT1-3A are removed. Bttl- 3AB comprises the sequence of KLKLKLT AKLKLK (SEQ ID NO: 12) (hairpin turn is underlined).• Btt1-3A11: terminal leucine-lysine of both side strands of BTT1-3A are removed Btt1-3A11comprises the sequence of LKLKL T AKLKL (SEQ ID NO: 13) (hairpin turn is underlined).• diF-Fis: both side strands of BTT1-3A are substituted with two phenylalanine residues at symmetric positions. diF-Fis comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO: 15) (hairpin turn is underlined).• F-Fn: terminal leucine-lysine of both side strands of BTT1-3A are removed, and both side strands of are substituted with one phenylalanine at symmetric positions. F-Fn comprises the sequence of FRVKLT AKVRF (SEQ ID NO: 17) (hairpin turn is underlined).• W-Wn: terminal leucine-lysine of both side strands of BTT1-3A are removed, and both side strands of are substituted with one tryptophan at symmetricpositions. W-Wn comprises the sequence of WRVKLTAKVRW (SEQ ID NO: 18) (hairpin turn is underlined).• E-EB: terminal leucine of both side strands of BTT1-3A are removed, and both side strands of BTT1-3A are substituted with one glutamic acid at symmetric positions. E-E comprises the sequence of EVRIKLT AKIRVE (SEQ ID NO: 19) (hairpin turn is underlined).W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21).W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22).W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23).W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24).W5 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 5- hydroxy-tryptophan (SEQ ID NO: 25).W6 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7-aza- tryptophan (SEQ ID NO: 26).W7 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 2,3- dihydro-2-oxo-tryptophan (SEQ ID NO: 27).Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28).F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29).F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30).F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienyl alanine (SEQ ID NO: 31).F5 and comprises the sequence of KXRXKLTAKXRXK, wherein X is Tyrosine (SEQ ID NO: 32)F6 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- furylalanine (SEQ ID NO: 33).• F7 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 5- thiazolylalanine (SEQ ID NO: 34).• F8 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3-(3- pyridyl)alanine (SEQ ID NO: 35).• E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO: 36)

[0040] Table 3A and 3B summarizes designs of the peptides of the present invention, and their physicochemical parameters.

[0041] Table 3A. Peptide designs and their physicochemical parameters. All peptides were amidated at C-terminus. The turn motif was underlined, and key residues are bold. Subscript numbers denote the length of the peptides.AveragePeptide Sequence Length Charge^’ „.......® Hydrophilicity(a)BTT1-3A LKLKLKLTAKLKLKL IS 7 0.301. Reduce lengthbtt1-3A₁₃ KLKLKLTAKLKLK 13 7 0.62btt1-3A₁₁ LKLKLTAKLKL 11 5 0.192. Increase aromaticityF-F₁₅ LKFRVKLTAKVRFKL 15 7 0.25diF-F₁₅ LKFRFKLTAKFRFKL 15 7 0.11F-F₁₃ KFRVKLTAKVRFK 13 7 0.56diF-F₁₃ KFRFKLTAKFRFK 13 7 0.41W-W₁₃ KWRVKLTAKVRWK 13 7 0.42F-F₁₁ FRVKLTAKVRF 11 5 0.12W-W₁₁ WRVKLTAKVRW 11 5 -0.053. Reduce positive chargeE-E₁₃ EVRIKLTA KIRVE 13 3 0.67E-E₁₃ASYMEVRIKLTAKIEVR 13 3 0.67(a) The charge and average hydrophilicity of peptide was calculated using Bachem peptide calculator. (https: / / www.bachem.com / knowledue-center / peptide- calculator / )

[0042] Table 3B. Peptide designs and their physicochemical parameters. All peptides were amidated at C-terminus. The turn motifs are underlined, and unnatural aromatic residues (X) are bold.Aromatic residues (X)Peptide Length Charge LogPL-Amino Acids Name LogP(a)Motif 1: KXRVKLTAKVRXK-NH₂W-W13Tryptophan Trp 13 7 -3.13 W1 3-Benzothienylalanine Bal 13 7 -1.67 W2 3-(1-Naphthyl)-alanine 1-Nal 13 7 -1.79 W3 7-fluoro-tryptophan Trp(7-F) 13 7 -2.86 W4 1-methyl-tryptophan Trp(1-Me) 13 7 -3.11 W5 5-Hydroxy-tryptophan Trp(5-OH) 13 7 -3.72 W6 7-aza-tryptophan Trp(7-Aza) 13 7 -4.34 W7 2,3-dihydro-2-oxo-tryptophan Oia 13 7 -4.35 Motif 2: KXRXKLTAKXRXK-NH₂diF-F13Phenylalanine Phe 13 7 -2.92 F1 Cyclohexylalanine Cha 13 7 -0.01 F2 3,4,5-trifluorophenylalanine Phe(3,4,5-F) 13 7 -1.25 F3 4-fluorophenylalanine Phe(4-F) 13 7 -2.37 F4 2-thienylalanine 2ThiAla 13 7 -2.68 F5 Tyrosine Tyr 13 7 -4.10 F6 2-furylalanine FurAla 13 7 -4.55 F7 5-thiazolylalanine 5Thiazo 13 7 -5.10 F8 3-(3-pyridyl)alanine 3-Pal 13 7 -5.34(a) Chemical structures were first drawn using ChemDraw and then converted to SMILES for Crippen logP calculation using RDKit.

[0043] In another example, the synthetic P-hairpin AMP as disclosed herein is W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3 -benzothienylalanine (SEQ ID NO: 21); W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22); W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7-fluoro-tryptophan (SEQ ID NO: 23); W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1-methyl-tryptophan (SEQ ID NO: 24); W5 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 5 -hydroxy -tryptophan (SEQ ID NO: 25); W6 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7-aza-tryptophan (SEQ ID NO: 26); W7 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 2,3-dihydro-2-oxo-tryptophan (SEQ ID NO: 27); Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28); F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5-trifluorophenylalanine (SEQ ID NO: 29), F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4-fluorophenylalanine (SEQ ID NO: 30); F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2-thienylalanine (SEQ ID NO: 31); F5 and comprises the sequence of KXRXKLTAKXRXK, wherein X is Tyrosine (SEQ ID NO: 32); F6 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2-furylalanine (SEQ ID NO: 33); F7 and comprises the sequence of KXRXKLT AKXRXK, wherein X is 5 -thiazolylalanine (SEQ ID NO: 34); F8 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3-(3-pyridyl)alanine (SEQ ID NO: 35); or E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO: 36).

[0044] Among the synthetic P-hairpin AMP as disclosed herein, diF-F₁₃ and W-W₁₃ were identified as the key candidates These two peptides, with similar physicochemical properties, interweave tightly and effectively trap and kill motile Gram-negative bacteria as well as non-motile bacteria. Among them, W-W₁₃ emerged as the lead candidate, demonstrating the best antibacterial activity against both Gram-positive and Gramnegative bacteria, superior bacterial trapping, and good cytocompatibility with skin cells.

[0045] The synthetic P-hairpin AMP as disclosed herein differs from the 15-mer BTT1-3A disclosed in the inventors' previous invention PCT / SG / 2023 / 050786 in the following ways:(a) Chemical features: BTT1-3A has Linear peptide sequence with repeating hydrophobic-cationic (Leu-Lys) residues, and forms beta-hairpin secondary structures when in contact with bacteria membranes components such as LPS or LTA. The synthetic P-hairpin AMP as disclosed herein has linear peptide sequence, but possesses unique side-strand optimization feature, either paired aromatic residues or asymmetrical anionic glutamic acids at both ends of the cross strands. They form beta-hairpin secondary structures when in contact with bacteria membranes.(b) Sequence Length: BTT1-3A has 15 amino acids length; while some of the synthetic P-hairpin AMP as disclosed herein, such as diF-F₁₃ and W-W₁₃ have a length of 13 amino acids to reduce production cost.(c) Nanonet morphology: BTT1-3A forms a nanonet with extensive entangled nanofibers in space between bacteria and lower bacterial surface coverage; while the synthetic P-hairpin AMP as disclosed herein such as diF-F₁₃ and W-W₁₃ forms tightly interwoven nanonet on or from bacterial surfaces, with high bacterial surface coverage.(d) Trapping efficiency: BTT1-3A forms a relatively loose nanonet which was relatively less effective in preventing the escape and spread of motile Gramnegative bacteria compared to the nanonet formed by the synthetic p -hairpin AMP as disclosed herein; The synthetic P-hairpin AMP as disclosed herein such as diF-F₁₃ and W-W₁₃ forms tight nanonets which effectively encage and significantly prevent the escape and spread of motile Gram-negative bacteria such as E. coli and P. aeruginosa.(e) Bioactivity: BTT1-3A are more effective against Gram-negative bacteria; while the synthetic p-hairpin AMP as disclosed herein, W-W₁₃ demonstrated improved bacterial killing against both Gram-negative and Gram-positive bacteria.

[0046] Additionally, analogs based on diF-F₁₃ and W-W₁₃, such as Wl, W2, W3, W4, F3 and F4, have demonstrated to be able to form tight nanonets, semi-tight nanonets or loose-entangled nanonets that are bacterial specific. Additionally diF-F₁₃, W-W₁₃, W1, W2, W3, W4, W5, W6, F1, F2, F3, F4, and F6, have also demonstrated antifungal activity.

[0047] There are some literature reports attempting to design synthetic anti-infective "nanonets". Those studies did not focus on the morphology of the bacteria-trapping nanofibers, or did they demonstrate the ability to directly prevent bacterial escape and trap bacteria, which is identified as a critical need to advance the field of synthetic nanonets. The present invention builds on the previous research on the BTT1 -3 A design However, the previous lead candidate BTT1-3A did not form extensive nanonets for effective bacterial trapping. The present invention highlights the significance of the Leu-Thr-Ala turn motif of BTT1-3A and the fine-tuning of side strands, which are crucial for forming tightly interwoven nanonets on or from bacterial surfaces to effectively trap and kill bacteria

[0048] The synthetic P-hairpin AMP as disclosed herein, upon contact with the PAMPs of a bacterium or fungus, forms interwoven nanonets on or from bacterial or fungal surfaces, which display both bacterial or fungal trapping and killing functionalities. The nanonets formed by peptide nanofibers interweaving on or from bacterial or fungal surfaces enhance trapping and limit microbial spread, as well as exhibit high antimicrobial activities. Hydrophobic residues at both cross ends of the P-hairpin peptides were symmetrically substituted with phenylalanine or tryptophan, known to enhance bacterial membrane or fungal cell wall or membrane interaction and bactericidal or fungicidal activity of typical AMPs. In one example, hydrophobic residues at both cross ends of theP-hairpin peptides were symmetrically substituted with two phenylalanine residues, resulting in diF-F₁₃ comprising the sequence of SEQ ID NO: 7. In another example, hydrophobic residues at both cross ends of the P-hairpin peptides were symmetrically substituted with one tryptophan, resulting in W-W13comprising the sequence of SEQ ID NO: 8. In another example, hydrophobic residues at both cross ends of the P-hairpin peptides were symmetrically substituted with one phenylalanine, resulting in F-F13comprising the sequence of SEQ ID NO: 16 (KFRVKLTAK VRFK). In another example, hydrophobic residues at both cross ends of the 15-mer P-hairpin peptide were symmetrically substituted with one phenylalanine, resulting in F-F15comprising the sequence of SEQ ID NO: 14 (LKFRVKLTAKVRFKL)

[0049] Among the peptides in Table 3A, surprisingly, adding more phenylalanine in diF-F15leads to loss of specificity towards bacterial membrane, forming random peptide hydrogels in PBS. diF-Fis forms spontaneous peptide hydrogels even in the absence of bacterial membranes. The two terminal leucine may contribute to the spontaneous formation of diF-F15hydrogels due to its extensive fibrillation and high overall hydrophobicity. Removing these terminal leucines results in diF-Fu which reduces its overall hydrophobicity, making it comparable to W-W13(Table 3A). This suggests that diF-F13and W-W13, with similar physicochemical properties (such as overall hydrophobicity and a four-backbone ring structure), are likely to fibrillate in a similar manner. diF-F13and W-W13interweave tightly and effectively trap motile Gram-negative bacteria and non-motile bacteria. Among them, W-W13 emerged as the lead candidate, demonstrating strong antifungal activity and antibacterial activity against both Grampositive and Gram-negative bacteria, superior bacterial trapping, and good cytocompatibility with skin cells. This demonstrates the importance of fine-tuning both the terminal end and aromaticity of the hydrophobic residue of the 13-mer P-hairpin peptide, which was crucial to enhance nanofibers fibrillation without turning into hydrogels. Hence, arriving at diF-F13and W-W13are not obvious. Further testing of diF-F13and W-W13analogs such as W1-W7 and F1-F8 further demonstrates this point.

[0050] Bacterial trapping and killing, prevention of post-trapping escape and fungicidal activity

[0051] The synthetic -hairpin AMP as disclosed herein display effective trapping and antimicrobial activity against a wide range of bacteria species because of the common occurrence and essentiality of LPS and LTA to the biology of Gram-negative and Grarn-positive bacteria respectively. The presence of these membrane components enables the formation of tightly interwoven nanonets on or from bacterial surfaces by the disclosed peptides, trapping and killing the bacteria. In one example, the synthetic p-hairpin AMP as disclosed herein can form tightly interwoven nanonets on or from bacterial surfaces to trap and kill a Gram-positive bacterium selected from any one or more of the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Listeria monocytogenes. In another example, the synthetic P-hairpin AMP as disclosed herein can form tightly interwoven nanonets on or from bacterial surfaces to trap and kill a Gramnegative bacterium selected from any one or more of the group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera. In another example, the synthetic -hairpin AMP as disclosed herein, upon contact with the PAMPs of cell wall or membrane component of a fungus, forms interwoven nanonets on or from fungal surfaces, which display both fungal trapping and killing functionalities. In one example, the fungus is selected from the group consisting of Candida, albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii. Preferably, Candida, albicans.

[0052] In another example, the synthetic P-hairpin AMP as disclosed herein kills or reduces bacterium, fungus, or both, and / or eliminates or reduces bacterial activity, fungal activity, or both. In another example, the synthetic P-hairpin AMP as disclosed herein kills or reduces bacterium or fungus and / or has antibacterial and / or antifungal activityagainst any one or more bacterium or fungi selected from one or more of the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Vibrio cholera, Candida, albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii. In another preferred example, the synthetic P -hairpin AMP as disclosed herein kills or reduces and / or has antifungal fungal activity against Candida albicans.

[0053] In another example, the synthetic P-hairpin AMP as disclosed herein can form tightly interwoven nanonets on or from bacterial surfaces which display potent antimicrobial and agglutinating and trapping activity against an antibiotic-resistant bacterium such as a clinical isolate. In another example, the antibiotic-resistant bacterium is methicillin-resistant Staphylococcus aureus. In another example, the methicillin-resistant Staphylococcus aureus is MRSA ATCC 1556, a clinical isolate which demonstrates resistance to multiple existing antibiotics. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem- resistant Escherichia coli. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Klebsiella aerogenes. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Pseudomonas aeruginosa. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Acinetobacter baumannii. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Klebsiella pneumonia. The synthetic p-hairpin AMP as disclosedherein are effective against clinical isolates with colistin and / or carbapenem resistance gene. The synthetic p-hairpin AMP as disclosed herein are effective against Gramnegative clinical isolates with carbapenem resistance gene because their resulting mutations do not cause any relevant change to the bacterial membranes.

[0054] Advantageously, the peptide nanonets formed by the synthetic P-hairpin AMP have distinct morphologies with either tightly interwoven nanofibers on or from the bacterial surface, or extensive nanonets with minimal bacteria membrane coverage. Recent studies on bacteria-trapping nanofibers have not focused on the morphology or fine-tuning of nanofiber interweaving patterns to improve trapping efficiency. The ability to control nanofiber interweaving patterns is crucial for tailoring nanonets to trap bacteria tightly or loosely, depending on therapeutic needs. Tightly interwoven nanonets on or from bacteria are particularly important for immobilizing and encaging bacteria, leading to more effective infection control and better clinical outcomes. The nature HD6 peptide nanonets in the small intestine share similar properties as E-E13ASYMof the present invention, as both loosely trap bacteria without killing them. In body environment with easy access by immune cells, such as small intestine, this peptide loosely trapping partially immobilizes bacteria, which control bacteria motility and might provide better interaction with immune cells. This may activate immune system to eliminate bacteria in regulated manner, reducing the risk of systemic infection. Additionally, loose nanonet allow better access of therapeutic compounds (eg: antibiotic, antimicrobial peptides or protein therapeutics) to interact with the trapped bacteria. On the other hand, W-W13and diF-F13offer additional advantage by forming peptide nanonets that alone can effectively kill and prevent bacterial post-trapping escape to spread. This is particularly beneficial in environments with lower immune cell access, such as in superficial skin or eye infections.

[0055] Advantageously, the present peptides such as W-W13and diF-F13effectively encage and kill motile Gram-negative bacteria, significantly reducing their motility and preventing escape to limit bacterial spread. In nature, motile Gram-negative bacteria can quickly escape from area with high local drug concentration to avoid being killed. While many studies show that bacteria-trapping nanofibers can agglutinate bacteria, they do not directly demonstrate the design's ability to reduce bacterial motility or contain them effectively in entrapment during in vitro assays. The disclosed peptides surpass conventional antibiotic by effectively encaging motile Gram-negative bacteria, reducing their motility and preventing escape, which enhances bacterial killing and limits spread.This novel approach provides better control over infections and directly addresses the challenge of bacterial escape, a gap often overlooked in traditional treatments.

[0056] Advantageously, the nanonets formed by the synthetic p-hairpin AMP as disclosed herein can simultaneously trap and kill bacteria or fungi. Dual antimicrobial function enables manipulation of the antimicrobial profile of the peptides to meet the clinical needs through rational design of the peptide sequence. It also enables more complete killing as entrapped microbes cannot escape from the tightly interweaved nanonets.

[0057] In one example, the synthetic P-hairpin AMP as disclosed herein can kill at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% bacteria or fungi that has been contacted by the synthetic P-hairpin AMP as disclosed herein. In another example, the synthetic P-hairpin AMP as disclosed herein has a minimum inhibitory concentration (MIC)90(the lowest concentration of at which 90% of the bacteria are inhibited) of 2-64 pM. In another example, the synthetic p-hairpin AMP as disclosed herein has a MIC90selected from the group consisting of about 2 μM, about 4 μM, about 6 pM, about 8 pM, about 10 pM, about 12 pM, about 14 pM, about 16 μM, about 18 pM, about 20 pM, about 22 pM, about 24 pM, about 26 pM, about 28 pM, about 30 pM, about 32 pM, about 30 pM, about 32 pM, about 34 pM, about 36 pM, about 38 pM, about 40 pM, about 42 pM, about 44 pM, about 46 pM, about 48 pM, about 50 pM, about 52 pM, about 54 pM, about 56 pM, about 58 pM, about 60 pM, about 62 pM, and about 64 pM.

[0058] In another example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% bacteria or fungi that has been contacted by the synthetic P-hairpin AMP is entrapped within the tightly interweaved nanonets formed by the synthetic P-hairpin AMP as disclosed herein. In another example, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 8%, less than 5%, less than 3%, less than 2%, or less than 1% bacteria or fungi after being entrapped within the tightly interweaved nanonets formed by the synthetic P-hairpin AMP as disclosed herein were able to escape the nanonet. In another example, the synthetic P-hairpin AMP has a trapping efficiency between about 36% to about 55%, such as, about 34%, about 36%, about 42%, about 44%, about 50% or about 55%.

[0059] In one example, the synthetic P-hairpin AMP as disclosed herein forms tightly interweaved nanonets to encage and kill a non-motile bacterium selected from any one or more of the group consisting of Staphylococcus aureus, Yersinia pestis, Klebsiella pneumoniae, Mycobacterium tuberculosis, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Corynebacterium diphtheriae, and Bacillus anthracis. In another example, the synthetic p-hairpin AMP as disclosed herein forms tightly interweaved nanonets to encage and kill a motile bacterium selected from any one or more of the group consisting of Escherichia coli, Pseudomonas aeruginosa Helicobacter pylori, Listeria monocytogenes, Bacillus cereus, Clostridium botulinum, Clostridium difficile, and Clostridium tetani. Non-motile bacteria are types of bacteria that don’t have the capabilities or physical makeup to move through their environment on their own. Non-motile bacteria only develop along the stab line when they are cultivated in stab tubes. This line will appear diffuse and spread into the media if the bacteria are mobile. The cilia and / or flagella of the motile bacteria are the components that enable movement or motility.

[0060] In another aspect, the present disclosure refers to a pharmaceutical or adjuvant composition comprising the synthetic P-hairpin AMP as disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0061] As used herein, "adjuvant composition" refers to a composition comprising the synthetic P-hairpin AMP as disclosed herein, which works synergistically with an antibiotic to treat a bacterial infection, or to delay or prevent the development of antibi otic-resi stance.

[0062] As used herein, the term "pharmaceutically acceptable carrier or diluent" refers to molecular entities and compositions that are compatible with the synthetic P-hairpin AMP as disclosed herein, physiologically tolerable and do not typically produce an allergic, toxic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; glycine; hyaluronic acid; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives suchas methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate, polyvinylpyrolidone; agar, gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0063] The pharmaceutical or adjuvant composition as disclosed herein may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. In one example, the pharmaceutical or adjuvant composition as disclosed herein is for intravenous injection. In another example, the pharmaceutical or adjuvant composition as disclosed herein is for intraperitoneal injection. In another example, the pharmaceutical or adjuvant composition as disclosed herein is for intratracheal administration. In another example, the pharmaceutical or adjuvant composition as disclosed herein is in a formulation selected from any one or more of the group consisting of a parenteral formulation, a nasal spray, an eye drop, a mouth wash or oral gel, and a topical ointment.

[0064] For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

[0065] Some examples of suitable carriers, diluents, excipients for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

[0066] Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and / or time delay agents. Suitable binders include gum acacia, gelatine, com starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include com starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0067] Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

[0068] Suspensions for oral administration may further comprise dispersing agents and / or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinylpyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or dioleate, -stearate or -laurate and the like.

[0069] The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

[0070] The topical formulations of the present invention, comprise an active ingredient together with one or more acceptable carriers, and optionally any othertherapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

[0071] Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and / or fungicidal agent and / or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

[0072] Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and / or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.

[0073] Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.

[0074] The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

[0075] The compositions may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.

[0076] In another aspect, the present disclosure refers to a method for treating a condition associated with a bacterial infection, fungal infection, or an immune-related disease in a subject in need thereof, comprising administering the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to the subject. In one example, the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or a bacterium or fungus to (a) trap and kill the bacterium or fungus; and / or (b) trap and reduce motility of the bacterium or the fungus.

[0077] In another aspect, the present disclosure refers to use of the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein, in the manufacture of a medicament for treating a condition associated with a bacterial or fungal infection, or an immune-related disease in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject. In one example, the synthetic P-hairpin AMP selfassembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from the a bacterium or a fungus to (a) trap and kill the bacterium or fungus; and / or (b) trap and reduce motility of the bacterium or fungus.

