Peptides with multivalent effects

Abstract

The present invention relates to thrombin-derived peptides that contain at least one internal covalent bond between the side chains of two non-adjacent internal amino acids. The peptides have anti-inflammatory activity.

Description

[Technical field]

[0001] The present invention is in the field of peptides for the treatment of inflammation and / or infections. In particular, the present invention provides peptides with good stability, high anti-inflammatory activity and / or antibacterial activity. [Background technology]

[0002] Sensing of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) is important in the early response to infection, and subsequent NF-κB activation triggers a variety of biological effects associated with sepsis and ARDS, including the release of cytokines, chemokines, and subsequent deleterious hemostatic disorders, leading to consumption of coagulation factors and other mediators. Interestingly, the major extracellular protein of SARS-CoV-2, the spike glycoprotein, promotes LPS responses in vitro and in animal models, providing a molecular explanation for the ARDS seen in COVID-19 (Petruk et al., JMCB, 2020), and uncontrolled LPS responses not only cause local excessive inflammation but also severe systemic responses to infection. Thus, although sensing of LPS is important for the early host defense response, clearance and control of this molecule are critical to avoid excessive inflammation and organ damage.

[0003] Current therapies based on antibiotics and antivirals target only microbes and not the associated overactivation of immune responses, as seen in sepsis. It is a leading cause of death in the United States alone, with an estimated 700,000+ cases annually, and a 30-50% mortality rate in patients with septic shock. Therefore, a therapeutic concept based on nature's unique innate defense strategy, targeting not only bacteria but also the overactive immune response, may have great therapeutic potential. Thrombin-derived C-terminal peptides (TCPs) of approximately 2 kDa have been demonstrated to exert antiendotoxin functions in vitro and in vivo. Such small peptides belong to a diverse family of host defense peptides (HDPs), including neutrophil-derived α-defensins and the cathelicidin LL-37, all known to exhibit immunomodulatory activity. TCP-25 (SEQ ID NO: 12), which encompasses the sequence of native TCP, has been shown to neutralize LPS in vitro and protect against P. aeruginosa sepsis and LPS-mediated shock in experimental animal models, primarily via reducing systemic cytokine responses (Kalle et al., PLOS One, 2011).

[0004] TCP-25 binds LPS and directly interacts with monocytes and macrophages, preventing CD14 signaling and TLR4 / MD2 dimerization, and thus inhibiting TLR4- and TLR2-induced NF-κB activation in response to microbial agonists and intact bacteria (Saravanan et al., Nat Comm, 2018). Apart from interacting with bacterial membranes and LPS, TCP also binds to the LPS-binding groove of CD14 (Saravanan et al., Nat Comm, 2018). The fact that TCP exerts multiple, relatively weak affinities for LPS and CD14, all in the μM range, allows for the modulation of the host response to infection. Multivalency, multiple targets, high off-rates, and μM-level K d Sharing many characteristics with value-defined transient agents, TCPs are of interest for the development of novel nature-inspired anti-inflammatory therapies.

[0005] However, like many peptide-based therapeutics, TCP-25 is degraded by endogenous proteases (Puthia et al., 2020). For diseases such as sepsis and ARDS that require systemic or inhaled administration, a rapidly degraded peptide would require very large doses and frequent administration. Furthermore, from a pharmacological point of view, improved affinity for its target receptor, CD14, would be desirable, which would reduce the required effective concentration. Finally, TCP-25 forms oligomers and aggregates at high concentrations, so improved solubility would be an advantage from a drug delivery point of view. Summary of the Invention

[0006] The present invention provides peptides that have several advantageous properties, including one or more of the following: High in vivo stability Increased stability in the presence of proteases such as human neutrophil elastase (HNE), Pseudomonas elastase (PE), and / or trypsin High anti-inflammatory activity (e.g., as determined by reduced release of pro-inflammatory cytokines such as TNF-α and / or IL-1β, or reduced NF-κB activity) Antibacterial activity, e.g. bactericidal activity against gram-negative and / or gram-positive bacteria Low hemolytic activity in blood Low hemolytic activity against red blood cells ·Low toxicity

[0007] In particular, the peptides of the invention have low hemolytic activity in blood at concentrations at which the peptides have high anti-inflammatory activity.