[0078] In another aspect, the present disclosure refers to the synthetic -hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein, for use in treating a condition associated with a bacterial or fungal infection, or an immune-related disease in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject. In one example, the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or PAMPsof cell wall or membrane component of the fungus, or a bacterium or a fungus to (a) trap and kill the bacterium or fungus; and / or (b) trap and reduce motility of the bacterium or fungus.

[0079] As used herein, the term "treating" and grammatical variations of that term, refer to administration of the synthetic P-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to a subject as described herein by any appropriate means as described herein. Such treatment includes any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

[0080] As used herein, the term "subject" refers to patients of human or other mammal and includes any individual in need of treatment of a condition, disorder or disease (such as a bacterial infection, or an immune-related disease) using the methods as disclosed herein. However, it will be understood that “patient” does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (eg. sheep, cows, horses, donkeys, pigs), laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters), companion animals (eg. cats, dogs) and captive wild animals (eg. foxes, deer, dingoes). In a particular example, the subject is a human.

[0081] As used herein, the term "administering" and variations thereof refers to contacting, applying, delivering or providing the synthetic p-hairpin AMP as disclosed herein, or the pharmaceutical or adjuvant composition as disclosed herein to the subject as disclosed herein, by appropriate means.

[0082] The tightly interweaving nanonets on or from bacterial or fungal surface formed by the synthetic -hairpin AMP as disclosed herein are highly effective in bacterial or fungal trapping, killing, and reducing mobility, as discussed herein, to thereby treat a condition associated with a bacterial or fungal infection, or an immune-related disease. In one example, the bacterial infection is caused by a Gram-positive bacteria selected from any one or more of the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria,and Listeria monocytogenes. In another example, the bacterial infection is caused by a Gram-negative bacteria selected from any one or more of the group consisting of Escherichia coli. Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera. In some examples, the bacterial infection is caused by an antibiotic-resistant bacterium. In a particular example, the antibiotic-resistant bacterium is colistin- and / or carbapenem- resistant Escherichia coli. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Klebsiella aerogenes. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Pseudomonas aeruginosa. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Acinetobacter baumannii. In another example, the antibiotic-resistant bacterium is colistin- and / or carbapenem-resistant Klebsiella pneumonia. In another example, the fungal infection is caused by any one or more fungi selected from the following group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpur eocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii. In a particular example, the fungal infection is caused by Candida albicans.

[0083] In some examples, the condition associated with abacterial infection is selected form any one or more of the group consisting of sepsis, septic arthritis, listeriosis, skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, abscesses, toxic shock syndrome (TSST-1), urinary tract infection (UTI), bloodstream infections, otitis media, sinusitis, meningitis, bacterial endocarditis, pharyngitis, cellulitis, impetigo, erysipelas, scarlet fever, necrotizing fasciitis, glomerulonephritis, rheumatic fever, myocarditis, arrhythmias, gastroenteritis, peritonitis, brucellosis, cat-scratch disease, cholera, Legionnaires' disease, pertussis, salmonella infections, shigellosis, tularemia, typhoid fever, acne, athlete's feet, onychomycosis, caries, periodontal disease, cold sore, rhinitis, and pink eye.

[0084] In certain examples, the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition as disclosed herein is administered to the patient after treatment of a condition associated with a bacterial or fungal infection, or an immune-related disease, either with the composition of the invention or with an alternative therapy, to prevent further recurrence.

[0085] Advantageously, the synthetic P-hairpin AMP as disclosed herein can form tightly interwoven nanonets on or from bacteria surface, to simultaneously trap and kill bacteria, and prevent the escape of motile bacteria out of the nanonets, enabling more complete killing before entrapped microbes can escape from the mesh. In addition, Gramnegative pathogens are recognized by the WHO to have high clinical priority, as the development pipeline for antibiotics against Gram-negative infections has been alarmingly lacking. The synthetic P-hairpin AMP as disclosed herein demonstrates effective antibacterial effect against Gram-negative bacterial pathogens such as E. coli and P. aeruginosa.

[0086] The synthetic P-hairpin AMP as disclosed herein, the 13-mer and 15-mer peptides as disclosed herein also show improved cytocompatibility and caused less membrane disruption in HaCaT keratinocytes compared to BTT1-3A, highlighting the potential application of these novel nanonets to trap bacteria and reduce chronic skin infections and inflammation In one example, the synthetic P-hairpin AMP as disclosed herein resulted in high viability of HaCaT keratinocytes when incubated with the HaCaT keratinocytes at concentrations up to 1000 pM. In another example, the 50% cytotoxicity concentration (CC50) of diF-Fu is above 60 pM, for example about 62.9 pM. In another example, the CC50 of W-W13 is above 69 pM, for example about 69.3 pM. In another example, the CC50 of F-F13 is above 120 pM, for example about 127.8 pM.

[0087] In one example, the immune-related disease is a bacterial or fungal infection-associated inflammatory disease. In one example, the bacterial or fungal infection-associated inflammatory disease is skin infection and inflammation. In another example, the bacterial or fungal infection-associated inflammatory disease is a chronic skin infection and inflammation. The synthetic P-hairpin AMP as disclosed herein is able to effective trap and kill bacteria or fungi what causes skin infection and inflammation to treat the disease, but in the meanwhile causes minimal toxicity to the skin cells Other bacterial or fungal infection associated inflammatory diseases may affect tissues that constantly expose to bacteria or fungi. In another example, the bacterial infection-associated inflammatory disease is cystic fibrosis-associated lung infection. In another example, the bacterial infection-associated inflammatory disease is bacterial conjunctivitis. In another example, the bacterial or fungal infection-associated inflammatory disease is chronic rhinosinusitis. In another example, the bacterial or fungal infection-associated inflammatory disease is inflammatory bowel diseases.

[0088] In addition, the synthetic P-hairpin antimicrobial peptides (AMPs) as disclosed herein has the ability to self-assemble into nanonets in response to bacterial endotoxins. The polycationicity in the side strands of the AMPs proved integral to facilitating interactions between the peptides and endotoxins. The disclosed fibrillating peptides could display anti-inflammatory functionality by capturing free endotoxins and pro-inflammatory cytokines in solution via electrostatic attractions. Endotoxin trapping can impede its engagement with host immune and endothelial cells, including activation of Toll-like receptor 4 (TLR4) and induction of nitric oxide production. By alleviating the damage of endotoxemia, the multifunctional nanonets formed by the synthetic P-hairpin AMPs as disclosed herein offer a holistic anti-infective therapy that mitigates both upstream and downstream ramifications of infectious pathogenesis.

[0089] In one example, the nanonets formed by the synthetic P-hairpin AMPs as disclosed herein trap an endotoxin LPS. Endotoxin trapping can impede its engagement with host immune and endothelial cells and ensuing unwanted immune response. In another example, the synthetic P-hairpin AMP that traps LPS is selected from any one or more of the group consisting of W-W13(SEQ ID NO: 8), diF-Fu (SEQ ID NO: 7), diF-F15 (SEQ ID NO: 15), F-F13(SEQ ID NO: 16), F-F15(SEQ ID NO: 14), E-E13ASYM(SEQ ID NO: 20), W1 (SEQ ID NO: 21), W2 (SEQ ID NO: 22), W3 (SEQ ID NO: 23), W4 (SEQ ID NO: 24), Fl (SEQ ID NO: 28), F2 (SEQ ID NO: 29), F3 (SEQ ID NO: 30), F4 (SEQ ID NO: 31) and E-E13ASYM(ch) (SEQ ID NO: 36). In another example, the synthetic p-hairpin AMP can trap LPS with an EC50 (the concentration of P-hairpin AMP that produces 50% of its maximum effect) of less than 1-10 pM. In another example, the synthetic p-hairpin AMP as disclosed herein has a EC50 selected from the group consisting of less than 10 pM, less than 8 pM, less than 6 pM, less than 5 pM, less than 4 pM, less than 3 pM, less than 2 pM, and less than 1 pM. In another example, after trapping and sequestering endotoxin such as LPS, antagonistic interactions between an antibiotic and extracellular LPS can be nullified, and the activity of the antibiotic can be restored.

[0090] In another example, the nanonets formed by the synthetic 0-hairpin AMPs as disclosed herein trap a pro-inflammatory cytokine selected from any one or more of the group consisting of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-12α (IL-12α), interleukin-12β (IL-12β), interleukin-18 (IL-18), and high mobility group box 1 (HMGB1). In another example, the nanonets formed by the synthetic 0-hairpin AMPs as disclosed herein do not trap an antiinflammatory cytokine selected from any one or more of the group consisting of interleukin-4 (IL-4), interleukin- 10 (IL- 10), interleukin- 11 (IL- 11), interleukin- 13 (IL-13), transforming growth factor beta 1 (TGF-β1), and interleukin-18BP (IL-18BP). Pro-inflammatory cytokines (e g TNF-a, IL-6, IL-la, IL-10, IL-12a, IL-120, IL-18 and HMGB 1) mostly carry negative charges, whereas many anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13, TGF-01 and IL-18BP) are positively charged. The apparent trapping selectivity of the nanonets formed by the synthetic 0-hairpin AMPs as disclosed herein towards the pro-inflammatory cytokines is indicative of electrostatic interactions mediated by the cationic Lys residues on the peptides.

[0091] The tightly interweaving nanonets formed by the synthetic 0-hairpin AMP as disclosed herein is able to trap and prevent bacterial spread. In one example, the synthetic 0-hairpin AMP as disclosed herein is able to inhibit spread of 60%-90% of motile bacteria (for example, Gram-negative bacteria selected from any one or more of the group consisting of E. coli and P. aeruginosa) at a concentration of 20-40 uM. In another example, about 20 pM W-W13 is able to inhibit spread of 60%-90% of motile bacteria such as E. coli and / '. aeruginosa, after about 30 min contact with the bacteria. In another example, about 40 pM diF-Fi3 is able to inhibit spread of 60%-90% of motile bacteria such as E. coli and P. aeruginosa, after about 30 min contact with the bacteria

[0092] In one example, the synthetic 0-hairpin AMP as disclosed herein may be coadministered with an antibiotic, to prevent or delay the development of antibiotic resistance, since the nanonets formed by the synthetic 0-hairpin AMP as disclosed herein tightly interweaves the bacterial surface, trapping, killing, and reducing the motility of the bacteria. The antibiotics may be of various classes. In another example, the antibiotic belongs to a class selected from any one or more of the group consisting of aminoglycoside, macrolide, fluoroquinolone, 0-lactam, glycopeptide, oxazolidinone, and ansamycin. In another example, aminoglycoside is selected from any one or more of the group consisting of amikacin, gentamicin, kanamycin, neomycin, plazomicin,streptomycin and tobramycin. In another example, macrolide is selected from any one or more of the group consisting of azithromycin, clarithromycin, erythromycin and fidaxomicin. In another example, fluoroquinolone is selected from any one or more of the group consisting of ciprofloxacin, delafloxacin, gemifloxacin, levofloxacin, moxifloxacin, norfloxacin, and ofloxacin. In another example, P-lactam is selected from the group consisting of carbapenem, cephalosporin, cephamycin, clavam, monobactam and penicillin. In another example, glycopeptide is selected from any one or more of the group consisting of dalbavancin, oritavancin, teicoplanin, telavancin and vancomycin. In another example, oxazolidinone is selected from any one or more of the group consisting of linezolid and tedizolid. In another example, ansamycin is selected from any one or more of the group consisting of streptovaricin, rifamycin, naphthomycin, geldanamycin and ansamitocin. In some examples, the antibiotic is selected from any one or more of the group consisting of colistin, ceftazidime, ciprofloxacin, streptomycin, clarithromycin, vancomycin, linezolid, rifampicin, and rifabutin. In a particular example, the antibiotic is colisitin. In another particular example, the antibiotic is rifampicin. In one example, synthetic P-hairpin AMP as disclosed herein may be administered simultaneously with an antibiotic as disclosed herein. In another example, the synthetic -hairpin AMP for coadministration is selected from any one or more of the group consisting of W-W13 (SEQ ID NO: 8), diF-F13(SEQ ID NO: 7), diF-F15(SEQ ID NO: 15), F-F13 (SEQ ID NO: 16), F-F15 (SEQ ID NO: 14), E-E13ASYM(SEQ ID NO: 20), W1 (SEQ ID NO: 21), W2 (SEQ ID NO: 22), W3 (SEQ ID NO: 23), W4 (SEQ ID NO: 24), Fl (SEQ ID NO: 28), F2 (SEQ ID NO: 29), F3 (SEQ ID NO: 30), F4 (SEQ ID NO: 31) and E-E13ASYM(ch) (SEQ ID NO: 36). For treating a condition associated with a bacterial infection, or an immune -related disease, the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg. In one example, the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 2.5-4 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 3-5 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 4-6 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 5-7 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 6-8 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 7-9 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of 8-10 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of about 2.5mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of about 5 mg / kg. In another example, the synthetic P-hairpin AMP is administered in a dose of about 10 mg / kg.

[0093] The minimum inhibitory concentration (MIC)90 (the lowest concentration of at which 90% of the bacteria or fungi are inhibited) of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 4-128 pM. In one example, the MIC of the synthetic p-hairpin AMP as disclosed herein towards a bacteria or fungus is 4-8 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 8-12 pM. In another example, the MIC of the synthetic p-hairpin AMP as disclosed herein towards a bacteria or fungus is 8-16 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 12-16 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 16-20 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 18-24 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 26-32 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 32-36 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 36-40 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 40-44 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 44-48 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 48-52 pM. In another example, the MIC of the synthetic p-hairpin AMP as disclosed herein towards a bacteria or fungus is 52-56 pM In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 56-60 pM. In another example, the MIC of the synthetic p-hairpin AMP as disclosed herein towards a bacteria or fungus is 60-64 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 64-96 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is 96-128 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 4 pM. In another example, the MIC of the synthetic β-hairpin AMP as disclosed herein towards a bacteria or fungus is about 8 μM. In another example, the MIC of the synthetic p-hairpin AMP as disclosedherein towards a bacteria or fungus is about 16 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 18 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 20 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 22 pM In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 24 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 26 pM. In another example, the MIC of the synthetic p-hairpin AMP as disclosed herein towards a bacteria or fungus is about 28 pM In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 30 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 32 pM. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 64 M. In another example, the MIC of the synthetic P-hairpin AMP as disclosed herein towards a bacteria or fungus is about 128 M.

[0094] To delay or prevent the development of antibiotic resistance, the synthetic P-hairpin AMP is administered in a sub -inhibitory concentration of 1-64 pM, l-32pM, 1-16 pM, 1-8 pM, 1-6 pM, or 1-4 pM. In one example, the sub-inhibitory concentration is about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 8 pM, about 16 pM, about 32pM or about 64pM. When used together with an antibiotics, to delay the development of antibiotic resistance, the synthetic p-hairpin AMP as disclosed herein is not administered as the main active therapeutic, but as an adjuvant in combination to support the function of the main antibiotic. Hence, it is given at sub-inhibitory concentration.

[0095] The synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered via a route selected from any one or more of the group consisting of intravenous, intraperitoneal, intratracheal, intramuscular, intradermal, subcutaneous, oral, topical, and intranasal administration.

[0096] The synthetic p-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered at a frequency depending on the specific disease to be treated and / or the administration frequency of an antibiotic. In some examples, the synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered at a frequency selected from the group consisting of one, two, three, four, or six times a day.

[0097] The synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered for a duration depending on the specific disease to be treated and / or the administration duration of an antibiotic. In some examples, the synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein may be administered for a duration selected from the group consisting of about 1 day, about 2 days, about 1 week, about 10 days, about 15 days, about 1 month, about 3 months, about 6 months, about 1 year, or greater than 1 year. The treatment may be continual for days, weeks, months, or even years.

[0098] In another aspect, the present disclosure refers to a kit comprising the pharmaceutical or adjuvant composition of as disclosed herein, and a dispenser and / or applicator. The dispenser and / or applicator facilitates dispensing and applying the synthetic P-hairpin AMP, the pharmaceutical or adjuvant composition as disclosed herein, and may be designed for convenient application and long-term storage of the AMP and compositions. The packaging or dispenser may include a bottle, tube, spray bottle, a syringe or other dispenser. In certain examples, the composition is packaged in a concentrated form, and diluted to a desired concentration upon use by the end user. In some examples, the composition is formulated and packaged in a manner suitable for long-term storage to maintain efficacy of the composition.

[0099] The kit may further include additional components to facilitate application of the composition to the affected area, such as, for example, a brush, sponge, cotton swab, syringe, needle, nebulizer, or the like.

[0100] Other components of the kit may include, but are not limited to, one or more antibiotics selected from the group consisting of colistin, ceftazidime, ciprofloxacin, streptomycin, clarithromycin, vancomycin, linezolid, rifampicin, and rifabutin.

[0101] In another aspect, the present disclosure refers to a method for trapping an endotoxin lipopolysaccharide to reduce inflammatory activity in a subject in need thereof, comprising administering the synthetic P-hairpin AMP as described herein, or the pharmaceutical or adjuvant composition of statement described herein to the subject. In one example, the administered synthetic P-hairpin AMP is selected from any one or more of the group consisting of W-W13 (SEQ ID NO: 8), diF-F13(SEQ ID NO: 7), F-F13 (SEQ ID NO: 16), F-F15 (SEQ ID NO: 14) and E-E13ASYM(SEQ ID NO: 20).

[0102] Antimicrobial peptide nanonet coating

[0103] In another aspect, the present disclosure refers to a coated substrate comprising the synthetic p-hairpin AMP as disclosed herein. In one example, the coated substrate comprises a substrate coated with a synthetic P-hairpin antimicrobial peptide (AMP) comprising the following functional modules: (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation; wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA (‘p’: d-Proline) (SEQ ID NO: 38); and (b) a structural module comprising a first side strand and a second side strand for P-sheet formation and molecular stacking during fibrillation, wherein the synthetic p-hairpin AMP comprises a sequence selected from any one or more of the group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X is an aromatic hydrophobic amino acid, or an analog or derivative thereof; c is a cationic amino acid, or an analog or derivative thereof; h is a non-aromatic hydrophobic amino acid, or an analog or derivative thereof; a is an anionic amino acid, or an analog or derivative thereof; and wherein the synthetic P-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to (i) trap and kill the bacterium or the fungus; and / or (ii) trap and reduce motility of the bacterium or the fungus.

[0104] In another aspect, the present disclosure refers to a medical device comprising the coated substrate as described herein.

[0105] In another aspect, the present disclosure refers to a method for preventing or reducing bacterial biofilm formation on a medical device surface, comprising conjugating a synthetic P-hairpin antimicrobial peptide (AMP) onto the medical device surface, wherein the synthetic P-hairpin AMP comprises the following functional modules: (a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation; wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA (‘p’:d-Proline) (SEQ ID NO: 38); and (b) a structural module comprising a first side strand and a second side strand for β-sheet formation and molecular stacking during fibrillation, wherein the synthetic P-hairpin AMP comprises a sequence selected from the any one or more of group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X is an aromatic hydrophobic amino acid, or an analog or derivative thereof; c is a cationic amino acid, or an analog or derivative thereof; h is a non-aromatic hydrophobic amino acid, or an analog or derivative thereof, a is an anionic amino acid, or an analog or derivative thereof; and wherein the synthetic p-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to (i) trap and kill the bacterium or the fungus; and / or (ii) trap and reduce motility of the bacterium or the fungus.In another aspect, the present disclosure refers to a method of preventing or treating a biofilm-related infection in a subject in need thereof, comprising implanting a medical device comprising the coated substrate as described herein, into the subject, wherein the synthetic P-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to (i) trap and kill the bacterium or the fungus; and / or (ii) trap and reduce motility of the bacterium or the fungus.

[0106] In another aspect, the present disclosure refers to a medical device comprising the coated substrate as described herein for use in treating preventing or treating a biofilm-related infection in a subject in need thereof, wherein the medical device is for implantation into the subject, wherein the synthetic p-hairpin AMP coated on the substrate self-assembles into nanonets or interwoven nanofibers on or from bacterial surface or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to (i) trapand kill the bacterium or fungus; and / or (ii) trap and reduce motility of the bacterium or fungus.

[0107] As one example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the the synthetic p-hairpin AMP is: BTT1-3A and comprises the sequence of LKLKLKLTAKLKLKL (SEQ ID NO: 5); BTT2-4A and comprise the sequence of LKLKLKVpPAKLKLKL (SEQ ID NO: 39). W-W13and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); F-FB and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); E-E13 and comprises the sequence of EVRIKLT AKIRVE (SEQ ID NO: 19); F-FB and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16), diF-Fis and comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO: 15); W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3 -benzothienylalanine (SEQ ID NO: 21); W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22); W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7-fluoro-try ptophan (SEQ ID NO: 23); W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1 -methyl-tryptophan (SEQ ID NO: 24); Fl and comprises the sequence of KhRliKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28); F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5-trifluorophenylalanine (SEQ ID NO: 29); F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4-fluorophenylalanine (SEQ ID NO: 30); F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2-thienylalanine (SEQ ID NO: 31); E-E13ASYM(C1I) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO: 36), or a combination thereof.

[0108] The substrate as described herein may include any solid material, article, or surface onto which one or more of the synthetic p-hairpin AMP peptides can be immobilized, grafted, or otherwise affixed, whether directly or through one or more intermediate layers, linkers, or surface-modification steps. The substrate may be a polymeric, metallic, ceramic, or composite material and includes both bulk materials and surface coatings on underlying articles. As one example, the substrate is selected from any one or more of the group consisting of a polyurethane (PU), hydrogel coated latex,silicone, latex, polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), plasticized polyvinyl chloride (pPVC), polyimide (PI), ethylene vinyl acetate (EVA), poly(tetrafluoroethylene) (PTFE), titanium dioxide (Ti02), Polyetheretherketone (PEEK) and silicon dioxide (Si02). As another particular example, the substrate is selected from any one or more of the group consisting of PU, PVC, PTFE, silicone and latex.

[0109] The substrate may be coated with the synthetic P-hairpin AMP peptides using any suitable technique known to a person skilled in the art, including without limitation physical adsorption, covalent grafting, layer-by-layer assembly, surface-initiated polymerization, or other chemical or physico-chemical immobilization strategies compatible with the substrate material and the intended use. By way of non-limiting example, for grafting peptides onto polymer surfaces such as polyurethane (PU), a representative process may include covalently conjugating the peptide to a PU film using a bifunctional cross-linker. In one such implementation, the polymer surface is first exposed to oxygen plasma to introduce oxygen-containing functionalities that enhance surface reactivity. The activated surface is then silanized, for instance with an aminosilane such as (3-Aminopropyl)triethoxysilane (APTES), to form covalent siloxane bonds and provide a stable functional interface bearing pendant amino groups. A dialdehyde crosslinker such as glutaraldehyde is subsequently applied so that its aldehyde moieties couple with available amino groups, enabling covalent attachment of the peptide to the functionalized surface. This approach yields a robust immobilization of the antimicrobial peptide on the polymer substrate while preserving the bioactivity of the peptide and providing a durable coating suitable for medical device applications. This method is applicant to various substrates, such as but not limited to, PVC, PTFE, silicone and latex.