[0008] Preferred peptides of the invention have all the aforementioned properties. High in vivo stability, increased stability against proteases, and low hemolytic activity make the peptides of the invention particularly useful for systemic administration. In particular, the invention provides peptides with low hemolytic activity against red blood cells (RBCs) at concentrations with high anti-inflammatory activity.

[0009] More specifically, the peptide of the present invention is based on thrombin-derived peptide, and its structure is locked by a covalent bond between two non-adjacent amino acids.Interestingly, the peptide of the present invention has some, preferably all, of the above-mentioned advantageous properties.Many of the linear peptides derived from thrombin have both anti-inflammatory and antibacterial activity, but generally have low in vivo stability.

[0010] The peptides of the present invention with stabilized structure generally contain helical structure(s). They have stabilized protease-resistant structure, exert antibacterial activity, and generally have improved anti-inflammatory effect. Thus, the peptides of the present invention are interesting primary anti-inflammatory peptidomimetics. In general, the peptides have a lower tendency to oligomerize compared to natural TCP. Drug oligomerization is a well-known phenomenon that can cause aggregation, reduce efficacy, and increase the risk of delayed immune response. Thus, the peptides of the present invention generally show significantly less oligomerization, which is advantageous from the drug point of view.

[0011] Endogenous TCP HVF18 exerts higher affinity for LPS at low pH. The preferred peptides of the present invention have increased polarity and charge at the N-terminus, for example, by the addition of cationic K and R residues. The present invention utilizes a combination of nuclear magnetic resonance spectroscopy (NMR), biophysics, mass spectrometry, microbiology, cellular, and in vivo studies to show that increased charge, especially +2 charge, can provide optimal efficacy and high therapeutic index, in contrast to peptides with longer cationic stretches that have high toxicity and reduced anti-inflammatory activity.

[0012] Stapling a peptide can improve its proteolytic stability, but surprisingly, stapling certain thrombin-derived peptides also causes undesirable effects. For example, stapling GKY25 at a single position leads to the production of peptides with high hemolytic activity, which is undesirable. Stapling GKY25 at a single position also leads to peptides with reduced anti-inflammatory effects compared to non-stapled GKY25.

[0013] Surprisingly, the present invention discloses that shorter thrombin-derived peptides having an overall length of 10-23 amino acids, such as 13-23 amino acids, as opposed to longer peptides such as GKY25, possess most, and often all, of the aforementioned advantageous properties.

[0014] Furthermore, the present invention discloses that stapling longer thrombin-derived peptides having a length of 24-40 amino acids at at least two positions provides peptides having some of the advantageous properties mentioned above.

[0015] The present invention further demonstrates that peptides containing additional positively charged amino acids have even greater anti-inflammatory effects.

[0016] Furthermore, the peptides of the present invention may also have anticoagulant activity. The present invention shows that peptides containing additional positively charged amino acids may have even greater anticoagulant activity.

[0017] The present invention provides a peptide comprising a continuous sequence of 10 to 23 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, the peptide comprising: i) having a total length of 10 to 40 amino acids, ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) further comprising at least the amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; However, when the peptide has a total length of 24-40 amino acids, it contains at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids, the amino acids of the first internal covalent bond are designated X1 and X2, and the amino acids of the second internal covalent bond are designated X3 and X4. [Brief description of the drawings]

[0018] [Figure 1] SDS-PAGE of intact and digested peptides with different proteases for different lengths of time. One representative image out of three independent experiments is shown (n=3). [Diagram 2] a) NF-κB activation and cell viability in THP1-XBlue-CD14 reporter cells 20 h after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of linear and stapled GKY25. Results are presented as mean ± SD of four experiments (n=4). Significance was established by regular two-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism software. b) Cytokines released from human blood 24 h after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of GKY25 and sGKY25. Results are presented as mean ± SEM. Blood from different donors was used each time (n=4). Significance was established by regular two-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism software. [Diagram 3]Left panel: NF-κB activation and cell viability in THP-1 monocytes 20 h after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of HVF18 and sHVF18. Results are presented as mean ± SD of four experiments (n = 4). Significance was established by regular two-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism software. Right panel: Released cytokines from human blood 24 h after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of HVF18 and sHVF18. Results are presented as mean ± SEM. Blood from different donors was used each time (n = 4). Significance was established by regular two-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism software. [Figure 4] Heat maps show the hemolytic activity of peptides in red blood cells (RBC) or whole blood. Numbers indicate % hemolytic activity. Data are presented as the average of three independent experiments, each performed using blood from a different donor (n=3). A) GKY25 and sGKY25, B) HVF18 and sHVF18. [Diagram 5] The effect of stapling on the anti-inflammatory activity of peptides is shown. a, Representative binding curves between CD14 and GKY25, HVF18 or sHVF18 in the presence or absence of NaCl obtained by MST. Kd was calculated from the MST curves. Data are presented as the mean ± SD of six different measurements (n = 6). Significance was established by ordinary one-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism software. [Figure 6]Anti-inflammatory activity of linear and stapled HVF18. a, NF-κB activation and cell viability in THP1-XBlue-CD14 reporter cells 20 h after stimulation with 100 ng ml-1 E. coli LPS (LPSEc), 1 μg ml-1 S. aureus LTA (LTASa), 1 μg ml-1 E. coli PGN (PGNEB), 1 μg ml-1 S. aureus PGN (PGNSa), and 10 μg ml-1 S. cerevisiae zymosan (ZymSc) in the presence or absence of 10 μM linear and stapled HVF18. Results are presented as the mean ± SD of four experiments (n = 4). Significance was established by conventional one-way ANOVA followed by Dunnett's multiple comparison test using GraphPad Prism software. [Figure 7] Anti-inflammatory activity of linear and stapled HVF18 in blood. a) Cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS mixed with increasing doses of HVF18 or sHVF18. b) Cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS for 30 min and then incubated with increasing doses of HVF18 or sHVF18. Results are presented as mean ± SEM. Blood from different donors was used each time (n=4). Significance was established by ordinary two-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism software. [Figure 8a]Figure 1 shows the effect of stapled peptides on endotoxin response in an experimental mouse model. Representative in vivo inflammation imaging by IVIS in NF-κB reporter mice. HVF18 or sHVF18 was mixed with LPS immediately before subcutaneous injection into the backs of transgenic BALB / c Tg(NF-κB-RE-luc)-Xen reporter mice. In vivo imaging was acquired using an IVIS Spectrum bioimaging system at 3, 6, and 24 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n=8 each group). P values ​​were determined using the Mann-Whitney U test. [Figure 8b] Figure 1 shows the effect of stapled peptides on endotoxin response in an experimental mouse model. Cytokine release from plasma collected 8 and 20 hours after stimulation of C57BL / 6 mice with a sublethal dose of LPS administered intraperitoneally (i.p.) and subsequently treated with sHVF18 i.p. Data are presented as mean ± SEM (each circle represents one mouse). P values ​​were determined using ordinary one-way ANOVA followed by Dunnett's multiple comparison test. [Figure 9]a, Effect of linear and stapled peptides on endotoxin response in an experimental mouse model. b, Representative in vivo inflammation imaging by IVIS in NF-κB reporter mice. 200 μg of HVF18 or sHVF18 were mixed with 25 μg of LPS immediately before subcutaneous injection into the backs of transgenic BALB / c Tg(NF-κB-RE-luc)-Xen reporter mice. In vivo imaging was acquired using an IVIS Spectrum bioimaging system at 3, 6, and 24 h after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 each group). P values ​​were determined using the Mann-Whitney U test. c, Cytokine release from plasma collected 20 h later from C57BL / 6 mice stimulated with a