[0110] As one example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the synthetic p-hairpin AMP is conjugated to the substrate or the medical device surface via glutaraldehyde cross-linking.

[0111] As another example, in any of the coated substrates, the medical devices compri sing the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing ortreating a biofilm-related infection in a subject in need thereof as described herein, the synthetic P-hairpin AMP retains fibrillation capacity after conjugation to the substrate.

[0112] As another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the synthetic p-hairpin AMP is coated at a concentration selected from the group consisting of 10-1000 pM / cm2, 50-900 pM / cm2, 100-800 pM / cm2, 200-700 pM / cm2, 300-600 pM / cm2, 400-500 pM / cm2, 10-100 pM / cm2, 50-200 pM / cm2, 100-300 pM / cm2, 200-400 pM / cm2, 300-500 pM / cm2, 400-600 pM / cm2, 500-700 pM / cm2, 600-800 pM / cm2, 700-900 pM / cm2, or 800-1000 pM / cm2. In a particular example, the synthetic β-hairpin AMP is coated at a concentration of about 100 pM / cm2.

[0113] A medical device as described herein may include any product, instrument, implant, material, or system that, in its intended use, comes in contact directly or indirectly with the body or bodily tissues and is intended to diagnose, monitor, prevent, or treat disease or injury; support or modify the body or a physiological process; or deliver or remove substances to or from the body. A medical device may include external or internal devices, invasive or non-invasive devices, temporary or long-term devices, passive or active devices, and single-use or reusable devices for use in a subject

[0114] In one example, in any of the coated substrates, the medical devices, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the medical device is selected from any one or more of the group consisting of a urinary catheter, a urinary stent, a joint implant, a vascular catheter, a polyurethane (PU) catheter, an intravenous line, a dental implant, a continuous glucose monitor, a wound dressing, a suture, an adhesive bandage, and a feeding tube.

[0115] In another example the medical device is coated with the synthetic P-hairpin AMP on both internal and external surfaces.

[0116] In another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the bacterial membrane component is selected from any one or more of the group consistingof lipoteichoic acid (LTA) of a Gram-positive bacterium and lipopolysaccharide (LPS) of a Gram-negative bacterium, and the bacterium is a Gram-positive bacterium or a Gramnegative bacterium. In a particular example, in any of the coated substrates, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofil -related infection in a subject in need thereof as described herein, the bacterium is one associated with urinary tract infection, selected from any one or more of the group consisting of uropathogenic E. coli, Klebsiella pneumoniae, Carbapenemase Producers (KpCP), Enterococcus faecalis, Proteus Mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp., Aeromonas spp., Serratia spp., Neisseria spp., Providencia spp., Acinetobacter spp., Veillonella spp., Citrobacter spp., Bacteroids spp., Staphylococcus saprophylicus, Streptococcus spp., Lactobacillus spp., Coryne bacterium spp., Aerococcus spp., Actinobaculum spp., Gardnerella spp., Propionibacterium spp., Ureaplasma spp., and Mycoplasma spp.

[0117] In another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the bacterium is an antibiotic-resistant bacterium or the bi ofi Im -related infection is an infection of an antibiotic-resistant bacterium. In a particular example, the antibioticresistant bacterium is selected from any one or more of the group consisting of colistin-and / or carbapenem- resistant Escherichia coli, colistin- and / or carbapenem-resistant Pseudomonas aeruginosa, and colistin- and / or carbapenem-resistant Klebsiella pneumonia.

[0118] In another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the fungus is or the fungal membrane component belongs to any one or more fungi selected from the following group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusilaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus. Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii. In a particular example, the fungus is Candida albicans.

[0119] In another example, in any the medical devices for use or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the biofilm-related infection is selected from any one or more of the group consisting of Catheter-Associated Urinary Tract Infections (CAUTIs), Central Line-Associated Bloodstream Infections (CLABSIs), Ventilator- Associated Pneumonia (VAP), Surgical Site Infections (SSIs), and Implant-Associated Infections (IAIS).

[0120] In another example, in any of the coated substrates, the medical devices compri sing the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the coated substrate or the medical device traps and kills the bacterium or fungus, reduces mobility of the bacterium or fungus, prevents or reduces biofouling, and delays or prevents the development of bacterial resistance compared to a conventional antibiotic selected from any one or more of the group consisting of Colistin, Vancomycin, Daptomycin, Linezolid, Teicoplanin, Cefazolin, Ceftriaxone, Meropenem, Ertapenem, Ciprofloxacin, Levofloxacin, Tigecycline, Tobramycin, Gentamicin, Naficillin, Oxacillin, Ceftazidime, Amoxicillin, Rifampin, Fosfomycin, Nitrofurantoin, Minocycline, and Clindamycin.

[0121] In another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the coated substrate or medical device maintains an antifouling and / or antimicrobial effect for up to 4-8 weeks. In a particular example, the medical device maintains an antifouling and / or antimicrobial effect for up to 4 weeks, 5 weeks, 6 weeks, or 8 weeks. In another particular example, the medical device maintains an antifouling and / or antimicrobial effect for up to four weeks.

[0122] In another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducingbacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the coated substrate or medical device results in at least 50% reduction in biofilm formation after 24 hours of implant, optionally compared to a medical device without the coated substrate. In another example, the coated substrate or medical device results in about 90% reduction in biofilm formation at the end of the first week of use.

[0123] In another example, in any of the medical devices comprising the coated substrate for use or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, an additional therapy is to be administered. In a particular example, the additional therapy is an antibiotic selected from any one or more of the group consisting of colistin, penicillin, amoxicillin, cephalexin, ceftriaxone, azithromycin, erythromycin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, tobramycin, ulfamethoxazole-trimethoprim, meropenem and imipenem. In another example, the additional therapy is to be administered before, during, or after the implantation of the medical device.

[0124] In another example, in any of the coated substrates, the medical devices comprising the coated substrate or the use thereof, the methods for preventing or reducing bacterial biofilm formation on a medical device surface or the methods of preventing or treating a biofilm-related infection in a subject in need thereof as described herein, the coated substrate or the medical device reduces oxidative stress and inflammatory responses associated with infections. In another example, the reduction in oxidative stress and inflammatory responses associated with infections is through reducing a pro-inflammatory cytokine. In a particular example, the coated substrate or medical device reduces a pro-inflammatory cytokine selected from any one or more of the group consisting of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin-1α (IL-1α), interleukin-ip (IL-lp), interleukin-12a (IL-12a), interleukin-12β (IL-12β), interleukin-18 (IL-18), and high mobility group box 1 (HMGB1). In another example, the medical device reduces the pro-inflammatory cytokine by at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, optionally compared to a medical device without the coated substrate. In a particular example, the pro-inflammatory cytokine is TNF-a.

[0125] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer” includes a plurality of primers, including mixtures and combinations thereof.

[0126] As used herein, the term “comprising” means “including.” Variations of the word "comprising", such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a composition “comprising” X may consist exclusively of X or may include one or more additional unrecited components.

[0127] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0128] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0129] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0130] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0131] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0132] Other embodiments are within the following claims and non-limiting examples

[0133] STATEMENTS

[0134] 1. A synthetic P-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising the following functional modules:(a) a recognition module comprising a hairpin turn having an amino acid sequence of LTA (SEQ ID NO: 6), for interacting with pathogen-associated molecular patterns (PAMPs) of a bacterium or PAMPs of cell wall or membrane component of a fungus to initiate amyloid nucleation; and(b) a structural module comprising a first side strand and a second side strand for P-sheet formation;wherein the synthetic -hairpin AMP comprises a sequence selected from any one or more of the group consisting of:cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO:4),wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;a = an anionic amino acid, or an analog or derivative thereof;andwherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or fungus, or on or from a bacterium or fungus.

[0135] 2. The synthetic P-hairpin AMP of statement 1, wherein the synthetic P-hairpin AMP is:a) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); b) W-W13and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); c) F-F13and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16); d) F-F15 and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);f) diF-F15and comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO: 15)g) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21);h) W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);i) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);j) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24);k) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);l) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);m) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);n) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienylalanine (SEQ ID NO: 31);o) E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO:36) orp) a combination thereof.

[0136] 3. The synthetic P-hairpin AMP of statement 1, wherein the PAMPs is selected from any one or more of the group consisting of lipoteichoic acid (LTA) of a Grampositive bacterium and lipopolysaccharide (LPS) of a Gram -negative bacterium, and the bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

[0137] 4. The synthetic P-hairpin AMP of statement 3, wherein the Gram-positive bacterium is selected from the any one or more of group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, / Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Listeria monocytogenes.

[0138] 5. The synthetic P-hairpin AMP of statement 3, wherein the Gram-negative bacterium is selected from the any one or more of group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera.

[0139] 6. The synthetic p-hairpin AMP of statement 1, wherein the fugus is selected from one or more of the group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusilaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fiimigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

[0140] 7. A pharmaceutical or adjuvant composition comprising the synthetic P- hairpin AMP of any one of statements 1-6, and a pharmaceutically acceptable carrier or diluent.

[0141] 8. The pharmaceutical or adjuvant composition of statement 7, wherein the composition is in a formulation selected from any one or more of the group consisting of a parenteral formulation, a nasal spray, an eye drop, a mouth wash or oral gel, and a topical ointment.

[0142] 9. A method for treating a condition associated with a bacterial infection, fungal infection, or an immune-related disease in a subject in need thereof, comprising administering the synthetic P-hairpin AMP of any one of statements 1-6, or the pharmaceutical or adjuvant composition of statement 7 or 8 to the subject,wherein the synthetic |3-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to(a) trap and kill the bacterium or fungus; and / or(b) trap and reduce motility of the bacterium or fungus.

[0143] 10. Use of the synthetic p-hairpin AMP of any one of statements 1-6, or the pharmacal or adjuvant composition of statement 7 or 8, in the manufacture of a medicament for treating a condition associated with a bacterial infection, fungal infection or an immune-related disease in a subject in need thereof, wherein the synthetic [3-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic [3-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to(a) trap and kill the bacterium or fungus; and / or(b) trap and reduce motility of the bacterium or fungus.

[0144] 11. The method of statement 9 or the use of statement 10, wherein the bacterial infection is an infection of a Gram-positive bacterium, a Gram-negative bacterium or a fungus selected from any one or more of the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis. Salmonella typhi, Vibrio cholera, Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides,Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus, and Paecilomyces variotii.

[0145] 12. The method of statement 9 or the use of statement 10, wherein the bacterial infection is an infection of an antibiotic-resistant bacteria.

[0146] 13. The method or the use of statement 15, wherein the antibiotic-resistant bacteria is selected from any one or more of the group consisting of colistin-and / or carbapenem- resistant Escherichia coll, colistin- and / or carbapenem- resistant Klebsiella aerogenes, colistin- and / or carbapenem-resistant Pseudomonas aeruginosa, colistin- and / or carbapenem-resistant Acinetobacter baumannii, and colistin- and / or carbapenem-resistant Klebsiella pneumonia.

[0147] 14. The method of statement 9 or the use of statement 10, wherein the condition associated with a bacterial or fungal infection is selected from any one or more of the group consisting of sepsis, septic arthritis, listeriosis, skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, abscesses, toxic shock syndrome (TSST-1), urinary tract infection (UTI), bloodstream infections, otitis media, sinusitis, meningitis, bacterial endocarditis, pharyngitis, cellulitis, impetigo, erysipelas, scarlet fever, necrotizing fasciitis, glomerulonephritis, rheumatic fever, myocarditis, arrhythmias, gastroenteritis, peritonitis, brucellosis, cat-scratch disease, cholera, Legionnaires' disease, pertussis, salmonella infections, shigellosis, tularemia, typhoid fever, acne, athlete's feet, onychomycosis, caries, periodontal disease, cold sore, rhinitis, and pink eye.

[0148] 15. The method of statement 9 or the use of statement 10, wherein the immune-related disease is a bacterial or fungal infection-associated inflammation disease.

[0149] 16. The method or use of statement 18, wherein the bacterial or fungal infection-associated inflammation disease is a chronic skin infection and inflammation.

[0150] 17. The method of any one of statements 9 and 11-16, or the use of any one of statements 10-16, wherein the synthetic -hairpin AMP or the composition is administered with an antibiotic.

[0151] 18. The method or use of statement 17, wherein the antibiotic is selected from any one or more of the group consisting of aminoglycoside, macrolide, fluoroquinolone, -lactam, glycopeptide, oxazolidinone, and ansamycin

[0152] 19. The method of any one of statements 9 and 11-18, or the use of any one of statements 10-18, wherein the subject is a human.

[0153] 20. The method of any one of statements 9 and 11-19, or the use of any one of statements 10-19, wherein the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg.

[0154] 21. The method or use of statement 20, wherein the synthetic P-hairpin AMP is administered at a sub-inhibitory concentration of 1-64 pM, preferably 1-8 pM or 1-4 pM.

[0155] 22. The method of any one of statements 9 and 11-21, or the use of any one of statements 10-21, wherein the synthetic P-hairpin AMP is administered via a route selected from any one or more of the group consisting of intravenous, intraperitoneal, intratracheal, intramuscular, intradermal, subcutaneous, oral, topical, and intranasal administration.

[0156] 23. The method of any one of statements 9 and 11 -22, or the use of any one of statements 10-22, wherein the synthetic P-hairpin AMP is administered at a frequency selected from the group consisting of one, two, three, four, and six times a day.

[0157] 24. The method of any one of statements 9 and 11-23, or the use of any one of statements 10-23, wherein the synthetic P-hairpin AMP is administered for a duration selected from the group consisting of about 1 day, about 2 days, about 1 week, about 10 days, about 15 days, about 1 month, about 3 months, about 6 months, about 1 year, or greater than 1 year.

[0158] 25. A kit comprising the pharmaceutical or adjuvant composition of statement 7 or 8, and a dispenser and / or applicator.

[0159] 26. A coated substrate, comprising: a substrate coated with a synthetic -hairpin antimicrobial peptide (AMP) comprising the following functional modules:(a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation; wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA fp’: d-Proline) (SEQ ID NO: 38); and(b) a structural module comprising a first side strand and a second side strand for - sheet formation and molecular stacking during fibrillation;wherein the synthetic P-hairpin AMP comprises a sequence selected from any one or more of the group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof;c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof; a = an anionic amino acid, or an analog or derivative thereof; andwherein the synthetic P-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to(i) trap and kill the bacterium or the fungus; and / or(ii) trap and reduce motility of the bacterium or the fungus.

[0160] 27. The coated substrate of statement 26, wherein the synthetic P-hairpin AMP is:a) BTT1-3A and comprises the sequence of LKLKLKLTAKLKLKL (SEQ ID NO:5);b) BTT2-4A and comprise the sequence of LKLKLKVpPAKLKLKL (SEQ ID NO:39).c) W-W13and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); d) F-F15 and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); f) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);g) F-F13and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16), h) diF-F₁₅ and comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO:15)i) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21);j) W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);k) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);l) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24);m) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);n) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);o) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);p) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienyl alanine (SEQ ID NO: 31);q) E-EirASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO: 36) or;a combination thereof

[0161] 28. The coated substrate of any one of statement 26 or 27, wherein the substrate is selected from any one or more of the group consisting of a polyurethane (PU), hydrogel coated latex, silicone, latex, polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), plasticized polyvinyl chloride (pPVC), polyimide (PI), ethylene vinyl acetate (EVA), poly(tetrafluoroethylene) (PTFE), titanium dioxide (TiO₂), Polyetheretherketone (PEEK) and silicon dioxide (SiO₂).

[0162] 29. The coated substrate of statement 28, wherein the substrate is or is part of a medical device selected from any one or more of the group consisting of a urinary catheter, a urinary stent, a joint implant, a vascular catheter, a polyurethane (PU) catheter, an intravenous line, a dental implant, a continuous glucose monitor, a wound dressing, a suture, an adhesive bandage, and a feeding tube.

[0163] 30. The coated substrate of statement 26, wherein the bacterial membrane component is selected from the any one or more of the group consisting of lipoteichoic acid (LTA) of a Gram-positive bacterium and lipopolysaccharide (LPS) of a Gramnegative bacterium, and the bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

[0164] 31. The coated substrate of statement 30, wherein the bacterium is one associated with urinary tract infection, selected from any one or more of the groupconsisting of uropathogenic E. coli, Klebsiella pneumoniae, Carbapenemase Producers (KpCP), Enterococcus faecalis, Proteus Mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp., Aeromonas spp., Serratia spp., Neisseria spp., Providencia spp., Acinetobacter spp., Veillonella spp., Citrobacter spp., Bacteroids spp., Staphylococcus saprophyticus, Streptococcus spp., Lactobacillus spp., Corynebacterium spp., Aerococcus spp., Actinobaculum spp., Gardnerella spp., Propionibacterium spp., Ureaplasma spp., and Mycoplasma spp.

[0165] 32. The coated substrate of statement 26, wherein the fungus is or the fungal membrane component belongs to any one or more fungi selected from the following group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur. Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

[0166] 33. The coated substrate of statement 30, wherein the bacterium is an antibiotic-resistant bacterium.

[0167] 34. The coated substrate of statement 33, wherein the antibiotic-resistant bacterium is selected from any one or more of the group consisting of colistin-and / or carbapenem- resistant Escherichia coli, colistin- and / or carbapenem-resistant Pseudomonas aeruginosa, and colistin- and / or carbapenem-resistant Klebsiella pneumonia.

[0168] 35. The coated substrate of any one of statements 26-34, wherein the synthetic β-hairpin AMP is conjugated to the substrate via glutaraldehyde cross-linking.

[0169] 36. The coated substrate of any one of statements 26-35, wherein the synthetic -hairpin AMP retains fibrillation capacity after conjugation to the substrate.

[0170] 37. The coated substrate of any one of statements 26-36, wherein the synthetic β-hairpin AMP is coated at a concentration selected from the group consisting of 10-1000 pM / cm2, 50-900 pM / cm2, 100-800 pM / cm2, 200-700 pM / cm2, 300-600 pM / cm2, 400- 500 pM / cm2, 10-100 pM / cm2, 50-200 pM / cm2, 100-300 pM / cm2, 200-400 pM / cm2, 300- 500 pM / cm2, 400-600 pM / cm2, 500-700 pM / cm2, 600-800 pM / cm2, 700-900 pM / cm2, or 800-1000 pM / cm2, preferably about 100 pM / cm2.

[0171] 38. A medical device, comprising the coated substrate of any one of statements 26-37.

[0172] 39. The medical device of statement 38, selected from any one or more of the group consisting of a urinary catheter, a urinary stent, a joint implant, a vascular catheter, a polyurethane (PU) catheter, an intravenous line, a dental implant, a continuous glucose monitor, a wound dressing, a suture, an adhesive bandage, and a feeding tube.

[0173] 40. The medical device of statement 39, wherein the medical device is coated with the synthetic p-hairpin AMP on both internal and external surfaces.

[0174] 41. A method for preventing or reducing bacterial biofilm formation on a medical device surface, comprising conjugating a synthetic P-hairpin antimicrobial peptide (AMP) onto the medical device surface, wherein the synthetic P-hairpin AMP comprises the following functional modules:(a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation; wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA (‘p’: d-Proline) (SEQ ID NO: 38); and(b) a structural module comprising a first side strand and a second side strand for P- sheet formation and molecular stacking during fibrillation;wherein the synthetic P-hairpin AMP comprises a sequence selected from the group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof;c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof; and a = an anionic amino acid, or an analog or derivative thereof,wherein the synthetic P-hairpin AMP self-assembles into nanonets or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of thebacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to(i) trap and kill the bacterium or the fungus; and / or(ii) trap and reduce motility of the bacterium or the fungus.

[0175] 42. The method of statement 41, wherein the synthetic 0-hairpin AMP is: a) BTT1-3A and comprises the sequence of LKLKLKLTAKLKLKL (SEQ ID NO:5);b) BTT2-4A and comprise the sequence of LKLKLKVpPAKLKLKL (SEQ ID NO:39).c) W-W₁₃ and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); d) F-F₁₅ and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); f) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);g) F-F₁₃ and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16); h) diF-F₁₅ and comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO:15)i) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21);j) W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);k) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);l) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24);m) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);n) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);o) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);p) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienylalanine (SEQ ID NO: 31);q) E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO: 36) or;r) a combination thereof.

[0176] 43. The method of statement 41, wherein the medical device surface belongs to a medical device selected from the group consisting of a urinary catheter, a urinary stent, a joint implant, a vascular catheter, a polyurethane (PU) catheter, an intravenous line, a dental implant, a continuous glucose monitor, a wound dressing, a suture, an adhesive bandage, and a feeding tube.

[0177] 44. The method of statement 41, wherein the bacterial membrane component is selected from the group consisting of lipoteichoic acid (LTA) of a Gram-positive bacterium and lipopolysaccharide (LPS) of a Gram-negative bacterium, and the bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

[0178] 45. The method of statement 44, wherein the bacterium is one associated with urinary tract infection, selected from the group consisting of uropathogenic E. coll, Klebsiella pneumoniae, Carbapenemase Producers (KpCP), Enterococcus faecalis, Proteus Mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp., Aeromonas spp., Serratia spp.. Neisseria spp., Providencia spp., Acinetobacter spp., Veillonella spp., Citrobacter spp., Bacteroids spp., Staphylococcus saprophylicus, Streptococcus spp., Lactobacillus spp., Coryne bacterium spp., Aerococcus spp., Actinobaculum spp., Gardnerella spp., Propionibacterium spp., Ureaplasma spp., and Mycoplasma spp.

[0179] 46. The method of statement 41, wherein the fungus is or the fungal cell wall or membrane component belongs to any one or more fungi selected from the following group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur. Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

[0180] 47. The method of statement 41, wherein the bacterium is an antibioticresistant bacterium.

[0181] 48. The method of statement 47, wherein the antibiotic-resistant bacterium is selected from the group consisting of colistin-and / or carbapenem- resistant Escherichia coli, colistin- and / or carbapenem-resistant Pseudomonas aeruginosa, and colistin- and / or carbapenem-resistant Klebsiella pneumonia.

[0182] 49. The method of statement 41, wherein the synthetic p-hairpin AMP is conjugated to the medical device surface via glutaraldehyde cross-linking.

[0183] 50. The method of statement 41, wherein the synthetic p-hairpin AMP retains fibrillation capacity after conjugation to the medical device surface.

[0184] 51. The method of statement 41, wherein the synthetic β-hairpin AMP is coated at a concentration selected from the group consisting of 10-1000 μM / cm2, 50-900 pM / cm2, 100-800 pM / cm2, 200-700 pM / cm2, 300-600 pM / cm2, 400-500 pM / cm2, 10-100 pM / cm2, 50-200 pM / cm2, 100-300 pM / cm2, 200-400 pM / cm2, 300-500 pM / cm2, 400-600 pM / cm2, 500-700 pM / cm2, 600-800 pM / cm2, 700-900 pM / cm2, or 800-1000 pM / cm2, preferably about 100 pM / cm2.