sublethal dose of LPS by i.p. and then treated with increasing doses of sHVF18 i.p. Data are presented as mean ± SEM (each circle represents one mouse). P values ​​were determined using ordinary one-way ANOVA followed by Dunnett's multiple comparison test. [Figure 10A] Figure 1 shows the effect of stapling on the antibacterial activity of HVF18. Heat maps show the antibacterial activity of increasing concentrations of HVF18 and sHVF18 as determined by RDA. Activity was assessed against E. coli, P. aeruginosa O1, and S. aureus in both the absence and presence of NaCl. Data are displayed as zones of clearance. The grey scale and the value of each box represent the mean (n=4). [Figure 10B] FIG. 1 shows the effect of stapling on the antibacterial activity of HVF18. Killing efficacy of HVF18 and sHVF18 alone or in Tris buffer supplemented with NaCl or 25% human plasma as assessed by VCA. Data are presented as mean±SEM (n=4). [Figure 10C] Figure 1 shows the effect of stapling on the antibacterial activity of HVF18. Killing effect of sHVF18 against S. aureus in Tris buffer as assessed by VCA. Data are presented as mean ± SEM (n=4). [Figure 10D] Figure 1 shows the effect of stapling on the antibacterial activity of HVF18. MIC values ​​for HVF18 and sHVF18 against E. coli, P. aeruginosa O1, and S. aureus. The experiment was repeated four times with similar results (n=4). [Figure 10E] Figure 1 shows the effect of stapling on the antibacterial activity of HVF18. MIC values ​​for clinical isolates of P. aeruginosa O1 and S. aureus. The experiment was repeated four times with similar results (n=4). [Figure 11] Figure 1 shows the secondary structure assessment of sHVF18 and its K and R mutants. All peptides were diluted from 1 mM stock solutions to a final concentration of 10 μM in 10 mM Tris, pH 7.4. Spectra were acquired at 25° C. Results are presented as the average of three different experiments. [Figure 12a] Figure 1 shows the evaluation of the hemolytic properties of different stapled peptides in vitro. The histograms show the hemolytic activity of sHVF18K and R mutants at different concentrations against red blood cells (a) or whole blood (b). Data are the mean ± SD of four independent experiments (shown as dots). In (c) the hemolytic activity of GKY25, sGKY25, and 2sGKY25 against whole blood is shown. Data are the mean ± SD of two independent experiments. In all graphs, the dashed line represents the hemolytic activity of 100 μM sHVF18, and the dotted line corresponds to 10% lysis. [Figure 12b] Figure 1 shows the evaluation of the hemolytic properties of different stapled peptides in vitro. The histograms show the hemolytic activity of sHVF18K and R mutants at different concentrations against red blood cells (a) or whole blood (b). Data are the mean ± SD of four independent experiments (shown as dots). In (c) the hemolytic activity of GKY25, sGKY25, and 2sGKY25 against whole blood is shown. Data are the mean ± SD of two independent experiments. In all graphs, the dashed line represents the hemolytic activity of 100 μM sHVF18, and the dotted line corresponds to 10% lysis. [Figure 12c]Figure 1 shows the evaluation of the hemolytic properties of different stapled peptides in vitro. The histograms show the hemolytic activity of sHVF18K and R mutants at different concentrations against red blood cells (a) or whole blood (b). Data are the mean ± SD of four independent experiments (shown as dots). In (c) the hemolytic activity of GKY25, sGKY25, and 2sGKY25 against whole blood is shown. Data are the mean ± SD of two independent experiments. In all graphs, the dashed line represents the hemolytic activity of 100 μM sHVF18, and the dotted line corresponds to 10% lysis. [Figure 13A] Figure 1 shows the evaluation of anti-inflammatory activity of sHVF18 and its K and R mutants in THP-1-XBlue-CD14 reporter cells. NF-κB activation and cell viability in THP-1 monocytes 20 h after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of K (a) and R (b) sHVF18 mutants. Results are presented as the mean ± SD of four experiments (n=4). 10 μM sHVF18 in the presence of LPS was used for comparison. [Figure 13B] Figure 1 shows the evaluation of anti-inflammatory activity of sHVF18 and its K and R mutants in THP-1-XBlue-CD14 reporter cells. NF-κB activation and cell viability in THP-1 monocytes 20 h after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of K (a) and R (b) sHVF18 mutants. Results are presented as the mean ± SD of four experiments (n=4). 