[0185] 52. A method of preventing or treating a biofilm-related infection in a subject in need thereof, comprising implanting a medical device comprising the coated substrate of any one of statements 26-37, or the medical device of any one of statements 38-40 into the subject,wherein the synthetic β-hairpin AMP coated on the substrate self-assembles into nanonets or interwoven nanofibers on or from bacterial surface or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to(i) trap and kill the bacterium or fungus; and / or(ii) trap and reduce motility of the bacterium or fungus.

[0186] 53. The method of statement 52, wherein the medical device is selected from the group consisting of a urinary catheter, a urinary stent, a joint implant, a vascular catheter, a polyurethane (PU) catheter, an intravenous line, a dental implant, a continuous glucose monitor, a wound dressing, a suture, an adhesive bandage, and a feeding tube.

[0187] 54. The method of statement 52, wherein the biofilm-related infection is selected form the group consisting of Catheter-Associated Urinary Tract Infections (CAUTls), Central Line-Associated Bloodstream Infections (CLABSls), Ventilator-Associated Pneumonia (VAP), Surgical Site Infections (SSIs), and Implant-Associated Infections (IAIS).

[0188] 55. The method of statement 54, wherein the CAUTIs is caused by a bacterium selected from the group consisting of uropathogenic E. coli, Klebsiella pneumoniae, Carbapenemase Producers (KpCP), Enterococcus faecalis, Proteus Mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp., Aeromonas spp., Serratia spp., Neisseria spp., Providencia spp., Acinetobacter spp., Veillonella spp., Citrobacter spp., Bacteroids spp., Staphylococcus saprophyticus, Streptococcus spp., Lactobacillus spp., Corynebacterium spp., Aerococcus spp., Actinobaculum spp., Gardnerella spp., Propionibacterium spp., Ureaplasma spp., and Mycoplasma spp.

[0189] 56. The method of statement 52, wherein the fungus is or the fungal membrane component belongs to any one of fungi selected from the following group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

[0190] 57. The method of statement 54, wherein the biofilm-related infection is an infection of an antibiotic-resistant bacterium.

[0191] 58. The method of statement 57, wherein the antibiotic-resistant bacterium is selected from the group consisting of colistin-and / or carbapenem- resistant Escherichia coli, colistin- and / or carbapenem-resistant Pseudomonas aeruginosa, and colistin- and / or carbapenem-resistant Klebsiella pneumonia.

[0192] 59. The method of statement 52, wherein the medical device comprising the coated substrate of any one of statements 26-37, or the medical device of any one of statements 38-40, traps and kills the bacterium or fungus, reduces mobility of the bacterium or fungus, prevents or reduces biofouling, and delays or prevents the development of bacterial resistance compared to a conventional antibiotic selected from the group consisting of Colistin, Vancomycin, Daptomycin, Linezolid, Teicoplanin, Cefazolin, Ceftriaxone, Meropenem, Ertapenem, Ciprofloxacin, Levofloxacin,Tigecycline, Tobramycin, Gentamicin, Naficillin, Oxacillin, Ceftazidime, Amoxicillin, Rifampin, Fosfomycin, Nitrofurantoin, Minocycline, and Clindamycin.

[0193] 60. The method of statement 59, the method of any one of statements 41-51, the medical device of any one of statements 38-40, or the coated substrate of any one of statements 26-37, wherein the coated substrate or medical device maintains an antifouling and / or antimicrobial effect for up to four weeks.

[0194] 61. The method of statement 59, the method of any one of statements 41-51, the medical device of any one of statements 38-40, or the coated substrate of any one of statements 26-37, wherein the coated substrate or medical device results in an about 90% reduction in biofilm formation at the end of the first week of use

[0195] 62. The method of statement 59, the method of any one of statements 41-51, the medical device of any one of statements 38-40, wherein the coated substrate or medical device results in at least 50% reduction in biofilm formation after 24 hours of implant, optionally compared to a medical device without the coated substrate.

[0196] 63. The method of statement 52, wherein the synthetic [3-hairpin AMP is conjugated to the medical device surface via glutaraldehyde cross-linking.

[0197] 64. The method of statement 52, wherein the synthetic p-hairpin AMP retains fibrillation capacity after conjugation to the medical device surface.

[0198] 65. The method of statement 52, wherein the synthetic β-hairpin AMP is coated at a concentration selected from the group consisting of 10-1000 pM / cm2, 50-900 pM / cm2, 100-800 pM / cm2, 200-700 pM / cm2, 300-600 pM / cm2, 400-500 pM / cm2, 10-100 pM / cm2, 50-200 pM / cm2, 100-300 pM / cm2, 200-400 pM / cm2, 300-500 pM / cm2, 400-600 pM / cm2, 500-700 pM / cm2, 600-800 pM / cm2, 700-900 pM / cm2, or 800-1000 pM / cm2, preferably about 100μM / cm2.

[0199] 66. The method of statement 52, wherein an additional therapy is to be administered.

[0200] 67. The method of statement 66, wherein the additional therapy is an antibiotic selected from the group consisting of colistin, penicillin, amoxicillin, cephalexin, ceftriaxone, azithromycin, erythromycin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, tobramycin, ulfamethoxazole-trimethoprim, meropenem and imipenem.

[0201] 68. The method statement 67, wherein the additional therapy is to be administered before, during, or after the implantation of the medical device.

[0202] 69. A method for trapping an endotoxin lipopolysaccharide to reduce inflammatory activity in a subject in need thereof, comprising administering the synthetic p-hairpin AMP of any one of statements 1-6, or the pharmaceutical or adjuvant composition of statement 7 or 8 to the subject.

[0203] 70. The method of statement 69, wherein the synthetic P-hairpin AMP is selected from the group consisting of W-W13 (SEQ ID NO: 8), di F-F 1 (SEQ ID NO: 7), F-F13 (SEQ ID NO: 16), F-F15 (SEQ ID NO: 14) and E-E13ASYM(SEQ ID NO: 20).

[0204] 71. The method or use of statement 18, wherein the antibiotic is rifampicin.

[0205] 72. The method of statement 52, wherein the medical device reduces oxidative stress and inflammatory responses associated with infections.

[0206] 73. The method of statement 72, wherein the medical device reduces a pro-inflammatory cytokine selected from the group consisting of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin- la (IL- la), interleukin- ip (IL- 1 P), interleukin-12a (1L-I2a), interleukin- 12p (IL-12P), interleukin- 18 (IL-18), and high mobility group box 1 (HMGB1).

[0207] 74. The method of statement 73, wherein the medical device reduces the pro-inflammatory cytokine by at least 40%, optionally compared to a medical device without the coated substrate.

[0208] 75. The method of statement 73 or 74, the pro-inflammatory cytokine is TNF-a.

[0209] 76. A synthetic p-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising a sequence selected from any one or more of the group consisting of:cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO:4),wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;a = an anionic amino acid, or an analog or derivative thereof;andwherein the AMP kills or reduces bacteria, fungus, or both; and / oreliminates or reduces bacterial activity, fungal activity or both.

[0210] 77. The synthetic P-hairpin AMP of statement 76, wherein the synthetic P-hairpin AMP is:a) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); b) W-W13and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); c) F-FB and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16), d) F-F15 and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);f) diF-F₁₅ comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO: 15); g) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21);h) W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);i) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);j) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24);k) W5 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 5- hydroxy-tryptophan (SEQ ID NO: 25),l) W6 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7-aza- tryptophan (SEQ ID NO: 26);m) W7 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 2,3- dihydro-2-oxo-tryptophan (SEQ ID NO: 27);n) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);o) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);p) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);q) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienylalanine (SEQ ID NO: 31),r) F5 and comprises the sequence of KXRXKLTAKXRXK, wherein X is Tyrosine (SEQ ID NO: 32);s) F6 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- furylalanine (SEQ ID NO: 33);t) F7 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 5- thiazolylalanine (SEQ ID NO: 34);u) E-E13ASYM(ch) and comprises the sequence of E1KVRLTARVEIK (SEQ ID NO:36); orv) a combination thereof.78. The synthetic P-hairpin AMP of statement 76, wherein the bacterium or fungus is selected from the following group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis. Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Listeria monocytogenes, Escherichia coll, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, Vibrio cholera, Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

[0211] 79. A pharmaceutical or adjuvant composition comprising the synthetic P-hairpin AMP of any one of statements 76-78, and a pharmaceutically acceptable carrier or diluent.

[0212] 80. A method for treating a condition associated with a fungal infection in a subject in need thereof, comprising administering the synthetic p-hairpin AMP of any oneof statements 76-78, or the pharmaceutical or adjuvant composition of statement 79 to the subject.

[0213] 81. Use of the synthetic P-hairpin AMP of any one of statements 76-78, or the pharmaceutical or adjuvant composition of statement 79 in the manufacture of a medicament for treating a condition associated with a bacterial infection, fungal infection or an immune-related disease in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject.

[0214] 82. The method of statement 80, or the use of statement 81, wherein the synthetic p-hairpin AMP is administered in a dose of 2.5-10 mg / kg.

[0215] 83. The method of statement 80 or the use of statement 81, wherein the bacterial infection is an infection of an antibiotic-resistant bacteria.EXAMPLES

[0216] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0217] Example 1 - Materials and Methods

[0218] Materials and Methods

[0219] Cation-adjusted Mueller Hinton broth (MHB), Luria broth base, Miller's modified (LB), LB Broth (Luria low salt), LPS from E. coli O111:B4, LTA from S. aureus, Vancomycin hydrochloride, Polymyxin B sulfate, Kanamycin sulfate, Penicillin-Streptomycin solution, Propidium Iodide (PI), Trypsin-EDTA solution, Dimethyl sulfoxide (DMSO) and Glutaraldehyde (grade I, for use as electron microscope fixative) were obtained from Sigma (St. Louis, MO). Osmium tetroxide (OSO4) was purchased from Ted Pella (Redding, CA). K114 dye was obtained from Santa Cruz (Dallas, TX).1 OX Phosphate Buffered Saline (PBS) was obtained from Vivantis Technologies Sdn Bhd (Shah Alam, Malaysia). LB Agar (Lennox L Agar) and BODIPY™ TR Cadaverine were purchased from ThermoFisher Scientific (Waltham, MA). HyClone™ Standard Fetal Bovine Serum (FBS) was obtained from Cytiva (Marlborough, MA). CellTiter-Glo®Luminescent Cell Viability Assay kit was purchased from Promega (Madison, WI). Triton X-100 was obtained from Bio-Rad Laboratories (Hercules, CA).

[0220] The peptides used in this study were synthesized by GL Biochem (Shanghai, China) and were confirmed to have a purity of at least 95%, as determined by the manufacturer using reversed-phase high-performance liquid chromatography (RP-HPLC). Analytical HPLC chromatograms and ESI (+) mass spectra are provided in the supporting figures.

[0221] Bacterial Strains

[0222] E. coli ATCC 25922, S. aureus ATCC 29737, and P. aeruginosa PAO1 were obtained from American Type Culture Collection (Manassas, VA). E. coli K12 BW25113 was obtained from Horizon Discovery (Waterbeach, UK). Additionally, the following bacterial strains were tested:coli M2, M6, M12, K. pneumoniae ATCC 700603 and S. aureus ATCC BAA1556 and BAA1717.

[0223] Mammalian Cell line

[0224] HaCaT keratinocytes were kind gift by Professor Paul Ho Chi Lui (NUS, Singapore). The cells were cultured in high-glucose DMEM supplemented with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin.

[0225] Prediction of Peptide Secondary Structures

[0226] The amino acid sequences of the designed peptide libraries were submitted to D-I-TASSER (https: / / seq2fun.dcmb.med.umich.edu / D-I-TASSER / ) to predict their secondary structures.

[0028] The predicted structures were then visualized using PyMol tool.

[0227] Circular Dichroism (CD) Spectroscopy

[0228] CD spectroscopy was used to evaluate the secondary structures of peptides under various treatments, following the method previously described.

[0029] In brief, CD spectra were recorded for each peptide at a concentration of 50 pM, either in lx PBS buffer (pH 7.4) alone or in the presence of 0.2 mg / mL of LPS or LT A. The measurements were conducted using a quartz cell with a 1 mm path length over a wavelength range of 190-260 nm, with a scanning speed of 60 nm / min on a Jasco CD Spectrometer (JASCO, Easton, MD). Each sample was measured three times, and the spectra were averaged. Mean residue ellipticity (θM) was calculated using the following equation:θM = θobs / 10 · MRW / (c·l)M~ 10 ' c. lθM represents the mean residue ellipticity (deg cm2dmol⁻¹), θobsis the observed ellipticity corrected for the blank at a given wavelength (mdeg), MRW is the residue molecular weight (A / JP divided by the number of amino acids), c is the peptide concentration (mg / mL), and I is the path length (cm).

[0229] KI 14 Fluorescence Assay

[0230] The fibrillation of peptides was quantified by assessing the binding of KI 14 fluorescence dye. A 10 mM KI 14 stock solution was prepared by dissolving the dye in DMSO. This stock was then diluted to 250 pM with sterile deionized water, then immediately mixed and incubated with 20 pM of peptides in the presence of 5 pg / rnL LPS, LTA, or lx PBS (pH 7.4) alone, at 37°C with shaking at 120 rpm. The fluorescence intensity of KI 14 (XEX = 360 nm, XEM = 550 nm) was measured at 0.5, 1.0, and 2.0 hours using a TECAN Infinite M200 plate reader (Mannedorf, Switzerland). The percentage increase in the KI 14 signal was calculated using the following equation:Percentage increase in K114 signal = (Fluorescence Intensity(With LPS or LTA)- Fluorescence Intensity(Without LPS or LTA)x 100%Fluorescence Intensity(Without LPS or LTA)BTT1-3A served as positive control whereas BTT4 that did not fibrillate serves as negative control. Peptides that have higher fluorescence signal than BTT4 were identified as fibrillating peptides and selected for subsequent experiments.

[0231] Con focal Microscopy

[0232] To verify the spontaneous self-assembly of diF-Fis, a solution of 64 pM diF-Fis and 20 pM K114 dye was prepared in lx PBS buffer (pH 7.4) and incubated at 37°C with shaking at 120 rpm for 30 minutes. The sample was then placed on a coverslip, and z-stack confocal images were captured at 5 pm intervals using a BC43 Andor confocal microscope to visualize the bound K114 dye with an excitation wavelength of 360 nm and an emission wavelength of 550 nm. The images were processed and visualized using Imaris software, with scale bars added using ImageJ software.

[0233] Field-Emission SEM

[0234] The preparation of SEM samples was conducted as described in our previous work,

[0030] with some minor modifications. Bacterial cultures in the mid-log phase were diluted in MHB to an initial OD600 of 0.07 Peptides were then added at a concentrationof 40 pM, except 100 pM for E-E13ASYMto the diluted bacteria culture at a final volume of 500 pL in 1.5 mL Eppendorf tubes. The mixtures were incubated at 37°C with shaking at 120 rpm for 2 hours to induce nanonet formation. After incubation, the samples were centrifuged at 6000g for 6 minutes, and the pellets were washed gently with lx PBS (pH 7.4), a process repeated three times. The samples were then fixed overnight at 4°C in 2.5% glutaraldehyde (in water). Following fixation, the samples were washed twice with lx PBS and resuspended in 500 μL of 1x PBS. The samples were then added to pre-coated poly-L-lysine coverslips for 30 minutes, and unbound bacteria were removed by washing with 1x PBS. The samples were post-fixed with 1% OsO4 for 30 minutes and washed twice with distilled water to remove any residual OsO4. The samples were then dehydrated through a series of ethanol washes (50%, 75%, 95%, and three times with 100%). Finally, the samples were sputter-coated with gold using a Leica EM ACE200 (Wetzlar, Germany) and imaged with a JEOL JSM-6701F scanning electron microscope (Tokyo, Japan). At least three independent fields were imaged for each sample to ensure representativeness. The final images were processed in ImageJ (National Institutes of Health, Bethesda, MD) to include scale bars.

[0235] Minimum Inhibitory Concentration (MIC) assay

[0236] The broth microdilution method was used to determine the MICs of the peptides. Stock solutions of the peptides and the positive control antibiotics (vancomycin and polymyxin B) were first diluted to twice the highest tested concentration in a 96-well plate using MHB. The peptides were then serially diluted two-fold to achieve the desired concentration range, with a final volume of 50 pL per well. Bacterial cultures in the midlog phase were diluted to an ODeoo of 0.07, then further diluted 100-fold in MHB to obtain an initial inoculum of 106colony-forming units (CFU) / mL. A 50 pL aliquot of this bacterial suspension was added to each well containing the peptides, followed by incubation at 37°C with shaking at 120 rpm for 18 hours. After incubation, the ODeoo was measured using a Tecan Infinite M200 microplate reader (Mannedorf, Switzerland) The MIC value was defined as the lowest peptide concentration that resulted in a more than 90% reduction in ODgoo compared to the drug-free control.

[0237] BODIPY-Cadaverine (BC) Displacement Assay

[0238] The experimental protocol was adapted from a previously described method.1311The relative binding affinities of the peptides to bacterial LPS or LTA wereassessed by evaluating their capacity to displace BC from LPS-BC or LTA-BC complexes. Displacement of BC, which binds strongly to LPS and LTA, results in dequenching and fluorescence of the free BC. Initially, 50 pg / mL of LPS or LTA was incubated with 5 pM BC in lx PBS buffer (pH 7.4) for 30 minutes at room temperature, protected from light. The peptides and polymyxin B control were prepared by diluting 1 mM stock solutions to twice the highest tested concentration in a Costar® 96-well black polystyrene plate using lx PBS buffer. Serial two-fold dilutions were then performed to achieve the desired concentration range, with a final volume of 50 pL per well. Subsequently, 50 pL of the LPS / LTA-BC mixtures were added to the wells containing the peptides and incubated at 37°C with shaking at 120 rpm for 30 minutes. After incubation, the fluorescence intensity of the BC samples (XEX = 580 nm, XEM = 620 nm) was measured using a TECAN Infinite M200 plate reader (Mannedorf, Switzerland). The BC Occupancy Factor was calculated using the following equation:BC Occupancy Factor = (Fmax − Fsample) / (Fmax − Fmin)(Fmax − Fmin)Where Fmaxrepresents the highest fluorescence intensity signal of among the samples, Fmin the lowest and F sample the fluorescence intensity signal of the test sample.The percentage of BC displaced was calculated using equation below:Percentage of BC displaced (%) = (1 — BC Occupancy factor x 100%

[0239] Agglutination Assay

[0240] The bacteria were initially cultured in MHB overnight to achieve a sufficient density. The cultures were then centrifuged at 3000 rpm for 7 minutes, after which the supernatant was discarded, and the bacterial pellet was resuspended in lx PBS buffer (pH 7.4). The bacterial suspension was further diluted with lx PBS to obtain a starting OD600 of 0.23, measured using a disposable plastic cuvette (12 * 12 * 4.5 mm) with 1 mL of the bacterial culture. A 1 mM stock solution of the peptide was then added to the bacterial suspension to achieve a final peptide concentration of 40 pM in a total volume of 1 mL. After thorough mixing, 1 mL of the sample was transferred to a disposable plastic cuvette, and the ODeoo was measured immediately at time T=0 h. The ODeoo was subsequentlyrecorded at hourly intervals for up to 8 hours at room temperature using a Tecan Infinite M200 microplate reader (Mannedorf, Switzerland).

[0241] Cell Viability Assay

[0242] HaCaT keratinocytes were seeded at a density of 10,000 cells per well in a 96-well plate and allowed to adhere for 24 hours at 37 °C with 5% CO2. After this period, the culture medium was replaced with fresh serum-free media containing peptides at desired concentrations of up to 1000 pM. For controls, 1% Triton X was used as a positive control, while serum-free medium served as a negative control. Following 24 hours of incubation, the cells were washed, and 110 pL of a lOx diluted CellTiter-Glo® reagent (Promega) in serum-free media was added to each well, followed by a 3 5-hour incubation at 37 °C with 5% CO2. Absorbance was then measured at 490 nm using a Tecan Infinite M200 microplate reader (Mannedorf, Switzerland). The absorbance reading from the 1% Triton X control was subtracted from each sample as a blank. The percentage of cell viability was calculated using the following equation:Percentage cell viability (%) = AbsorbancePeptide-treated cells / AbsorbanceUntreated cellsx 100%Absorbance Untreated cellsCytotoxicity curves were plotted, and CC50 values were determined using non-linear regression analysis with GraphPad Prism 9.5 software.

[0243] HaCaT Keratinocyte Membrane Damage Assay

[0244] HaCaT keratinocytes were seeded at a density of 10,000 cells per well in a 96-well plate and allowed to adhere for 24 hours at 37 °C with 5% CO2, reaching 70-80% confluency. The culture medium was then replaced with fresh serum-free media containing peptides at the desired concentrations, and the cells were incubated for 1 hour under the same conditions. Following incubation, the cells were washed once with lx PBS buffer (pH 7.4) and stained with a 50 pg / mL PI solution in lx PBS at 37°C for 15 minutes. After staining, the cells were washed twice with lx PBS, and serum-free media were added to each well. The EVOS™ M7000 Imaging System (Life Technologies Corporation, Bothell, WA) was used to observe cell morphology and Pl-stained nuclei. Final images were processed in ImageJ (National Institutes of Health, Bethesda, MD) to include scale bars.

[0245] Soft Agar Motility Assay

[0246] All bacterial strains were cultured in LB medium until reaching mid-log phase, except for non-motile E. coli AflhD mutant, which was cultured in low-salt LB supplemented with 25 pg / mL kanamycin. The bacterial cultures were then diluted to 107colony-forming units (CFU) / mL in LB and incubated with peptides at the desired concentrations in a final volume of 60 pL in 1.5 mL Eppendorf tubes. The samples were thoroughly mixed by pipetting and incubated at 37 °C with shaking at 120 rpm for 30 minutes. Subsequently, 2 pL of each sample was seeded into 0.3% w / v LB agar plates and incubated at room temperature for 14 hours. After incubation, images of the agar plates were captured. Each sample was performed in independent triplicate. The diameter of the bacterial spreading zones on the plates was measured using ImageJ (National Institutes of Health, Bethesda, MD). The percentage inhibition of bacterial spreading was calculated using the following equation:Percentage Inhibition (%) = Diameter(Untreated bacteria) − Diameter(Peptide-treated bacteria) / Diameter(Untreated bacteria) x 100%Diameter(Untreated bacteria)

[0247] Statistical Analysis

[0248] Differences between groups were analyzed using one-way or two-way ANOVA, followed by Dunnett’s multiple comparisons test to compare each experimental group to the control group, (ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). GraphPad Prism 9.5 was used for statistical tests, graph drawing, and curve fitting.

[0249] FITC-LPS Trapping Assay

[0250] Peptides at varying concentrations were mixed with a fixed concentration of LPS (2.00 pg / mL) in lx PBS buffer (final volume: 500 pL) in 1.5 mL Eppendorf tubes. The mixtures were incubated at 37 °C with shaking at 120 rpm for 1 hour to allow nanonet formation. Subsequently, FITC-labeled LPS (2.00 pg / mL) was added to each tube, followed by an additional 1-hour incubation under the same conditions to enable FITC-LPS trapping by the nanonets. After incubation, the samples were centrifuged at 13,000 x g for 10 minutes. Then, 100 pL of the supernatant was carefully transferred to a black 96-well plate, and the fluorescence intensity of untrapped FITC-LPS (λEX = 492 nm, λEM = 525 nm) was measured using a TECAN Infinite M200 plate reader. The FITC- LPS trapping index was calculated using the following equation:Fsample — FblankFITC − LPS trapping index = 1 −Fcontrol — Fblank where:Fsample is the fluorescence intensity of the test sample,Fblank is the fluorescence of the PBS-only blank,Fcontrol is the fluorescence of the peptide-free control treated identically

[0251] NPN Uptake Assay

[0252] E. coli M2 strain was cultured in MHB until early exponential phase, then pelleted by centrifugation and resuspended in 5 mM HEPES buffer (containing 5 mM glucose, pH 7.0) to an optical density at 600 nm (ODeoo) of 0.1-0.2. Peptides were serially diluted in the same buffer to twice the intended final concentrations (f.c = 8, 16, 32, and 64 pM) at 50 pL per well in black 96-well plate. Separately, the bacterial suspension was mixed with NPN (f.c: 20 pM), and 50 pL of this mixture was added to each well containing peptide, resulting in the desired final peptide concentrations. After incubation at 37 °C with shaking at 120 rpm for 1 hour, fluorescence intensity was measured using a TECAN Infinite M200 plate reader (kex= 355 nm, Xem= 405 nm). The increase in NPN fluorescence, indicative of outer membrane disruption, was calculated using the following equation:

[0253] NPN fluorescence intensity’ = F - If where:F is the fluorescence of the peptide-treated sample,F0 is the fluorescence of the untreated bacterial suspension (NPN + bacteria without peptide).

[0254] PI Uptake Assay

[0255] E. coli M2 was grown in MHB to early-log phase, pelleted by centrifugation, and resuspended in lx PBS to an ODeoo of 0.1-0.2. Peptides were serially diluted in 1x PBSat twice the desired final concentrations (8, 16, 32, and 64 pM) at 50 pL per well in black 96-well plate. The bacterial suspension was mixed with PI to a final dye concentration of 50 pg / mL, and 50 pL of this mix was added to each peptide-containing well, yielding the target peptide concentrations. Plates were incubated at 37 °C with shaking at 120 rpm. Then, PI fluorescence which reflect inner membrane permeabilization was recorded on a TECAN Infinite M200 reader (Xex= 544 nm, λem= 612 nm). Inner membrane-damage-associated fluorescence was determined as:

[0256] PI fluorescence intensity’ = F -Fo where:F is the fluorescence of the peptide-treated sample,F0 is the fluorescence of the untreated bacterial suspension (PI + bacteria without peptide). Polymyxin B was used as a positive control for bacterial inner membrane disruption.

[0257] Antimicrobial Checkerboard Assay

[0258] To assess the interaction between peptides and rifampicin, each compound was first subjected to two-fold serial dilutions in MHB using separate 96-well plates. Equal volumes of the diluted peptide and antibiotic solutions were then combined to yield a final volume of 100 pL per well. Subsequently, 100 pL of bacterial inoculum (106CFU / mL) was added to each well, bringing the total volume to 200 pL per well. The plates were incubated at 37 °C with shaking (120 rpm) for 18-20 hours. Following incubation, bacterial growth was quantified by measuring ODsoo using a TECAN Infinite M200 plate reader. The degree of synergy between the peptides and antibiotic was evaluated using the fractional inhibitory concentration index (FICI), calculated as:FICI = MICA / (A+B) / MICA + MICB / (A+B) / MICBMICAMICBWhere:MICA / (A+B) and MICB / (A+B) refer to the MIC value of compounds A and B when used together,MICA and MICB refer to their respective MICs when tested individually.A FICI value of ≤ 0.5 was interpreted as evidence of synergistic interaction between the peptide and antibiotic.

[0259] Serial Passage Assay

[0260] Mid-log E. coli M2 cultures were diluted in MHB to ~1 × 106CFU ml-1. Rifampicin solutions covering 0.25 × to 2 × MIC were added to separate 2mL Eppendorf tubes, with or without the adjuvant peptide at a sub-MIC level in total volume of 300 uL. Tubes were shaken for 24 h at 37 °C (120 rpm). ODsoo was then recorded on a TECAN Infinite M200 to determine the new MIC for that passage Within each treatment set, the culture showing growth at the highest peptide concentration was diluted back to 1 × 106CFU / mL in fresh MHB and used to seed the next passage. This daily cycle of inoculation, incubation, and measurement was repeated throughout the serial -passage experiment for a total 18 days. The fold change in MIC was calculated by dividing the MIC at each passage by the initial MIC on day 0 These values were plotted using GraphPad Prism. A fold increase greater than 16 was considered indicative of resistance development, reflecting a significant reduction in antibiotic susceptibility.

[0261] Antifungal MIC assay

[0262] The antifungal activity of the peptides was evaluated using the broth microdilution method according to the CLSI M27 standard. Stock solutions of each peptide and the positive control amphotericin B were first prepared at twice the highest tested concentration in a 96-well plate using RPMI medium supplemented with MOPS (pH 7.0). The peptides were then serially diluted two-fold to obtain the desired concentration range, with a final volume of 50 pL per well. C. albicans was cultured on Sabouraud Dextrose Agar (SDA) plates for 24 hours at 30°C in a non-shaking incubator. A few colonies were then resuspended in RPMI + MOPS (pH 7.0), adjusted to an ODeoo of 0.1, and diluted 1,000-fold. A 50 pL aliquot of this fungal suspension was added to each well containing the peptide solutions, resulting in a final inoculum of approximately 5 x 103CFU / mL. The plates were incubated at 35°C without shaking for 24 hours. After incubation, fungal growth was assessed by measuring the OD492 using a Tecan Infinite M200 microplate reader (Mannedorf, Switzerland), and verified by microscopy. The MIC was defined as the lowest peptide concentration that produced >90% reduction in OD492 compared to the drug-free control.

[0263] Example 2- Results

[0264] Precise control over peptide nanonet architecture is instrumental in advancing the development of antibacterial nanonets. Here, it is presented a novel design strategy to control bacteria nanonet morphology through rational modification of the p-hairpin sidestrands, leveraging the unique chemical properties of amino acid side chains. By finetuning both the terminal ends and aromaticity of the hydrophobic residue, the W-W13 peptide was engineered with increased nanofibers interweaving on bacterial surfaces, forming a tightly interwoven nanonet that effectively trapped and killed both E. coll and 5’. aureus. Tn contrast, asymmetric glutamic acid substitutions on the cationic residues of the E-E13ASYMpeptide redirected the nanofibers to self-interweave, forming extensive nanonets with minimal bacterial coverage and no antibacterial activity. Using these nanonets with distinct morphologies and function, it was demonstrated that the formation of tightly interwoven nanonets on bacterial surfaces significantly reduced the spread of motile E. coli and P. aeruginosa, outperforming both loosely trapping nanonets and conventional potent antibiotic. The present findings pave the way for the development of novel peptide-based nanonets, offering a promising strategy to target bacterial motility and prevent spreading of bacteria.

[0265] In the inventors' previous work, a series of [3-hairpin peptides with novel turn motifs that exhibited potent antibacterial activity was designed.[lfilIntroducing the novel turn motif (Leu-Thr-Ala) in the 15-mer BTT1-3A enabled it to self-assemble into nanonets specifically in the presence of bacteria, rather than forming non-specific peptide hydrogels.1171In this invention, to achieve better control of peptide nanofibers interweaving on bacterial surfaces to enhance trapping and limit bacteria spread, the inventors strategically substituted residues on the side strands with either aromatic or negatively charged residues, and created nanonets with distinct morphologies: either tightly interwoven nanonets on bacterial surfaces or extensive hydrogel-like nanonets with minimal bacterial coverage. These tightly interwoven nanonets were shown to effectively encage motile bacteria, significantly reducing their spread compared to loosely trapping nanonets. A library of BTT1-3A analogues were designed, which retained the turn motif (Leu-Thr-Ala) but were systematically modified at side strands via three major approaches to control bacteria nanonet morphology while preserving bacterial specificity: 1) shortening the peptides from both termini, 2) fine-tuning aromaticity or 3) reducing the overall positive charge (Figure IB).

[0266] Determine minimum side strand lengths for fibrillation

[0267] First, the peptides were shortened from both ends to identify the minimum side strand length required for fibrillation. Next, to create tightly interwoven nanonets on bacteria surfaces for trapping and killing them, hydrophobic residues at both cross endsof the P-hairpin peptides were symmetrically substituted with phenylalanine or tryptophan, known to enhance bacterial membrane interaction and bactericidal activity of typical AMPs.[19–22]Crucially, the overall hydrophobicity of the peptides was carefully modulated to stabilize the P-hairpin and promote nanofiber fibrillation through intramolecular and intermolecular π-π stacking, while preventing spontaneous selfassembly into peptide hydrogels. Seven peptides were designed with varying combinations of length and symmetrical cross-end aromatic pair motifs (F-F, diF-F or W-W) to determine the optimal configuration (Table 3A). Conversely, to demonstrate that peptide nanonets without antibacterial activity can trap bacteria but are less effective at reducing bacteria spread, nanofibers that self-interweave to form extensive nanonets rather than interweaving on the bacterial surface were designed. This was achieved by substituting lysine with glutamic acid at both ends to reduce the net positive charge. This modification was devised to stabilize the P-hairpin and enhance nanofiber fibrillation through electrostatic attractions. However, careful placement of glutamic acids may be important to avoid destabilizing the P-hairpin due to negative charge repulsion; therefore, two peptides with different glutamic acid positions were designed. Additional modifications included substituting leucine with valine to reduce hydrophobicity while retaining high P-sheet-forming propensity / 231Leucine with flexible side chain was substituted with rigid isoleucine to prevent a-helix formation and promote P-sheet formation / 241Some lysine residues were replaced with arginine to enhance antimicrobial activity.l251The LC-MS analytical characterization of the peptides listed in Table 3A are shown in Figure 13-28.

[0268] Ab initio secondary structure prediction using D-I-TASSER indicated that all peptides were likely to adopt extended conformations with a high propensity to fold into P-hairpins (Figure 5) / 261Circular dichroism (CD) spectroscopy was performed to examine the peptide secondary structures. All peptides remained random-coiled in phosphate-buffered saline (PBS) (Figure 2A) and folded into P-sheets upon binding to bacterial PAMPs such as LPS and LTA (Figure 2B, C). The only exception was di F-F 15, which formed P-sheets in PBS alone, possibly due to its extensive fibrillation and high overall hydrophobicity. Then the extent of amyloid fibrillation was assessed by measuring the fluorescence of amyloid-specific K114 dye in the presence of bacterial PAMPs BTT1-3A, F-F15, F-FB, diF-Fu, W-Wn and E-E13ASYMfibrillated rapidly within 30 minutes, showing significantly higher K114 signals compared to the non-fibrillatingBTT4 baseline control (Figure 2D, E). Removing two terminal leucine residues in BTT1-3 A significantly decreased fibrillation. Further shortening to 11-mer peptide abolished fibrillation, as indicated by the negligible fluorescence signals of Btt1-3A11. Since incorporating F-F or W-W motifs into the 11-mer peptides did not enhance fibrillation, the 13- and 15-mer peptides was selected to be focused on, to study the impact of different side strand modifications on controlling the peptide fibrillation.

[0269] Forming interweaving nanonet with high bacteria surface coverage

[0270] Next, it was shown that F-F13, diF-F13, and W-W13, with symmetrical substitutions of F-F, diF-F, and W-W motifs, respectively, enhanced the fibrillation of the 13-mer peptide significantly. Interestingly, the 15-mer peptide F-F15, with a F-F motif, showed lower fibrillation compared to BTT1-3A. However, the additional phenylalanine pairs in diF-F15significantly increased the K114 fluorescence signal, even in PBS alone (Figure 6A). Using confocal and scanning electron microscopy (SEM), the spontaneous and uncontrolled self-assembly of di F-F 15 into a peptide hydrogel was visualized (Figure 6B, C). Since diF-F13, which had the same sequence as diF-F15but lacked the terminal leucine, did not formed 0-sheet in PBS alone (Figure 2A), the present findings suggest that fine-tuning both the terminal end and aromaticity of the hydrophobic residue of the 13-mer |3-hairpin peptide was crucial to enhance nanofibers fibrillation without turning into hydrogels Removing the terminal leucines in diF-F15, results in diF-F13, which reduces its overall hydrophobicity, making it comparable to W-W13(Table 3A). This suggests that diF-F13and W-W13, with similar physicochemical properties (such as overall hydrophobicity and a four-backbone ring structure), are likely to fibrillate in a similar manner.

[0271] Moreover, it was that asymmetrical substitution of glutamic acids at both ends of the 13-mer E-E13ASYMimproved fibrillation, while E-E13with symmetrical placement did not. This observation supports the hypothesis that proper positioning of glutamic acids was crucial to promote fibrillation through electrostatic attraction while minimizing negative charge repulsion. Overall, CD spectra and K114 fibrillation assays suggested that the rational design of peptides capable of selectively undergoing conformational changes and fibrillation upon binding to bacterial PAMPs is crucial for developing bacteria-responsive nanonets, rather than non-specific peptide hydrogels.

[0272] SEM images revealed that the fibrillating peptides formed nanonets of distinct morphologies when incubated with the Gram-negative E. coli and the Gram-positive S.aureus (Figure 3 A). W-W13 self-assembled into thin nanofibers which densely interwove around individual bacteria, forming a tight nanonet with higher surface coverage on E. coli than S. aureus, possibly due to differences in bacterial membrane composition. In contrast, the nanofibers of E-E13ASYMinterwove tightly among themselves to form extensive nanonets in the spaces between the bacteria, with minimal membrane contact and low surface coverage to loosely trap both E. coli and S. aureus.

[0273] Others fibrillating peptides formed nanonets with varying degree of extensiveness and surface coverage against both bacteria strains (Figure 3B, 7, 8). Btt1-3A13formed less extensive nanonets with low surface coverage around E. coli or random aggregated structures around S. aureus, consistent with its low K114 signal (Figure 2D, E). It was observed that BTT1-3A and F-F15 nanofibers tended to aggregate and clump, creating extensive nanonets with moderate surface coverage that loosely trapped both E. coli and S. aureus. When the terminal leucine was removed, F-F13nanofibers showed less aggregation and interwove extensively to loosely trap E. coli with moderate surface coverage. Conversely, F-F13slightly increased surface coverage on S. aureus but with less extensive nanonets. The additional phenylalanine pairs in diF-F13promoted tighter interweaving of nanofibers on the surface of E. coli, similar to W-W13with high bacterial surface coverage. However, diF-F13formed nanonets similar to F-F15 and BTT1-3A against S. aureus but with less aggregated structures. Overall, these nanonets demonstrated the ability to agglutinate both E. coli and S. aureus with varying effectiveness (Figure 9, 10).

[0274] Taken together, the present findings suggest that to develop tightly interwoven nanonet on bacteria surfaces, removing terminal leucine may prevent undesirable nanofibers aggregation, potentially redirecting them to interweave on bacterial membrane. Moreover, incorporating W-W or diF-F motifs of similar overall hydrophobicity into the 13-mer peptide (Table 3 A) can increase nanofiber interweaving on E. coli membrane. However, such tight nanonets only formed on S. aureus with W-W13, likely due different structures of LTA and LPS.120,271Conversely, to create extensive hydrogel-like nanonet with minimal bacterial coverage, asymmetrical substitution of lysine with glutamic acids can reduce the net positive charge of the peptide, enhancing self-interweaving of nanofibers through electrostatic attraction while minimizing binding to the negatively charged LPS or LTA on bacterial membranes.

[0275] Antibacterial activity

[0276] Next, the antibacterial activity of the newly designed peptide nanonets was assessed (Table 4; Figure 11 A). These nanonets with different morphologies were able to trap-and-kill both E. coli and S. aureus with antibacterial activity comparable to the parent peptide BTT1-3A, except E-E13ASYM. Notably, W-W13and F-F15showed a two-fold improvement in bacterial killing Tn contrast, E-E13ASYMformed a trap-only nanonet without antibacterial activity, likely due to its reduced binding to bacterial PAMPs (Figure 11B, C). Additionally, the 13-mer peptides showed improved cytocompatibility and caused less membrane disruption in HaCaT keratinocytes compared to BTT1-3A (Figure 12, 13), highlighting the potential application of these novel nanonets to trap bacteria and reduce chronic skin infections and inflammation.

[0277] Table 4. MIC90 values of antibiotic controls against E. coli ATCC 25922 or S. aureus ATCC 29737 All experiments were repeated at least three times.Bacteria Antibiotic MIC90(μM)E.coli ATCC 25 922 Polymyxin B 2.0S. aureus ATCC 29 737 Vancomycin 1.0

[0278] Trapping and preventing bacterial spread

[0279] Finally, the effectiveness of nanonet with different morphologies in trapping and preventing bacterial spread was evaluated. Since S. aureus is non-motile, it was speculated that the present nanonets with improved antibacterial activity, such as W-W13 and F-F15, would effectively trap-and-kill the bacteria within the net, preventing their spread compared to existing nanofibers that lack bactericidal activity.[7,8]Next, using soft-agar motility assay that better represents the body environment, where motile Gramnegative bacteria like E. coli and P. aeruginosa move freely and proliferate to cause infection, the trapping efficiency of different nanonets in preventing motile bacteria to escape from entrapment and spread was examined. For this study, W-W13 and diF-F13were selected, which formed tight nanonets that encage and kill E. coli, BTT1-3A, which formed loose nanonet and kill E. coli, and E-E13ASYM, which formed loose nanonets without antibacterial activity. It was first confirmed that these peptides formed nanonets with similar morphology and killing efficiency against P. aeruginosa as observed with E. coli (Figure 14; Table 5).

[0280] Table 5. MIC90 of peptides against P. aeruginosa PAO 1. All experiments were repeated at least three times.Peptides MIC90(μM)BTT1-3A 32.0diF-F1332.0W-W1316.0E-E13ASYM>128.0Polymyxin B 2.0

[0281] In this assay, bacteria were first treated with peptides for only 30 minutes to allow nanonet formation. The nanonet-trapped bacteria were then inoculated into soft-agar plates to evaluate their ability to prevent bacterial escape and spreading after 14 hours. Figure 4A and 4B showed that both E. coli and P. aeruginosa actively spread, forming clear motility halos. Treatment with potent antibiotic polymyxin B did not reduce the diameter of motility halos (Figure 15), indicating that motile bacteria can escape from areas with high local drug concentrations, thereby avoiding death. When both bacteria were treated with equal concentrations of BTT1-3A and diF-F13, which have similar antibacterial efficiency (Figure 11 A; Table 3 A), only the tight diF-F13nanonet significantly inhibited bacterial spreading, whereas the loose BTT1-3A nanonet did not (Figure 4C, D). The tight W-W 13 nanonet with improved antibacterial activity also effectively inhibited bacterial spread at lower concentrations. The present results clearly demonstrated that tightly interwoven nanonets on bacterial membrane was crucial for encaging and killing bacteria, preventing their escape and spread. Moreover, E-E13ASYMpartially prevented bacterial spread by forming extensive nanonets at high concentration, but without killing the bacteria, they eventually outgrew and escaped from entrapment.

[0282] Using an E. coli AflhD mutant with impaired motility, it was demonstrated that without bacteria movement to escape from areas with high local drug concentrations or from loosely trapped nanonets, non-motile bacteria were more susceptible to killing by polymyxin B or the loose BTT1-3A nanonets (Figure 16). W-W13and diF-F13effectively prevented the growth of non-motile bacteria. Lastly, it was confirmed that E-E13ASYMnanonets without antibacterial activity could not prevent the bacterial outgrowth within the nanonets, posing a risk of potential spread. Overall, the present findings indicated that bacterial motility can significantly reduce the effectiveness of potent antibiotics.13,41This implies that focusing solely on developing more potent antibiotics may not be the most effective approach. Instead, creating nanonets that tightly encage motile bacteria andrestrict their movement could offer a promising strategy to prevent bacterial spread and infection.

[0283] In summary, the design principles of peptide nanonets were explored, which enabled to control the peptide nanofibers interweaving on bacterial membranes to form nanonets of different morphologies This was achieved through rational modification of the P-hairpin side strands by leveraging the unique chemical properties of amino acid side chain. By carefully controlling both the terminal ends and the aromaticity of the hydrophobic residue in -hairpin peptide, W-W13formed densely interwoven nanofibers on bacteria surfaces, forming tight nanonets that effectively reduced bacterial spread. In contrast, asymmetrical substitution with glutamic acids on both ends resulted in E-E13ASYMthat formed extensive self-interwoven nanonet with minimal bacterial coverage and no antibacterial activity, which was insufficient to inhibit bacterial spread. Thus, having the ability to control the nanonet morphology is crucial for developing novel nanonets that can tightly encage bacteria, preventing their spread and infection. Although only canonical amino acids were utilized in these designs, this strategy can be extended to the wide array of non-canonical amino acid libraries. This could further enhance the potency, proteolytic stability, and cytocompatibility of the nanonets, potentially addressing specific clinical requirements.

[0284] EXAMPLE 3- Peptide Nanonets for Sequestration of Pro-inflammatory LPS

[0285] Gram-negative bacterial infections, if not treated promptly, can lead to bacteremia, during which LPS from the bacterial surface or released as free LPS can trigger a strong inflammatory response.

[0033] This can escalate into sepsis, potentially resulting in systemic shock, organ failure, and death. Currently, there is no effective therapy specifically designed to remove LPS and reduce the risk of sepsis. Although many AMPs can bind and neutralize LPS,[341their stoichiometric binding might limit their efficiency. Previously, the inventors demonstrated that our peptide nanonets can effectively trap free LPS through electrostatic interactions, thereby preventing the activation and secretion of pro-inflammatory cytokines, as shown using a FITC-LPS trapping assay — highlighting their anti-inflammatory potential.[181Here, the inventors designed new peptide nanonets with varying morphologies and evaluated their ability to trap free LPS (Figure 30A). The results showed that peptides with an overall +7 charge (BTT1-3A, W-W13, diF-Fn, F-F13, and F-F15) effectively sequestered LPS, with trappingefficiencies ranging from -42% to 55% (Figure 30B, C). Among them, the parent BTT1-3A that formed extensive nanonets achieved the highest Emax, up to 55%. Notably, the lead tight nanonet-forming peptide, W-W13, exhibited the lowest EC50, indicating it can trap more LPS at lower concentrations than the others. Additionally, although E-E13ASYM, a non-bactericidal peptide with a lower charge (+3), could still trap LPS, its efficiency was reduced compared to the more positively charged peptides. Overall, the results demonstrate that peptide nanonets of diverse morphologies and activities can effectively sequester free LPS, highlighting their therapeutic potential in preventing systemic inflammation and sepsis.