10 μM sHVF18 in the presence of LPS was used for comparison. [Figure 14A] Figure 1 shows the evaluation of anti-inflammatory activity of stapled peptides in human blood. Cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of K (a) and R (b) sHVF18 mutants, or GKY25 and its stapled mutants (c). Results are presented as mean ± SEM. Blood from different donors was used each time (n=4). [Figure 14B]Figure 1 shows the evaluation of anti-inflammatory activity of stapled peptides in human blood. Cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of K (a) and R (b) sHVF18 mutants, or GKY25 and its stapled mutants (c). Results are presented as mean ± SEM. Blood from different donors was used each time (n=4). [Figure 14C] Figure 1 shows the evaluation of anti-inflammatory activity of stapled peptides in human blood. Cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of K (a) and R (b) sHVF18 mutants, or GKY25 and its stapled mutants (c). Results are presented as mean ± SEM. Blood from different donors was used each time (n=4). [Figure 15] Figure 1 shows the evaluation of anti-inflammatory activity of sHVF18 and its K and R mutants in a mouse endotoxin model. C57BL / 6 mice were challenged with a sublethal dose of LPS and treated 30 min later with 10 μg of sHVF18, sKVF18, sKKVF18, sRVF18 or sRRVF18. After 20 h, mice were deeply anesthetized with isoflurane and blood was collected by cardiac puncture. Cytokine release profiles are shown as histograms. Data are presented as mean ± SEM (n = 7, sHVF18 and LPS fraction n = 13, and untreated n = 2). P values ​​were determined using ordinary one-way ANOVA followed by Dunnett's multiple comparison test. [Figure 16] Figure 1 shows the evaluation of antibacterial activity of sHVF18K and R mutants. Heat maps show the antibacterial activity of increasing concentrations of peptides as determined by RDA. Activity was evaluated against E. coli, P. aeruginosa O1 (PAO1), and S. aureus both in the absence (a) and presence (b) of NaCl. Data are displayed as clearance zones expressed in mm. Grey scale and each box value represents the mean (n=4). [Figure 17]Figure 1 shows the evaluation of antibacterial activity of sHVF18K and R mutants in solution. Bactericidal effect of sHVF18 and its mutants against E. coli, P. aeruginosa O1 (PAO1), and S. aureus alone (a) or supplemented with NaCl (b) in 10 mM Tris at pH 7.4, as assessed by VCA. Data are presented as mean ± SEM (n=4). [Figure 18] Figure 1 shows the effect of different peptides on coagulation. Activated partial thrombin time (aPTT) and prothrombin time (PT) were determined after adding increasing concentrations of different stapled peptides as indicated to human citrated plasma (n=2). [Figure 19] The hydrodynamic radius (Rh) of the different peptides is shown as a measure of oligomerization. Peptides were resuspended in 10 mM Tris, pH 7.4 or 10 mM NaOAc, pH 5.0, at a final concentration of 1 mM. 30 μL of each sample was used to measure the hydrodynamic radius (in nm) of the particles in solution. For each sample, spectra were recorded three times with 10 subruns using multimodal mode. Each experiment was performed in triplicate (n=3). P values ​​were determined using Tukey's multiple comparison test with one-way ANOVA. **P≦0.01, ****P≦0.0001. [Figure 20A]Selection of stapled peptides with improved anti-inflammatory activity is shown. a, Heat map showing hemolytic activity of peptides on whole blood or red blood cells (RBC). Data of experiments performed on red blood cells or blood from four different donors are displayed as averages (n=4). b, Heat map showing cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of different peptides. Results are displayed as averages. Blood from different donors was used each time (n=4). c, Graph obtained by combining data from (a) and (b) showing hemolytic activity of peptides as a function of IC50 of peptides against different cytokines as indicated. For peptides with IC50>10, hemolytic activity at 20 μM is shown. d, Hemolytic activity of peptides as a function of IC50 of peptides against TNF-α. Graph summarizing results obtained as described in Examples 1 and 2. For peptides with IC50>10 μM, the exact IC50 was unknown, but they were selected to show hemolytic activity at 50 μM. [Figure 20B]Selection of stapled peptides with improved anti-inflammatory activity is shown. a, Heat map showing hemolytic activity of peptides on whole blood or red blood cells (RBC). Data of experiments performed on red blood cells or blood from four different donors are displayed as averages (n=4). b, Heat map showing cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of different peptides. Results are displayed as averages. Blood from different donors was used each time (n=4). c, Graph obtained by combining data from (a) and (b) showing hemolytic activity of peptides as a function of IC50 of peptides against different cytokines as indicated. For peptides with IC50>10, hemolytic activity at 20 μM is shown. d, Hemolytic activity of peptides as a function of IC50 of peptides against TNF-α. Graph summarizing results obtained as described in Examples 1 and 2. For peptides with IC50>10 μM, the exact IC50 was unknown, but they were selected to show hemolytic activity at 50 μM. [Figure 20C]Selection of stapled peptides with improved anti-inflammatory activity is shown. a, Heat map showing hemolytic activity of peptides on whole blood or red blood cells (RBC). Data of experiments performed on red blood cells or blood from four different donors are displayed as averages (n=4). b, Heat map showing cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of different peptides. Results are displayed as averages. Blood from different donors