[0286] EXAMPLE 4- Peptide Nanonets Retain Potency Against Antibiotic Resistant Clinical Isolates

[0287] To further assess the peptide nanonets’ clinical relevance, the inventors expanded the MIC90 screening to include various antibiotic-resistant clinical isolates. The results showed that the trap-and-kill peptide nanonets retained antibacterial activity against E. coll strains carrying the MCR-1 mutation, as well as various Gram-positive S. aureus USA300 clinical isolates, including those resistant to conventional antibiotics. Several nanonets even exhibited enhanced killing potency compared to the parent peptide BTT1-3A (Figure 31A). Notably, the lead tight peptide nanonet W-W13 consistently demonstrated superior antibacterial activity against all tested clinical strains, highlighting its strong therapeutic potential. In contrast, the trap-only peptide E-E13ASYMremained non-bactericidal across all strains, underscoring the importance of the killing function in therapeutic applications.

[0288] Interestingly, E. coli M2, a strain harboring the MCR-1 mutation that reduces the negative charge of LPS — expected to decrease peptide binding on bacterial outer membrane — was paradoxically more sensitive to killing by the peptides. To investigate this unexpected result, the inventors examined the mechanism of action using membrane damage assays. First, outer membrane integrity was assessed using NPN uptake. W-W13, F-F 13, F-F15, and diF-F13induced outer membrane damage comparable to the antibiotic polymyxin B (Figure 3 IB). The parent peptide BTT1-3A also exhibited the strongest outer membrane-disrupting activity. Inner membrane damage, assessed using propidium iodide uptake, revealed that all trap-and-kill peptide nanonets were capable of disrupting the inner membrane, again comparable to polymyxin B (Figure 31C). Although W-W13, F-F13, F-F15, and diF-F13caused slightly weaker outer membrane damage than BTT1-3A,they achieved efficient inner membrane disruption at lower concentrations (8 pM), demonstrating potent bactericidal activity. Interestingly, the trap-only E-E13ASYMcaused significant outer membrane damage but failed to disrupt the inner membrane, further confirming that inner membrane disruption is essential for bacterial killing. Together, these findings demonstrate that the newly designed trap-and-kill peptide nanonets can effectively kill antibiotic-resistant clinical isolates by compromising both outer and inner bacterial membranes. Their ability to match the membrane-disrupting potency of polymyxin B, a last-resort antibiotic, underscores their strong potential as alternative therapeutics for combating multidrug-resistant infections in clinical settings.

[0289] EXAMPLE 5- Peptide nanonets enhance antibiotic potency and prevent resistance development

[0290] Current antibiotics are highly potent in killing bacteria, but their widespread misuse has led to the emergence of antibiotic-resistant strains. Therefore, innovative strategies must not only enhance antibiotic efficacy but also suppress the development of resistance during prolonged use. Given that the peptide nanonets both trap bacteria and disrupt their membranes, the inventors next evaluated their synergistic effect with rifampicin — an intracellular-targeting antibiotic with poor outer membrane penetration in Gram-negative bacteria — against the E. coll M2 strain. The inventors explored how nanonets with differing morphologies and antibacterial potencies affect antibiotic synergy and resistance suppression. Using a chequerboard assay (Figure 32A, B), it was found that tight nanonets such as W-W13and diF-F13synergized with rifampicin, yielding FICI values of 0.5. W-W13 reduced the required rifampicin concentration 4-fold to 8 pM with just 2 pM peptide, while diF-F13achieved the same with 4 μM peptide. Loose trap-and-kill nanonets (F-F13 and F-F15) showed additive effects Specifically, 4 pM F-F13 reduced rifampicin MIC by 32-fold to 1 pM, and 4 pM F-F15 achieved a 4-fold reduction to 8 pM. Even the trap-only peptide E-E13ASYMreduced rifampicin MIC by 2-fold to 16 pM, though at a much higher peptide concentration (64 pM). These results highlight the critical role of inner membrane disruption in enhancing antibiotic uptake and potency.

[0291] Next, the inventors conducted a serial passage experiment over 18 days using the peptide and rifampicin concentrations determined from the chequerboard assay to assess resistance development in E. coll M2. When treated with rifampicin alone, E. coll M2 rapidly developed resistance, with a 16-fold increase in MIC observed by day 4 (Figure 32C). Similarly, the trap-only peptide E-E13ASYMfailed to prevent resistance,showing the same trend as rifampicin alone. All trap-and-kill nanonets — regardless of their morphology — effectively suppressed resistance development, with MIC values fluctuating by less than 2-fold over the entire 18-day period. These findings indicate that the ability of the nanonets to damage bacterial membranes and trap bacteria is crucial for preventing the emergence of resistance. In conclusion, peptide nanonets not only enhance the efficacy of intracellular-targeting antibiotics through membrane disruption and bacterial entrapment but also prevent the rapid development of resistance. This highlights their therapeutic potential as a next-generation strategy to both potentiate antibiotic activity and mitigate the rise of antibiotic-resistant infections.

[0292] EXAMPLE 6- Structure-Activity Study of Aromatic P-Hairpin Peptides Reveals Tunable Window for Antibacterial Nanonet Activity

[0293] Background

[0294] The ability to control peptide nanofiber fibrillation and interweaving on bacterial surface membranes offers a novel strategy to regulate bacterial motility and infection. W-Wis and diF-Fn peptides exploit the aromatic properties of tryptophan and phenylalanine residues, whose indole and phenyl rings, respectively, play key roles in promoting peptide self-assembly and membrane binding.1201To further fine-tune the activity of peptide nanonets, non-canonical amino acids such as unnatural amino acids (UAAs), offer a powerful means of modifying the physicochemical properties of these aromatic scaffolds. For example, fluorination can enhance hydrophobicity, methylation can increase both hydrophobicity and peptide stability; while hydroxylation or the incorporation of polar aromatic groups such as -pyridylalanine can improve solubility and biocompatibility.[35–38]UAAs are increasingly employed to enhance the therapeutic performance of peptides by improving their stability, activity, or specificity. In the context of self-assembling AMPs, positive charge and high hydrophobicity is typically required for aggregation and membrane binding. However, exceeding a certain hydrophobicity threshold can lead to non-specific hydrogel formation, rather than bacteria-responsive nanonets. Despite the widespread use of UAAs, how subtle modifications to aromatic rings — affecting both physicochemical properties and electron density — influence supramolecular self-assembly, antibacterial activity, and cytocompatibility remains poorly understood, particularly in nanonet-forming peptides. While most UAA studies focus on improving peptide stability or receptor binding, few have systematicallyexplored how specific UAA side chain chemistries influence nanoscale morphology and biological function. By linking the chemical structure of aromatic UAAs to nanonet architecture and biofunction, this work fills a key knowledge and design gap in the development of self-assembling antimicrobial nanomaterials.

[0295] Results

[0296] Peptide Design Rationale for Modulating Nanonet Morphology’ and Activity with UAAs

[0297] To investigate how aromatic ring chemistry affects nanonet formation and function, the inventors designed W-W13and diF-F13analogs by substituting their Trp and Phe residues with UAAs to fine-tune hydrophobicity, as estimated by calculated logP values, while keeping sequence length and net positive charge (Figure 33 and Table 3A). In W-W13analogs, Trp was replaced with Trp(l-Me), Trp(7-F), 1-Nal, and Bal to increase hydrophobicity. Trp(l-Me) adds N1 methylation, enhancing steric bulk and hydrophobicity; Trp(7-F) introduces fluorination, affecting both hydrophobicity and ring electronics; 1-Nal increases aromatic surface area via a naphthyl ring; Bal replaces the indole NH with sulfur, altering hydrogen bonding and electron distribution. These hydrophobic substitutions are intended to maintain the formation of tight nanonets while improving bacterial selectivity. In contrast, hydrophilic variants include Trp(5-OH), which adds polarity via hydroxylation; Trp(7-Aza), which introduces a ring nitrogen and alters electronic distribution; and Oia, which inserts a carbonyl group, disrupting planarity and hydrogen bonding. While hydrophilic modifications are known to improve cytocompatibility, whether their additional hydrogen bonding capacity can promote peptide self-assembly remains to be determined.

[0298] For diF-F13analogs, all four Phe residues were substituted with hydrophobic UAAs such as Phe(4-F) and Phe(3,4,5-F), which introduce progressive fluorination to increase hydrophobicity; and 2ThiAla, which adds a thiophene ring that retains aromaticity but modifies polarity and electron distribution. The inventors also included Cha (cyclohexylalanine), a non-aromatic bulky hydrophobic residue, enabling the inventors to examine the role of ring planarity and K-stacking interactions in nanonet formation. Hydrophilic substitutions include Tyr (phenol), FurAla (furan ring), 5-Thiazo (thiazole), and 3-Pal (pyridine), each introducing varying degrees of polarity, electrondensity, or hydrogen-bonding capacity. Similar to W-W13 analogs, these analogs help assess whether hydrophilic modifications can improve biocompatibility without compromising nanonet function.

[0299] Overall, these peptide modifications were intended to minimally disrupt the existing antibacterial and self-assembling activity. The inventors hypothesized that moderate increases in hydrophobicity may enhance membrane binding and antibacterial activity, while excessive hydrophobicity could compromise bacterial selectivity and promote undesired spontaneous self-assembly in buffer. In contrast, more hydrophilic substitutions may improve biocompatibility while retaining antibacterial function and self-assembly via additional hydrogen bonding formation, provided that charge and folding are maintained.

[0300] Bacteria-Responsive Self-Assembly of UAA-Containing Peptide Analogs

[0301] The newly designed peptides were expected to maintain bacteria-responsiveness by remaining in a random coil conformation in buffer and folding into β-sheet structures only upon binding to bacterial PAMPs such as LPS or LTA. To test this, circular dichroism (CD) spectroscopy was used to assess the secondary structure of each peptide in the presence or absence of bacterial PAMPs. All W-W13 analogs exhibited a single negative peak around -200 nm in PBS, characteristic of a random coil structure (Figure 34A). Upon addition of LPS or LTA, these peptides adopted a β-sheet conformation, displaying a positive peak at -195 nm and a negative peak at 215-220 nm. A similar bacteria-induced P-sheet transition was observed for most diF-F 13 analogs, with the exception of Fl and F2, which displayed β-sheet features in PBS alone (Figure 34B), indicating spontaneous self-assembly likely due to excessive hydrophobicity. Interestingly, Fl exhibited an increase in β-sheet intensity upon PAMPs binding, suggesting partial retention of bacteria-responsiveness, whereas F2 showed no further structural change, implying a complete loss ofPAMP specificity. Overall, W-W13 analogs showed more consistent CD profiles compared to diF-F13 analogs, suggesting that modifications to the two indole rings in W-W13 exerted more subtle effects on peptide folding than substitutions on the four phenyl rings in diF-F13. These differences may also stem from the intrinsic structural properties of indole versus phenyl rings. Nevertheless, the CD data indicated that both hydrophilic and hydrophobic analog substitutions largely preserved the peptide’s ability to fold into β-sheet structures upon bacterial PAMPsrecognition, except when overall hydrophobicity exceeds a threshold that may promote spontaneous self-assembly.

[0302] To further investigate whether β-sheet folding promotes self-assembly, K114 dye assays was performed, which detect cross-β amyloid structures based on fluorescence emission upon binding. BTT1-3A served as a positive control for peptide self-assembly triggered by bacterial PAMPs, while BTT4, a non-fibrillating analog, served as a negative control. Peptides that exhibited a higher increase in KI 14 fluorescence than BTT4 were considered candidates for PAMP -responsive self-assembly. Among W-W13 analogs, W-W13, W3, and W4 exhibited low fibrillation (-20%), while W1 and W2 showed stronger self-assembly in response to both LPS and LTA (Figure 34C, D). In contrast, hydrophilic analogs W5, W6, and W7 failed to self-assemble despite forming 0-sheets, suggesting that hydrophilic modifications to the indole ring may disrupt key interactions required for nanonet formation. For the diF-F13 analogs, F3 and F4 exhibited clear self-assembly in response to both LPS and LTA (Figure 34E, F). Fl and F2 exhibited high K114 signals even in PBS alone, indicating spontaneous self-assembly (Figure 35A). SEM analysis further confirmed the formation of dense, hydrogel-like nanofiber networks for these peptides (Figure 35B, C). Notably, Fl still showed increased KI 14 fluorescence upon PAMP exposure, whereas F2 did not, aligning with CD data that suggest F 1 retains partial PAMP-responsiveness Similarly, the addition of a hydroxyl group to the phenyl ring in F5 abolished its ability to self-assemble. However, unlike the W-W13 analogs, several hydrophilic diF-F13 analogs (F6, F7, and F8) may still self-assemble upon binding to bacterial PAMPs. This may be because their heteroaromatic rings retain enough aromaticity and planarity to facilitate π-π stacking and self-assembly, exhibiting different properties compared to indole ring analogs. Together, the CD and KI 14 data indicate that fine-tuning the hydrophobicity of aromatic residues can modulate bacteria-responsive self-assembly, as excessive hydrophobicity promotes non-specific spontaneous aggregation while overly hydrophilic modifications may hinder self-assembly.

[0303] Effect of Aromatic UAA Substitutions on Nanonet Assembly and Morphology

[0304] To evaluate how UAA substitutions affect nanonet formation and morphology, SEM imaging was performed on the peptides against E. coll. Hydrophobic modifications to the indole rings largely preserved the ability of peptides to form nanofibers that wrap around bacterial membranes, although differences in morphology were observed (Figure36). W3 and W4 formed tight nanonets similar to the parent W-W13, where peptide nanofibers densely wrapped individual bacteria with minimal self-entangled fibres. In contrast, Wl and W2 formed semi -tight nanonets, showing reduced nanonet coverage on bacterial surfaces and increased self-entangled nanofibers in the surrounding space. This difference may be due to their higher fibrillation propensity with LPS observed in K114 assays (Figure 34C), suggesting a preference for peptide-peptide interactions over peptide-bacteria binding. Notably, replacing the indole NH with sulfur (Wl) or introducing N1 -methylation (W4) did not disrupt nanonet formation, suggesting thatNH-mediated hydrogen bonding is not essential for wrapping. For the diF-F13 analogs, F1, which retained partial PAMP -responsiveness, formed semi-tight nanonets that densely wrapped individual E. coli, along with extensive aggregate-like nanofibers between bacteria that closely resembled the spontaneous assemblies observed in buffer alone (Figure 35B). F3 and F4 displayed a loose-entangled morphology, with nanofibers favoring self-entanglement and leaving more bacterial surfaces exposed. Based on these observations, the inventors propose that W-W13, W3, and W4 that form nanonets tightly wrapping individual bacteria may be suitable for controlling infections by motile bacteria in blood circulation without risking vascular blockage. In contrast, peptides that form extensive nanonets (Wl, W2, Fl, F3, and F4) may be more appropriate for localized treatments, such as chronic infected wound management

[0305] Conversely, hydrophilic modifications such as hydroxylation of the indole (W5) or phenyl (F5) rings completely abolished nanonet formation even at higher peptide concentrations, consistent with their lack of K114 signal (Figure 34C-F). Similarly, F7 and F8, which were substituted with nitrogen-containing heteroaromatic rings, failed to form nanonets even at 100 pM. Although these peptides exhibited some fibrillation in solution with free bacterial PAMPs (Figure 34E, F), their altered π-π stacking likely led to unstable nanofibers that were disrupted during harsh SEM sample preparation. Regardless, these results suggest that the nanofibers formed by such analogs are weak and unsuitable for bacterial trapping.

[0306] Overall, these results reveal that aromatic side-chain hydrophobicity is a key determinant of nanonet formation and morphology. Subtle polar modifications that alter electron distribution within the aromatic ring (e g, Trp to Trp(5-OH); Phe to 3-Pal; 2ThiAla to 5Thiazo) can abolish nanonet formation despite maintaining peptide’s net positive charge and sequence motif.

[0307] Narrow Hydrophobicity Range Modulates Peptide Nanonet Activity and Biocompatibility

[0308]

[0309] Next, the MICs of each peptide were determined against a panel of pathogenic microorganisms, including antibiotic-resistant clinical isolates such as E. coli M2 and S. aureus BAA1556, as well as the fungal strain C. albicans. (Figure 37A). In general, analogs W1–W4 retained the broad-spectrum potency of the parent peptide W-W13, with W3 and W4 showing enhanced activity by lowering the MIC against E. coli to 8 pM. A subtle modification in W1 led to a further 2-fold increase in antibacterial activity against both Gram-negative and Gram-positive bacteria, which may be attributed to the sulfur substitution on the indole ring that potentially enhanced peptide-bacteria interactions. In contrast, hydrophilic substitutions in W5-W7 generally impaired antibacterial activity. An exception was W6, which may be Gram-negative selective, as it retained activity against E. coli but lost effectiveness against S. aureus. Most importantly, the findings demonstrate that W-W13 and its analogs (W1–W4) exhibit broad-spectrum activity against ESKAPE pathogen, including effectiveness against difficult-to-treat pathogens such as P. aeruginosa and K. pneumoniae, which possess thick polysaccharide capsules that contribute to resistance. The diF-F13 series displayed a similar trend. F3 and F4 analogs maintained antibacterial activity comparable to the parent diF-F13, but when all four phenyl rings were modified with polar functional groups, the peptides lost both nanonet formation and antibacterial activity (Figure 34E, F; Figure 36). Although Fl and F2 are highly hydrophobic, they showed reduced antibacterial potency. This may be due to their spontaneous self-assembly in buffer, which decreases the amount of free peptide available to interact with bacterial membranes, consistent with our previous finding that there is a trade-off between extensive peptide fibrillation and antibacterial killing.

[0017] When evaluating the antifungal properties of the peptides against C. albicans, BTT1-3A without an aromatic group showed poor antifungal activity, whereas aromatic-substituted peptides exhibited improved activity. Notably, hydrophobic aromatic UAA modifications were still required for effective fungal killing (Figure 37A). The W–W13 and W1–W4 analogs displayed comparable antimicrobial activity against both bacteria and fungi. Interestingly, diF–F13 and its analogs demonstrated strong antifungal activity, particularlyFl despite exhibiting weak antibacterial effects. These findings highlight the potential of modulating the chemical space of aromatic UAAs to fine-tune antimicrobial selectivity and potency.

[0310] Next, the cytotoxicity of each peptide analog was evaluated against HaCaT keratinocytes using the MTS assay (Figure 37B). W-W13, W3, and W4 with comparable hydrophobicity exhibited similar levels of cytotoxicity. W1 and W2 which have increased hydrophobicity, showed reduced CC50 values, indicating higher cytotoxicity. Conversely, W5-W7, which contain hydrophilic modifications, exhibited significantly improved biocompatibility, highlighting the trade-off between hydrophobicity and safety. For diF-F13 analogs, F3 and F4 showed improved CC50values while retaining antibacterial activity compared to the parent peptide, suggesting that specific structural modifications can enhance safety without compromising function. Fl also displayed improved CC50 despite its high hydrophobicity, but this may be attributed to its spontaneous selfassembly, which likely reduces the concentration of free peptide available to interact with and disrupt HaCaT cell membranes. Finally, F7 and F8 exhibited the highest biocompatibility, consistent with their high overall hydrophilicity.

[0311] Combining the MIC and MTS assay results, a general trend emerged: increasing peptide hydrophobicity tend to enhance antimicrobial activity but compromised biocompatibility, and vice versa. This observation aligns with existing literature, which reports an inverse relationship between the hydrophobicity of potent AMPs and their safety profiles. However, many studies have shown that incorporating UAAs can fine-tune the physicochemical properties of AMPs, leading to improved bacterial selectivity and reduced cytotoxicity.

[0039] Notably, in this study, W4 — featuring N1 -methylation on the indole ring — showed slight improvements in both MIC and CC50 while maintaining its ability to form tight nanonets (Figure 36), highlighting a promising design strategy for balancing activity and safety. Finally, plotting hydrophobicity against MIC and CC50 in three-dimensional space (Figure 37C, D) revealed a narrow physicochemical window in which peptides can be modified to form bacteria-responsive nanonets, kill bacteria, and remain biocompatible. Outside this window, excessive hydrophobicity triggers spontaneous self-assembly, whereas excessive hydrophilicity significantly improved biocompatibility but abolishes nanonet function and antibacterial activity. A list of Trp and Phe analogs was compiled with side chain modifications thatpotentially preserve nanonet formation and antibacterial activity if the overall peptide physiochemical properties are optimized within this range (Figure 39 and 40).

[0312] Conclusion

[0313] In this study, the incorporation of UAAs was explored to fine-tune the physicochemical properties of novel P-hairpin peptide motifs, aiming to understand the design principles that govern the interplay between antibacterial activity, bacteria-responsive self-assembly, and biocompatibility. The results show that minimal modifications to aromatic residues significantly influence peptide behavior, with increased hydrophobicity promoting nanonet formation and bacterial killing at the expense of biocompatibility, while enhanced hydrophilicity improves safety significantly but abolishes both nanonet assembly and antimicrobial function. Importantly, the inventors revealed a narrow physicochemical window in which peptides can exhibit bacteria-responsive self-assembly, strong antibacterial activity, and moderate cytotoxicity. Notably, the W4 analog, featuring N1 -methylation of the indole ring, enhanced both antibacterial activity and safety while maintaining tight nanonet formation. These findings advance the understanding of the delicate balance between peptide hydrophobicity, self-assembly, and function, providing a valuable framework for rational UAA substitution.

[0314] Example 7: Minimal Impact of Non-Key Residue Substitution on Peptide Nanonet Activity

[0315] [3-Hairpin peptides rely on an alternating pattern of hydrophobic and charged residues to create an amphiphilic surface that drives self-assembly and antibacterial activity.

[0014] the above studies showed that introducing key side-strand residues can modulate nanonet morphology and function. Here, the inventors asked whether the nonkey hydrophobic or charged residues mainly serve a structural role to maintain the p-hairpin fold without decisively controlling self-assembly or antimicrobial activity. If so, they should be replaceable with residues of similar physicochemical character without compromising nanonet performance. To test this, E-E13ASYMwas selected, which contains two asymmetrically placed Glu residues critical for its structure. This peptide forms loose-dispersed nanonets characterized by extensive nanofibers between bacterial cells but low coverage on bacterial surfaces, resulting in minimal antibacterial activity. Leaving the turn motif and the two key Glu residues intact, the remaining charged and non-aromatichydrophobic positions were exchanged for alternative amino acids of comparable polarity or hydrophobicity, generating E-E13ASYM(ch) (Figure 38 A). This analog retained the same net charge and molecular weight but presented subtly different side-chain geometries.

[0316] First, it was confirmed that the secondary structure of E-E13ASYM(ch) was not affected by the residue substitutions CD spectroscopy showed that the analog retained bacteria-responsive folding, remaining in a random coil conformation in PBS and transitioning to a β-sheet structure upon exposure to LPS or LTA (Figure 38B, C). Next, KI 14 fluorescence assays indicated that E-E13ASYM(ch) remained capable of PAMP-triggered self-assembly, although its fibrillation intensity was lower than that of the parent peptide upon LPS binding (Figure 38D, E). Despite this reduction, SEM imaging confirmed that both peptides formed comparable nanonet morphologies around E. coli (Figure 38F, G). Finally, functional assays demonstrated that E-E13ASYM(ch) retained antibacterial activity similar to the parent peptide while exhibiting improved biocompatibility toward HaCaT keratinocytes, as measured by MIC and MTS assays, respectively. Collectively, these data demonstrate that substituting non-essential residues with natural or UAAs of similar physicochemical properties has minimal impact on the peptide’s secondary structure, bacteria-responsive self-assembly, and nanonet function. Importantly, this strategy provides a valuable design principle for enabling the rational incorporation of UAAs to fine-tune side-strand stability and improve cytocompatibility, without compromising the core functional properties of the nanonets. The inventors compiled a list of positively charged (c) or non-aromatic hydrophobic (h) analogs with modified side chains that may be substituted without disrupting nanonet formation. (Figure 29B, C).

[0317] Example 8: Engineering a versatile antimicrobial peptide nanonet coating and develop the prototype

[0318] In this work, the inventors hypothesize that incorporating unique, bacteria-responsive nanonet-forming AMPs onto urinary catheters provides multi-functionality in terms of trapping and killing pathogens, thereby preventing catheter-associated UTIs. The key specific aims are 1) Devise a strategy for incorporating a nanonet-forming AMP onto the surface of a urinary catheter: applying AMPs as coatings on urinary catheter surfaces can gradually release the peptide and inhibit pathogen growth, improving catheter longevity and patient outcomes by reducing the risk of infection. An effective coatinghelps extend the period of catheter usage without compromising patient safety; 2) evaluate the in-vitro anti-infective efficacy of the nanonet-forming AMPs against uropathogenic ATCC strains and biofilms: CAUTIs are frequently caused by uropathogenic bacteria that form resilient biofilms. These biofilms create a protective barrier that renders infections difficult to treat with conventional antibiotics, increasing the likelihood of chronic and recurrent infections. Biofilm-associated infections are notably resistant to both immune responses and antimicrobial treatments, often leading to prolonged hospital stays, increased healthcare costs, and elevated morbidity. Evaluating the efficacy of peptides against uropathogenic strains and biofilms is essential to understanding their capacity to prevent and treat CAUTI. This assessment is critical for developing effective anti-infective treatments that can address the limitations of current antibiotics and reduce the clinical burden of CAUTI Furthermore, the AMP coating inhibits bacterial adhesion and biofilm formation, a key advantage given the high resistance of biofilms to antibiotics.

[0319] For grafting peptides onto polymer surfaces, polyurethane film was used as the model polymer since most urinary catheters are made of PU and their derivatives. The process involved conjugating peptides onto a PU surface using glutaraldehyde, a widely used method for immobilizing bioactive molecules. The multi-step process began with oxygen plasma irradiation of the polymer to introduce oxygen functional groups, such as hydroxyls, which enhance surface reactivity. Following the plasma treatment, the surface was silanized using APTES, forming covalent siloxane bonds with the activated surface to create a stable functional interface39. Glutaraldehyde, a bifunctional cross-linker, was then applied to the silanized surface. Its aldehyde groups reacted with the amino groups on the peptides, facilitating their covalent attachment (as shown in Figure 50).

[0320] This approach successfully conjugated peptides onto the polymer surface, enhancing the material's bioactivity for potential biomedical applications. The conjugation was characterized using ATR spectroscopy (Figure 50C). The presence of the PU surface correlates with the secondary amide N-H stretching observed at 1528 cm-1. The -NH- stretch at 3332 cm-1is characteristic of a primary amine functional group. The secondary amide bonds within the peptide molecule are indicated by the C=O stretch at 1625 cm-1, while the characteristic C-0 ether stretch corresponding to PU, present at 1058 cm-1, can be seen in both PU and peptide-conjugated PU. Analysis of the spectra for both PU and peptides reveals key characteristic peaks from both components in thePeptide-Conjugated PU spectra, suggesting the successful attachment of peptides to the PU surface.

[0321] Example 9: Peptide-coated PU Tubes can inhibit biofilm formation

[0322] To facilitate clinical translation, the peptides were conjugated onto curved PU surfaces using the same method as applied for PU Films, and the biofilm inhibition analyses were repeated. The results demonstrated that the peptide coatings maintained their efficacy on curved surfaces, effectively inhibiting biofilm formation. These findings suggest that the peptide coatings are adaptable to the geometry of medical devices such as catheters, further supporting their potential as an effective antimicrobial coating for preventing biofilm-related infections in clinical applications.

[0323] Bacterial biofilm formation was evaluated using the Crystal Violet assay, with biofilm quantified by measuring absorbance at 595 nm, comparing samples incubated with Uropathogenic E. coli (UPEC) to a control sample of PU incubated in lx PBS solution. Both qualitative and quantitative data indicated that the untreated PU substrate incubated in PBS for seven days showed the lowest absorbance levels, whereas the untreated PU substrate incubated with UPEC exhibited the highest biofilm formation (Figure 51) Importantly, biofilm formation on the PC-PU substrate was significantly reduced compared to the untreated PU substrate.

[0324] Additionally, time-kill curves were employed to evaluate bactericidal activity by comparing the PC-PU surface to Colistin. Both the PC-PU surface and Colistin exhibited bactericidal effects, achieving a 3 -log reduction in CFU / rnl of UPEC after 4 hours of treatment (Figure 5 ID). These results indicate that the PC-PU surface not only inhibits biofilm formation but also retains potent antimicrobial properties, comparable to those of standard antibiotics like Colistin. This dual functionality of bacterial trapping and killing establishes PC-PU as a promising material for mitigating bacterial colonization and biofilm-related infections, particularly in medical devices such as catheters.

[0325] To assess the effect of peptides conjugated to the PU surface on the membrane integrity of Uropathogenic E. coli (UPEC) cells, confocal microscopy was used to visualize the bacterial cells co-stained with Syto™ 9 and Propidium Iodide (PI). Bacteria incubated with the uncoated PU substrate exhibited uniform green fluorescence,indicating intact membranes (Figure 52A). In contrast, cells incubated with the PC-PU substrate showed significant red fluorescence (Figure 52C), indicative of membrane damage. This differential staining is explained by the permeability properties of the dyes: Syto™ 9 stains all bacterial membranes green, while PI only penetrates damaged membranes, staining the DNA red. These observations suggest that incubation with the PC-PU substrate compromises bacterial membrane integrity, leading to cell damage.

[0326] A mix of live (green) and dead (red) cells was observed within these clusters, suggesting a correlation between bacterial aggregation and membrane damage upon exposure to the PC-PU substrate.

[0327] The Soft Agar Motility assay was utilized to assess the ability of the peptides to trap bacteria by measuring colony diameters. In this assay, motile bacteria spread through the media, and greater motility is associated with larger colony diameters. Bacteria exposed to the PC-PU substrate showed an average colony diameter of 10.12 mm (Fig 1C), significantly smaller than the 47.04 mm observed for bacteria exposed to untreated PU substrates, indicating a strong bacterial trapping effect. Thus, the PC-PU substrate restricts bacterial motility and induces significant membrane damage in UPEC cells.

[0328] Scanning electron microscopy (SEM) analysis of Uropathogenic E. coli cells revealed significant morphological changes upon incubation with the PC-PU substrate. UPEC cells exposed to the PC-PU surface exhibited pronounced membrane blebbing and the formation of multiple nanofibrils that envelope the bacteria from the PU Surface (Figure 53C). In contrast, UPEC cells incubated with untreated PU displayed smooth, continuous surfaces (Figure 53B). These findings indicate that the conjugated peptides can trap and kill the bacteria, with the peptides capable of capturing and immobilizing bacteria while adhering to the PU surface. This highlights the potential of the PC-PU coating in disrupting bacterial membranes and forming nanonet structures, which play a key role in reducing bacterial colonization and biofilm formation.

[0329] In summary, the inventors have demonstrated that nanonet-forming peptides can be effectively conjugated onto PU surfaces through a straightforward chemical conjugation process. The results from this study are consistent with preliminaryIllexperiments where peptides were conjugated onto flat PU sheets, indicating that the coating performs similarly across different geometries.

[0330] Example 10: Peptide-coated catheters display anti-microbial activity for up to 7 days

[0331] Peptide-coated catheters display anti-microbial activity for up to 7 days: Microbiological analysis was conducted on the samples to assess their efficacy in preventing bacterial colonization and growth. The PC-PU segments were immersed in a bacterial suspension containing E. coli, K. pneumonia, and S. aureus at a concentration of 106 CFU / ml for 2 hours. The supernatant was then collected and measured for colonyforming units. Subsequent tests for antimicrobial activity were carried out after 7 days (Figure 62B-D). The results indicated that the PC-PU segments were able to effectively eliminate surrounding bacteria for up to 7 days.

[0332] Example 11: Peptide coated Polymers can trap the bacteria onto the surface:

[0333] Scanning Electron Microscopy (SEM) was utilized to conduct an in-depth analysis of the morphological changes occurring on the polymer surfaces, with a particular focus on examining the effects of peptides on bacterial membranes. SEM imaging of the PU segments (Figure 54A) revealed bacterial adhesion (after 1 day of incubation), with bacteria maintaining their structural integrity, indicating that the PU surface allowed for bacterial colonization without compromising the morphology of the bacterial cells. In contrast, the analysis of the PC-PU segments demonstrated a strikingly different interaction. The SEM images revealed the formation of peptide nanonets emerging from the PC-PU surface. These nanonets were observed to envelop and immobilize the bacterial cells, physically trapping them on the polymer surface. This entrapment appeared to be potentially effective in restricting bacterial movement on polymer surfaces and preventing further colonization. The emergence of peptide nanonets highlights the importance of maintaining a certain degree of flexibility and mobility in peptide design, as it appears to be a crucial factor for enabling the formation of bacteria-responsive nanonets. These results suggest that the conjugation of peptides tosurfaces can still support the dynamic assembly of structures that are critical for antimicrobial activity.

[0334] Example 12: Peptide coating is robust on various polymer substrates

[0335] To assess this versatility, the coating was applied to several polymers commonly used in biomedical device manufacturing, including PVC, PTFE, silicone, and latex, in addition to PU (Figure 54B). The results showed robust and uniform functionalization across all materials. Both coated and uncoated controls were then exposed to UPEC at a concentration of 106CFU / mL and incubated for 4 weeks. After incubation, biofilm burden was quantified using confocal laser scanning microscopy. The segments were stained with Syto™ 9, which labels all bacteria green, and Propidium Iodide (PI), which stains dead bacteria red. The coated substrates demonstrated significant suppression of biofilm biomass and viable adherent cells compared to their uncoated counterparts, indicating effective prevention of biofilm establishment throughout the testing period. These findings support both the robustness of the coating and its applicability to diverse device-relevant substrates.

[0336] Example 13: Evaluate the in vitro efficacy against bacteria models and clinical isolates from various biological: Nanonet-forming BTT peptides are potent against uropathogenic strains

[0337] The bacteria responsible for urinary tract infections (UTIs) are known as uropathogenic strains. Notably, Uropathogenic e. coli, klebsiella pneumoniae Carbapenemase Producers (KpCP), enterococcus faecalis. and Proteus mirabilis are frequently identified in patients with CAUTI. UPEC strains are the most common pathogens in community-acquired UTIs, accounting for 70% to 90% of cases, and constitute approximately 50% of the strains encountered in nosocomial UTIs. e. coli is the leading causative agent of UTIs in outpatients, representing 90% of all cases. KpCP is also prevalent in the urine of infected patients, contributing to 32% of infections. enterococcus faecalis causes more than 30% of CAUTls and is a significant healthcare-associated infection risk39, 40-43This is compounded by recent increases in multidrug resistance due to antibiotic misuse, particularly in enterococci. Additionally, infections from enterococci are challenging due to their ability to endure extreme environments, intrinsic antimicrobial resistance, and genomic variability, p. mirabilis, while less prevalent, ranks as the third most common cause of complicated UTIs (12%) and thesecond in catheter-associated bacteriuria among long-term catheterized patients (15%) 39,44

[0338] The inventors screened the most active nanonet-forming peptides for antimicrobial activity against the gram-negative uropathogenic strains in liquid media (Figure 55). BTT1-3A exhibited the highest antimicrobial potency, demonstrated by a low minimum inhibitory concentration (MIC). The MIC represents the lowest concentration of a drug required to inhibit microbial growth. Against both UPEC and KpCP, BTT1-3A had an MIC comparable to that of the control drug (colistin for UPEC and KpCP and Ciprofloxacin for p. mirabils), with values ranging from 8 to 16 pM, indicating its effectiveness against uropathogenic strains.

[0339] Example 14: Peptide coatings have demonstrated promising anti-fouling activity for up to 4 weeks

[0340] Biofilm formation is a significant and common outcome of CAUTI, making its prevention critically important. To investigate this, biofilm formation was initiated by incubating both PC-PU and uncoated polymer segments with uropathogenic E. coli. The growth media were replenished every 48 hours to sustain bacterial growth. To evaluate the extent of biofilm formation, live / dead staining was performed using confocal microscopy at the end of each week. The segments were stained with Syto™ 9, which labels all bacteria green, and Propidium Iodide (PI), which stains dead bacteria red. Over the 4-week incubation period, distinct differences in biofilm formation were observed between the uncoated polymer and the PC-PU segments. On the uncoated polymer surfaces, confocal microscopy revealed the development of thick, mature biofilms, characterized by a dense, continuous layer of bacteria adhering to the surface. The live / dead staining showed that the bacteria in these biofilms were viable, as evidenced by the dominant green fluorescence from Syto™ 9 (Figure 56A).

[0341] In contrast, the PC-PU-coated segments displayed a markedly different pattern. Instead of forming a continuous biofilm, the bacteria on these surfaces were observed in small, isolated clusters. These clusters were significantly less dense and widespread compared to the biofilms on the uncoated surfaces. Notably, the absence of large-scale biofilm formation on PC-PU segments suggests that the surface modification inhibited the ability of E coli to adhere and establish a biofilm. Furthermore, the live / dead staining indicated that the majority of the bacteria within these clusters were dead, as evidenced by the predominance of red fluorescence. At the end of the fourth week, observationsrevealed that the bacteria clusters exhibited a combination of green and red fluorescence, suggesting the coexistence of both live and dead bacteria. This indicates that while the peptides within the cluster are effective at capturing the bacteria, they are unable to kill them completely. To further investigate the differences in biofilm morphology, SEM was conducted at the end of the first and fourth weeks of biofilm incubation. SEM imaging of the PU segments at the end of the first week revealed the initial stages of biofilm formation, with a single layer of bacteria adhered to the surface. By the end of the fourth week, the PU surfaces exhibited a well-developed, thick, and dense bacterial biofilm, indicative of robust colonization and biofilm maturation (Figure 56B).

[0342] In contrast, SEM images of the PC-PU segments showed no signs of bacterial adhesion throughout the incubation period. Instead, at both time points, peptide nanonets were observed emerging from the PC-PU surface, enveloping and trapping bacterial cells These nanonets physically immobilized the bacteria, preventing them from adhering to the polymer surface and preventing biofilm formation.

[0343] This observation highlights a key functional difference between the two surfaces. While the PC-PU segments did not exhibit bactericidal activity beyond the first week, as evidenced by the persistence of bacteria in the surrounding environment seen previously, the peptide nanonets effectively inhibited bacterial adhesion on the surface. This suggests that the PC-PU surface, though not directly killing bacteria over extended periods, functions as an effective antifouling agent by preventing biofilm formation through the physical entrapment of bacteria. Thus, the peptide coating demonstrates a potential to reduce biofilm-associated infections by inhibiting the initial adhesion and colonization processes critical for biofilm development.

[0344] To standardize the biofilm analysis, biofilm formation studies were conducted in accordance with ISO 4768:2023, utilizing the crystal violet staining method to assess the anti-biofilm activity of the PC-PU-coated segments. The anti-biofilm activity was quantified as a percentage and calculated by comparing the optical density of crystal violet-stained biofilms between the coated and uncoated segments. In alignment with the results from the live / dead staining assay, these findings demonstrate that the PC-PU coating exhibits strong initial anti -biofilm activity, achieving a -90% reduction in biofilm formation at the end of the first week (Figure 56C). Although there is a gradual decline in efficacy, with activity decreasing to 45% by the fourth week, the coating continues to provide significant biofilm inhibition over an extended period. The ability of PC-PU tomaintain substantial anti-biofilm activity suggests that it holds promise as an effective strategy for reducing biofilm-related complications in CAUTI, particularly in the early stages of catheter use.

[0345] Example 15: Trapped bacteria in biofilms show a reduction in motility at the end of 4 weeks

[0346] Although the anti-biofilm activity of the PC-PU coating was observed to decrease over four weeks, dropping to 50%, confocal microscopy revealed that bacteria remained trapped in clusters due to the nanonet-forming properties of the peptide coating. To further investigate this phenomenon, the bacteria from the biofilm were dislodged using vortexing, diluted, and subsequently introduced into soft agar for motility analysis. Bacteria from biofilms formed on uncoated segments exhibited widespread motility, with an average spread diameter of 5 cm.

[0347] In contrast, bacteria from the PC-PU-coated segments showed significantly reduced motility, with a spread diameter limited to 2 cm (Figure 57). This result indicates that while the coating may not completely eliminate the bacteria, it effectively inhibits bacterial motility by trapping them in clusters. This reduced motility likely prevents the bacteria from spreading throughout the catheter and into the urinary tract, thereby lowering the risk of CAUTI. These findings suggest that the PC-PU coating, through its nanonet-forming capability, provides a dual mechanism of action by both limiting biofilm formation and restricting bacterial motility, making it a promising approach for reducing infection risk in clinical settings.

[0348] Example 16: Trapped bacteria in biofilms show a reduction in motility at the end of 4 weeks

[0349] Using BTTl-3A-coated polyurethane (PC-PU), the inventors assessed the 1-week antifouling performance against three representative clinical isolates: E. coli, K. pneumoniae and E. faecalis. From the confocal images, the live / dead assay illustrates (Figure 58) that the uncoated PU demonstrated substantial bacterial adhesion, with numerous viable cells attached to its surface, indicating its inability to inhibit colonization by these clinical isolates. The PC-PU coating exhibited significant antifouling activity against three representative clinical strains. It can be further seen that only a few bacterial clusters remained, with the majority of cells stained red, indicating they were dead. These findings suggest that the functionalized polymer can maintain strong antifouling activity against multiple clinical isolates.

[0350] Example 17: Peptide-coated Polymers can reduce infection associated oxidative stress and inflammatory response

[0351] Peptide-coated polymers can reduce oxidative stress and inflammatory responses associated with infections. Endotoxins, such as LPS, released during infections can induce oxidative stress through the production of ROS and trigger macrophages to release pro-inflammatory cytokines like TNF-a. Therefore, it is essential to assess the ability of the peptide-coated polymer to mitigate ROS generation and lower the secretion of pro-inflammatory cytokines.

[0352] To evaluate the antioxidant potential of the PC-PU, HEK293T-hTLR4 cells were co-incubated with LPS and PC-PU for 4 hours. ROS production was measured using the DCFH-DA probe, and intracellular fluorescence signals were visualized through confocal microscopy. As shown (Figure 59C), stimulation with LPS significantly increased ROS levels compared to control, serving as the positive control. In contrast, treatment with PC-PU resulted in a marked reduction in ROS fluorescence intensity, bringing levels close to those observed in the positive control group (NAC). This indicates that PC-PU effectively suppresses ROS generation.

[0353] To further investigate the anti-inflammatory properties of PC-PU, RAW264.7 and HEK293T-hTLR4 were co-incubated with LPS and PC-PU for 24 hours. The supernatants were analyzed for TNF-a secretion via ELISA Results from both RAW264.7 and HEK293T-hTLR4 cells demonstrated a significant decrease in TNF-a levels following co-incubation with PC-PU compared to the LPS-stimulated controls (Figure 59A and 59B).

[0354] In summary, these findings indicate that BTTl-3A-functionalized PU significantly reduces LPS-induced oxidative stress and pro-inflammatory cytokine secretion, highlighting its strong anti-inflammatory potential.

[0355] Example 18: Evaluate the ex-vivo efficacy in porcine urinary bladder (PUB) model:

[0356] To assess the efficacy of the peptide coating in a clinically relevant environment, ex vivo studies were conducted using porcine urinary bladder tissue. A 3.5 mm peptide-coated PU tube was used for this evaluation. After thoroughly washing the bladder, UPEC at a concentration of 106CFU / mL was introduced into the urethra, followed by the insertion and sealing of the coated tube (Figure 60 A). After 24 hours of incubation, both the coated tube and the urethral tissue in direct contact with the peptide-coated surface were harvested and washed to remove planktonic bacteria. The samples were then analyzed using three methods: (i) a colony -forming unit (CFU) counting assay to quantify viable bacterial load, (ii) biofilm quantification through crystal violet staining, and (iii) SEM to visualize surface morphology and bacterial colonization.

[0357] The CFU assay indicated a significant reduction in bacterial load on both the tissue and the coated tubing compared to uncoated controls (Figure 60B). Similarly, crystal violet staining demonstrated a substantial decrease in biofilm biomass on the peptide-coated samples relative to controls (Figure 60C). SEM imaging supported these findings, showing dense bacterial adhesion and mature biofilm structures on control tissues and coated surfaces, while the peptide-coated samples showed minimal to no bacterial colonization (Figure 60D). Collectively, these preliminary results underscore the strong potential of the peptide nanonet coating to effectively prevent biofilm formation and device-associated infections. Importantly, the coating demonstrates versatility for application across a wide range of biomaterials, emphasizing its promise as a platform for advanced antimicrobial coatings for biomedical devices.

[0358] Example 19: Evaluation of Fibrillating Peptide Coating on Polymer Surfaces

[0359] To investigate the coating behaviour of other fibrillating peptides from RE Lab’s peptide library on polymer substrates, two representative peptides, W-W13 and F-F15, were selected as model systems. W-W13 is known to exhibit a strong bacterial-binding tendency through tight wrapping, whereas F-F15 forms loosely associated nanonets. These peptides were immobilised onto polymer surfaces following the established conjugation protocol.

[0360] Attenuated Total Reflectance (ATR) analysis confirmed successful peptide conjugation. The appearance of a broad peak at 1036 cm1in the peptide-coated polyurethane (PC-PU) corresponds to the C-H stretching of aromatic moieties, indicating conjugation. Furthermore, broadening of the O-H stretch bands at 3335 cm1for W-W13 and 3326 cm1for F-F15 suggests effective peptide attachment. Notably, shifts in the amide I bands from 1610 to 1643 cm1for W-W13 and from 1610 to 1688 cm1for F-F15 further support the formation of peptide-polymer linkages (Figure 61 A).