was used each time (n=4). c, Graph obtained by combining data from (a) and (b) showing hemolytic activity of peptides as a function of IC50 of peptides against different cytokines as indicated. For peptides with IC50>10, hemolytic activity at 20 μM is shown. d, Hemolytic activity of peptides as a function of IC50 of peptides against TNF-α. Graph summarizing results obtained as described in Examples 1 and 2. For peptides with IC50>10 μM, the exact IC50 was unknown, but they were selected to show hemolytic activity at 50 μM. [Figure 20D]Selection of stapled peptides with improved anti-inflammatory activity is shown. a, Heat map showing hemolytic activity of peptides on whole blood or red blood cells (RBC). Data of experiments performed on red blood cells or blood from four different donors are displayed as averages (n=4). b, Heat map showing cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of different peptides. Results are displayed as averages. Blood from different donors was used each time (n=4). c, Graph obtained by combining data from (a) and (b) showing hemolytic activity of peptides as a function of IC50 of peptides against different cytokines as indicated. For peptides with IC50>10, hemolytic activity at 20 μM is shown. d, Hemolytic activity of peptides as a function of IC50 of peptides against TNF-α. Graph summarizing results obtained as described in Examples 1 and 2. For peptides with IC50>10 μM, the exact IC50 was unknown, but they were selected to show hemolytic activity at 50 μM. [Figure 21] Hemolytic and anti-inflammatory activity of K and R mutants of sKKW13. a, Heat map showing hemolytic activity of peptides on whole blood. Data from experiments performed on blood from 4 different donors are displayed as averages (n=4). b, Heat map showing cytokines released from human blood 24 hours after stimulation with 100 ng ml-1 E. coli LPS in the presence or absence of increasing concentrations of different peptides. Results are displayed as averages. Blood from different donors was used each time (n=4). [Figure 22A]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 22B]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 22C]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 22D]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 22E]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 22F]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 22G]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Fig. 22H]Intravenous administration of sHVF18 improved the sepsis-like state in pigs and prevented the progression of severe ARDS. The figures show the results of pigs with acute lung injury and ARDS treated with or without sHVF18. (a) The figure shows an overview of the experimental setup. Pigs were anesthetized with a ventilator and continuously monitored using an arterial line and a Swan-Ganz catheter. LPS was administered intravenously and treated animals were administered sHVF18. Hemodynamics, vitals, and pulmonary gas exchange were continuously followed during the experimental period. (b) Pulmonary gas exchange between treated pigs (n=5) and untreated pigs (n=5) as PaO2 FiO2 -1 ratio. All untreated animals developed mild to moderate ARDS. A significant increase in cardiac output (c), a significant decrease in urine output (d), a significant increase in the need for inotropic support (e), and a significant increase in lactate levels (f) were seen in untreated but not treated animals, indicating a severe stage of a sepsis-like condition. (g) Images representing n=15 samples of hematoxylin and eosin (H&E) histology of healthy control (n=5) (left), untreated (middle) (n=5) and sHVF18-treated (right) (n=5) lungs. Scale bar in large image represents 0.5 mm. Balloons indicate enlarged sections of tissue, scale bar represents 0.2 mm. (h) Results of cumulative blinded scoring of histology. Statistical significance between untreated and treated groups was tested by two-tailed Student's T-test and within groups by two-tailed ANOVA. *p<0.05, **p<0.01, ***p<0.001. All values ​​represent mean ± standard deviation. [Figure 23]In silico analysis of staple positions is shown. (a) HVF18 was docked to CD14 and the N-terminal GKYGFYT residues were modeled to form the GKY25 peptide. The binding energy of GKY25 to CD14 was calculated using MMPBSA. The indicated amino acids were replaced with pentenylalanine and staples were added to connect residues i and i+3 along the sequence of the peptide. MMPBSA was used to calculate the binding energy of the stapled peptide to CD14. The graph shows the difference in binding energy between non-stapled and stapled GKY25 at all staple positions. Positive values ​​indicate worse binding and negative values ​​indicate better binding of the stapled peptide. (b) A similar analysis was performed by adding a staple to connect residues i and i+4. (c and d) A similar analysis was performed for the shorter HVF18 peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition As used herein, the term "amino acid" refers to any amino acid, including any standard amino acid and non-standard amino acids.