[0361] To assess the peptides’ ability to fibrillate on the surface, scanning electron microscopy (SEM) was performed to examine the surface morphology. Both W-W13 and F-F15 coated polymers exhibited markedly reduced surface peptide density compared touncoated controls, indicative of antifouling characteristics. Morphological analysis of bacterial interactions revealed that the surface-coated W-W13 promoted tight bacterial wrapping, consistent with its known binding behaviour, while F-F15 formed extended nanonet structures entrapping bacterial cells (Figure 6 IB). These results demonstrate that peptide fibrillation and resultant bacterial interaction patterns are directly influenced by the intrinsic self-assembly behaviour of each peptide upon surface conjugation.

[0362] Industrial Applicability

[0363] Antibiotic resistance is a major global healthcare challenge, significantly diminishing the effectiveness of existing antibiotics This invention introduces improved peptide nanonets designed to address this issue directly, offering a better alternative for targeting bacterial resistance and meeting clinical needs.

[0364] The invention specifically targets the motility of Gram-negative bacteria, which enables them to escape areas of high drug concentration and contributes to antibiotic resistance. It also improves the trapping and killing of Gram-positive bacteria and fungi addressing the problem of internalization that leads to resistance against current antibiotics. The peptide nanonets disclosed herein effectively prevent the escape and spread of motile Gram-negative bacteria, showing superior performance compared to traditional antibiotics. This indicates that creating nanonets that tightly encage and restrict bacterial movement may be a more effective strategy than solely developing more potent antibiotics.

[0365] Additionally, this invention with reduced length also helps lower manufacturing costs, providing an added advantage for commercial production.

[0366] Additionally, the invention includes substrates and medical devices that are coated with the synthetic nanonet forming peptides that provides antifouling and antimicrobial properties.

[0367] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.Sequence listingSEQ ID NO: 1Amino acid sequence of the synthetic β-hairpin AMP (hairpin turn is underlined) cXchcLTAchcXcwherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;SEQ ID NO: 2Amino acid sequence of the synthetic β-hairpin AMP (hairpin turn is underlined) cXcXcLTAcXcXcwherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;SEQ ID NO: 3Amino acid sequence of the synthetic β-hairpin AMP (hairpin turn is underlined) hcXchcLTAchcXchwherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;SEQ ID NO: 4Amino acid sequence of the synthetic β-hairpin AMP (hairpin turn is underlined) ahchcLTAchahcwherein a = an anionic amino acid, or an analog or derivative thereof;c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;SEQ ID NO: 5synthetic β-hairpin AMP BTT1-3A (hairpin turn is underlined) LKLKLKLTAKLKLKL SEQ ID NO: 6hairpin turn of synthetic β-hairpin AMP BTT1-3ALTA SEQ ID NO: 7Amino acid sequence of the synthetic β-hairpin AMP diF-F13 (hairpin turn is underlined)KFRFKLTAKFRFK SEQ ID NO: 8Amino acid sequence of the synthetic β-hairpin AMP W-W13 (hairpin turn is underlined)KWRVKLTAKVRWK SEQ ID NO: 9first side strand at the N-terminal of, or second side strand at the C terminal of the synthetic β-hairpin AMP diF-F13KFRFK SEQ ID NO: 10first side strand at the N-terminal of the synthetic β-hairpin AMP W-W13 KWRVK SEQ ID NO: 11second side strand at the C terminal of the synthetic β-hairpin AMP W-W13 KVRWK SEQ ID NO: 12Amino acid sequence of the synthetic β-hairpin AMP Btt1-3A13 (hairpin turn is underlined)KLKLKLTAKLKLK SEQ ID NO: 13Amino acid sequence of the synthetic P-hairpin AMP Btt1-3A11(hairpin turn is underlined)LKLKLTAKLKL SEQ ID NO: 14Amino acid sequence of the synthetic β-hairpin AMP F-F15 (hairpin turn is underlined) LKFRVKLTAKVRFKL SEQ ID NO: 15Amino acid sequence of the synthetic P-hairpin AMP diF-F15 (hairpin turn is underlined)LKFRFKLTAKFRFKL SEQ ID NO: 16Amino acid sequence of the synthetic P-hairpin AMP F-F13 (hairpin turn is underlined) KFRVKLTAKVRFK SEQ ID NO: 17Amino acid sequence of the synthetic P-hairpin AMP F-F11 (hairpin turn is underlined) FRVKLTAKVRF SEQ ID NO: 18Amino acid sequence of the synthetic β-hairpin AMP W-W13 (hairpin turn is underlined)WRVKLTAKVRW SEQ ID NO: 19Amino acid sequence of the synthetic p-hairpin AMP E-E13 (hairpin turn is underlined) EVRIKLTAKIRVESEQ ID NO: 20Amino acid sequence of the synthetic p-hairpin AMP E-E13ASYM(hairpin turn is underlined)EVRIKLTAKIEVR SEQ ID NO: 21Amino acid sequence of the synthetic p-hairpin AMP W1 KXRVKLTAKVRXK, wherein X is 3 -benzothienylalanineSEQ ID NO: 22Amino acid sequence of the synthetic P-hairpin AMP W2 KXRVKLTAKVRXK, wherein X is 3-(l-naphthyl)-alanineSEQ ID NO: 23Amino acid sequence of the synthetic P-hairpin AMP W3 KXRVKLTAKVRXK, wherein X is 7-fluoro-tryptophanSEQ ID NO: 24Amino acid sequence of the synthetic P-hairpin AMP W4 KXRVKLTAKVRXK, wherein X is 1-methyl-tryptophanSEQ ID NO: 25Amino acid sequence of the synthetic P-hairpin AMP W5 KXRVKLTAKVRXK, wherein X is 5 -hydroxy -try ptophanSEQ ID NO: 26Amino acid sequence of the synthetic P-hairpin AMP W6KXRVKLTAKVRXK, wherein X is 7-aza-try ptophanSEQ ID NO: 27Amino acid sequence of the synthetic [3-hairpin AMP W7 KXRVKLTAKVRXK, wherein X is 2,3-dihydro-2-oxo-tryptophan SEQ ID NO: 28Amino acid sequence of the synthetic [3-hairpin AMP Fl KhRhKLTAKhRhK, wherein h is cyclohexylalanineSEQ ID NO: 29Amino acid sequence of the synthetic [3-hairpin AMP F2 KXRXKLT AKXRXK, wherein X is 3,4,5-trifluorophenylalanine SEQ ID NO: 30Amino acid sequence of the synthetic [3-hairpin AMP F3 KXRXKLT AKXRXK, wherein X is 4-fluorophenylalanineSEQ ID NO: 31Amino acid sequence of the synthetic [3-hairpin AMP F4 KXRXKLTAKXRXK, wherein X is 2-thienylalanineSEQ ID NO: 32Amino acid sequence of the synthetic [3-hairpin AMP F5 KXRXKLTAKXRXK, wherein X is TyrosineSEQ ID NO: 33Amino acid sequence of the synthetic [3-hairpin AMP F6 KXRXKLTAKXRXK, wherein X is 2-furylalanineSEQ ID NO: 34Amino acid sequence of the synthetic [3-hairpin AMP F7 KXRXKLTAKXRXK, wherein X is 5 -thiazolylalanineSEQ ID NO: 35Amino acid sequence of the synthetic [3-hairpin AMP F8 KXRXKLTAKXRXK, wherein X is 3-(3-pyridyl)alanineSEQ ID NO: 36Amino acid sequence of the synthetic [3-hairpin AMP E-EuAS™(ch) EIKVRLTARVEIK SEQ ID NO: 37Amino acid sequence of the synthetic [3-hairpin AMP E-E13ASYM(C1I) EIKVRLTARVEIKSEQ ID NO: 38hairpin turn of synthetic 0-hairpin AMP BTT2-4A VpPA wherein “p” is d-ProlineSEQ ID NO: 39Amino acid sequence of the synthetic 0-hairpin BTT2-4A LKLKLKVpPAKLKLKL wherein “p” is d-ProlineReference[1] F. 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Claims

Claims1. A synthetic P-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising the following functional modules:(a) a recognition module comprising a hairpin turn having an amino acid sequence of LTA (SEQ ID NO: 6), for interacting with pathogen-associated molecular patterns (PAMPs) of a bacterium or PAMPs of cell wall or membrane component of a fungus to initiate amyloid nucleation; and(b) a structural module comprising a first side strand and a second side strand for -sheet formation;wherein the synthetic P-hairpin AMP comprises a sequence selected from any one or more of the group consisting of:cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof, a = an anionic amino acid, or an analog or derivative thereof;andwherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or fungus, or on or from a bacterium or fungus.

2. The synthetic P-hairpin AMP of claim 1, wherein the synthetic P-hairpin AMP is: a) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); b) W-WB and comprises the sequence of K WR VKLTAK VRWK (SEQ ID NO: 8); c) F-FB and comprises the sequence of KFR VKLTAK VRFK (SEQ ID NO: 16), d) F-F15and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);f) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-benzothienylalanine (SEQ ID NO: 21);g) W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);h) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);i) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1 - methyl-tryptophan (SEQ ID NO: 24);j) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);k) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);l) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);m) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienylalanine (SEQ ID NO: 31);n) E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO:36) oro) a combination thereof.

3. The synthetic P-hairpin AMP of claim 1, wherein the PAMPs is selected from any one or more of the group consisting of lipoteichoic acid (LT A) of a Gram-positive bacterium and lipopolysaccharide (LPS) of a Gram-negative bacterium, and the bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

4. The synthetic P-hairpin AMP of claim 3, wherein the Gram-positive bacterium is selected from the any one or more of group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, and Listeria monocytogenes,' and / orwherein the Gram-negative bacterium is selected from the any one or more of group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae,Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera.

5. The synthetic P-hairpin AMP of claim 1, wherein the fugus is selected from one or more of the group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinell aides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

6. A pharmaceutical or adjuvant composition comprising the synthetic P-hairpin AMP of any one of claims 1-5, and a pharmaceutically acceptable carrier or diluent.

7. A method for treating a condition associated with a bacterial infection, fungal infection, or an immune-related disease in a subject in need thereof, comprising administering the synthetic P-hairpin AMP of any one of claims 1-5, or the pharmaceutical or adjuvant composition of claim 7 to the subject,wherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to(a) trap and kill the bacterium or fungus; and / or(b) trap and reduce motility of the bacterium or fungus.

8. Use of the synthetic P-hairpin AMP of any one of claims 1-5, or the pharmaceutical or adjuvant composition of claim 6 in the manufacture of a medicament for treating a condition associated with a bacterial infection, fungal infection or an immune-related disease in a subject in need thereof, wherein the synthetic P-hairpin AMP or the pharmaceutical or adjuvant composition is to be administered to the subject, wherein the synthetic P-hairpin AMP self-assembles into interwoven nanofibers on or from bacterial or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to(a) trap and kill the bacterium or fungus; and / or(b) trap and reduce motility of the bacterium or fungus.

9. The method of claim 7 or the use of claim 8, wherein the bacterial infection is an infection of an antibiotic-resistant bacteria.

10. The method of claim 7 or 9, or the use of claim 8 or 9, wherein the synthetic P-hairpin AMP or the composition is administered with an antibiotic11. The method of any one of claims 7 and 9-10, or the use of any one of claims 8-10, wherein the synthetic P-hairpin AMP is administered in a dose of 2.5-10 mg / kg.

12. A method for trapping an endotoxin lipopolysaccharide to reduce inflammatory activity in a subject in need thereof, comprising administering the synthetic P- hairpin AMP of any one of claims 1-5, or the pharmaceutical or adjuvant composition of claim 6 to the subject.

13. A kit comprising the pharmaceutical or adjuvant composition of claim 6, and a dispenser and / or applicator.

14. A coated substrate, comprising: a substrate coated with a synthetic P-hairpin antimicrobial peptide (AMP) comprising the following functional modules:(a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation; wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA fp’: d-Proline) (SEQ ID NO: 38); and(b) a structural module comprising a first side strand and a second side strand for P- sheet formation and molecular stacking during fibrillation;wherein the synthetic P-hairpin AMP comprises a sequence selected from any one or more of the group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof;c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof;a = an anionic amino acid, or an analog or derivative thereof; andwherein the synthetic P-hairpin AMP self-assembles into nanonets and / or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of the bacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to(i) trap and kill the bacterium or the fungus; and / or(ii) trap and reduce motility of the bacterium or the fungus.

15. A method for preventing or reducing bacterial biofilm formation on a medical device surface, comprising conjugating a synthetic p-hairpin antimicrobial peptide (AMP) onto the medical device surface, wherein the synthetic P-hairpin AMP comprises the following functional modules:(a) a recognition module comprising a hairpin turn for interacting with a bacterial membrane component or a fungal cell wall or membrane component to initiate amyloid nucleation; wherein the hairpin turn comprises the sequence of LTA (SEQ ID NO: 7) or VpPA (‘p’: d-Proline) (SEQ ID NO: 38); and(b) a structural module comprising a first side strand and a second side strand for P- sheet formation and molecular stacking during fibrillation;wherein the synthetic P-hairpin AMP comprises a sequence selected from the group consisting of:LKLKLKLTAKLKLKL (SEQ ID NO: 5),LKLKLKVpPAKLKLKL (SEQ ID NO: 39),cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO: 4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof;c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof; and a = an anionic amino acid, or an analog or derivative thereof,wherein the synthetic P-hairpin AMP self-assembles into nanonets or interwoven nanofibers on or from bacterial surface or fungal surface, in the presence of thebacterial membrane component or the fungal cell wall or membrane component, or on or from a bacterium or a fungus to(i) trap and kill the bacterium or the fungus; and / or(ii) trap and reduce motility of the bacterium or the fungus.

16. The coated substrate of claim 14, or the method of claim 15, wherein the synthetic - hairpin AMP is:a) BTT1-3A and comprises the sequence of LKLKLKLTAKLKLKL (SEQ ID NO:5);b) BTT2-4A and comprise the sequence of LKLKLKVpPAKLKLKL (SEQ ID NO:39).c) W-W13and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); d) F-F₁₅ and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); f) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);g) F-F13and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16); h) diF-F₁₅ and comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO:15)i) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21);j) W2 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);k) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);l) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24);m) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);n) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);o) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);p) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienylalanine (SEQ ID NO: 31);q) E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO: 36) or;a) a combination thereof.

17. The coated substrate of claim 14 or 16, wherein the substrate is selected from any one or more of the group consisting of a polyurethane (PU), hydrogel coated latex, silicone, latex, polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), plasticized polyvinyl chloride (pPVC), polyimide (PI), ethylene vinyl acetate (EVA), poly(tetrafluoroethylene) (PTFE), titanium dioxide (TiO2), Polyetheretherketone (PEEK) and silicon dioxide (SiO₂).

18. The coated substrate of claim 17, or the method of claim 15 or 16, wherein the substrate is or is part of a medical device, or the medical device surface belongs to a medical device, selected from any one or more of the group consisting of a urinary catheter, a urinary stent, a joint implant, a vascular catheter, a polyurethane (PU) catheter, an intravenous line, a dental implant, a continuous glucose monitor, a wound dressing, a suture, an adhesive bandage, and a feeding tube.

19. The coated substrate of claim 14, or the method of claim 15, wherein the bacterial membrane component is selected from the any one or more of the group consisting of lipoteichoic acid (LTA) of a Gram-positive bacterium and lipopolysaccharide (LPS) of a Gram-negative bacterium, and the bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

20. The coated substrate of claim 19, or the method of claim 19, wherein the bacterium is one associated with urinary tract infection, selected from any one or more of the group consisting of uropathogenic E. coli, Klebsiella pneumoniae, Carbapenemase Producers (KpCP), Enterococcus faecalis, Proteus Mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella spp., Aeromonas spp., Serratia spp., Neisseria spp., Providencia spp., Acinetobacter spp., Veillonella spp., Citrobacter spp., Bacteroids spp., Staphylococcus saprophyticus, Streptococcus spp., Lactobacillus spp., Corynebacterium spp., Aerococcus spp., Actinobaculum spp., Gardnerella spp., Propionibacterium spp., Ureaplasma spp., and Mycoplasma spp and or wherein the fungus is or the fungal membrane component belongs to any one or more fungi selected from the following group consisting of Candida albicans, Candidaauris, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida dubliniensis, Candida lusitaniae, Trichosporon asahii, Malassezia furfur, Saccharomyces cerevisiae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Rhizopus arrhizus (Rhizopus oryzae), Mucor circinelloides, Rhizomucor pusillus, Fusarium solani, Scedosporium apiospermum, Lomentospora prolificans, Purpureocillium lilacinum (Paecilomyces lilacinus), and Paecilomyces variotii.

21. The coated substrate of claim 19, or the method of claim 19, wherein the bacterium is an antibiotic-resistant bacterium.

22. The coated substrate of any one of claims 14 and 16-21, or the method of 15, wherein the synthetic β-hairpin AMP is conjugated to the substrate via glutaraldehyde crosslinking.

23. The coated substrate of any one of claims 14 and 16-22, or the method of any one of claims 15 and 16-22, wherein the synthetic β-hairpin AMP is coated at a concentration selected from the group consisting of 10-1000 μM / cm2, 50-900 μM / cm2, 100-800 μM / cm2, 200-700 μM / cm2, 300-600 μM / cm2, 400-500 μM / cm2, 10-100 μM / cm2, 50- 200 μM / cm2, 100-300 μM / cm2, 200-400 μM / cm2, 300-500 μM / cm2, 400-600 μM / cm2, 500-700 μM / cm2, 600-800 μM / cm2, 700-900 μM / cm2, or 800-1000 μM / cm2, preferably about 100 μM / cm2.

24. A medical device, comprising the coated substrate of any one of claims 14 and 16- 23.

25. A method of preventing or treating a biofilm-related infection in a subject in need thereof, comprising implanting a medical device comprising the coated substrate of any one of claims 14 and 16-23, or the medical device claim 24 into the subject, wherein the synthetic β-hairpin AMP coated on the substrate self-assembles into nanonets or interwoven nanofibers on or from bacterial surface or fungal surface in the presence of the PAMPs of the bacterium or the PAMPs of cell wall or membrane component of the fungus, or on or from a bacterium or fungus to(i) trap and kill the bacterium or fungus; and / or(ii) trap and reduce motility of the bacterium or fungus.

26. The method of claim 25, wherein the medical device comprising the coated substrate of any one of claims 14 and 16-23 or the medical device of claim 24, traps and kills the bacterium or fungus, reduces mobility of the bacterium or fungus, prevents or reduces biofouling, and delays or prevents the development of bacterial resistancecompared to a conventional antibiotic selected from the group consisting of Colistin, Vancomycin, Daptomycin, Linezolid, Teicoplanin, Cefazolin, Ceftriaxone, Meropenem, Ertapenem, Ciprofloxacin, Levofloxacin, Tigecycline, Tobramycin, Gentamicin, Naficillin, Oxacillin, Ceftazidime, Amoxicillin, Rifampin, Fosfomycin, Nitrofurantoin, Minocycline, and Clindamycin.

27. The method of any one of claims 15-23 and 26, the medical device of claim 24, or the coated substrate of any one of statements 14 and 16-23, wherein the coated substrate or medical device:- maintains an antifouling and / or antimicrobial effect for up to four weeks;results in an about 90% reduction in biofilm formation at the end of the first week of use, and / orresults in at least 50% reduction in biofilm formation after 24 hours of implant, optionally compared to a medical device without the coated substrate.

28. The method of claim 25, wherein the medical device reduces oxidative stress and inflammatory responses associated with infections.

29. The method of claim 28, wherein the medical device reduces a pro-inflammatory cytokine selected from the group consisting of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-12a (IL-12a), interleukin-12β (IL-12β), interleukin- 18 (IL-18), and high mobility group box 1 (HMGB1).

30. The method of claim 29, wherein the medical device reduces the pro-inflammatory cytokine by at least 40%, optionally compared to a medical device without the coated substrate.

31. A synthetic β-hairpin antimicrobial peptide (AMP) with a length of 13 or 15 amino acids comprising a sequence selected from any one or more of the group consisting of: cXchcLTAchcXc (SEQ ID NO: 1),cXcXcLTAcXcXc (SEQ ID NO: 2),hcXchcLTAchcXch (SEQ ID NO: 3), andahchcLTAchahc (SEQ ID NO:4);wherein X = an aromatic hydrophobic amino acid, or an analog or derivative thereof; c = a cationic amino acid, or an analog or derivative thereof;h = a non-aromatic hydrophobic amino acid, or an analog or derivative thereof,a = an anionic amino acid, or an analog or derivative thereof;andwherein the AMP kills or reduces bacteria, fungus, or both; and / oreliminates or reduces bacterial activity, fungal activity or both.

32. The synthetic β-hairpin AMP of claim 31, wherein the synthetic β-hairpin AMP is:a) diF-F₁₃ and comprises the sequence of KFRFKLTAKFRFK (SEQ ID NO: 7); b) W-W13and comprises the sequence of KWRVKLTAKVRWK (SEQ ID NO: 8); c) F-F13and comprises the sequence of KFRVKLTAKVRFK (SEQ ID NO: 16); d) F-F15and comprises the sequence of LKFRVKLTAKVRFKL (SEQ ID NO: 14); e) E-E13ASYMand comprises the sequence of EVRIKLTAKIEVR (SEQ ID NO:20);f) diF-F₁₅ comprises the sequence of LKFRFKLTAKFRFKL (SEQ ID NO: 15); g) W1 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 3- benzothienylalanine (SEQ ID NO: 21);h) W2 and comprises the sequence of KXR VKLTAK VRXK, wherein X is 3-(1- naphthyl)-alanine (SEQ ID NO: 22);i) W3 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7- fluoro-tryptophan (SEQ ID NO: 23);j) W4 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 1- methyl-tryptophan (SEQ ID NO: 24);k) W5 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 5- hydroxy-tryptophan (SEQ ID NO: 25);l) W6 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 7-aza- tryptophan (SEQ ID NO: 26);m) W7 and comprises the sequence of KXRVKLTAKVRXK, wherein X is 2,3- dihydro-2-oxo-tryptophan (SEQ ID NO: 27);n) Fl and comprises the sequence of KhRhKLTAKhRhK, wherein h is cyclohexylalanine (SEQ ID NO: 28);o) F2 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 3,4,5- trifluorophenylalanine (SEQ ID NO: 29);p) F3 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 4- fluorophenylalanine (SEQ ID NO: 30);q) F4 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- thienylalanine (SEQ ID NO: 31);r) F5 and comprises the sequence of KXRXKLTAKXRXK, wherein X is Tyrosine (SEQ ID NO: 32);s) F6 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 2- furylalanine (SEQ ID NO: 33);t) F7 and comprises the sequence of KXRXKLTAKXRXK, wherein X is 5- thiazolylalanine (SEQ ID NO: 34);u) E-E13ASYM(ch) and comprises the sequence of EIKVRLTARVEIK (SEQ ID NO:36); orv) a combination thereof.

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