[0020] The term "standard amino acid" as used herein refers to a proteinogenic amino acid. Preferably, the proteinogenic amino acid is one of the 20 amino acids encoded by the standard genetic code. The IUPAC one-letter code and three-letter code are used for the name of the amino acid.

[0021] As used herein, the term "covalent bond" between two side chains of amino acids refers to either a covalent bond between said side chains, or that said side chains are covalently attached to each end of a linker, such that all bonds connecting the side chains are covalent.

[0022] The term "hydrocarbon staple" as used herein refers to an alkyl or alkenyl moiety attached to an amino acid side chain. Typically, a "hydrocarbon staple" is a C 6~16 It is an alkenyl moiety.

[0023] The term "internal" as used herein with respect to an amino acid within a peptide refers to an amino acid that is not positioned as the most N-terminal or the most C-terminal amino acid in the primary sequence of the peptide.

[0024] The term "non-adjacent" as used herein with respect to amino acids in a peptide refers to two amino acids that are not located next to each other in the primary sequence of the peptide.

[0025] The term "position n" as used herein with respect to an amino acid in a peptide refers to the position in the primary sequence where the most N-terminal amino acid has position n.

[0026] As used herein, the term "stapled peptide" refers to a peptide that contains at least one covalent bond between the side chains of two non-adjacent internal amino acids. In particular, a stapled peptide may contain a hydrocarbon staple.

[0027] Peptides containing internal covalent bonds The present invention provides a peptide comprising a continuous sequence of 10 to 23 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, the peptide comprising: i) has a total length of 10 to 40 amino acids, preferably a total length of 10 to 23, more preferably a total length of 13 to 23; ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) further comprising at least the amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; However, when the peptide has a total length of 24-40 amino acids, it contains at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids, the amino acids of the first internal covalent bond are designated X1 and X2, and the amino acids of the second internal covalent bond are designated X3 and X4.

[0028] The present invention further provides a peptide comprising a continuous sequence of 10 to 40 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, the peptide comprising: i) has a total length of 10 to 40 amino acids, preferably a total length of 10 to 23, more preferably a total length of 13 to 23; ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) further comprising at least the amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; However, when the peptide has a total length of 24-40 amino acids, it contains at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids, the amino acids of the first internal covalent bond are designated X1 and X2, and the amino acids of the second internal covalent bond are designated X3 and X4.

[0029] The present invention further provides a peptide comprising or consisting of a consecutive sequence of amino acids derived from thrombin of SEQ ID NO: 1 containing up to six amino acid substitutions, said peptide comprising: i) having a total length of 10 to 23 amino acids; ii) comprising at least one covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being denoted X1 and X2; iii) further comprising at least amino acids K247, K248, K252 of thrombin of SEQ ID NO:1.

[0030] The present invention further provides a peptide comprising or consisting of a consecutive sequence of amino acids derived from thrombin of SEQ ID NO: 1 containing up to six amino acid substitutions, said peptide comprising: i) having a total length of 13 to 23 amino acids; ii) comprising at least one covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being denoted X1 and X2; iii) further comprising at least amino acids K247, K248, K252 of thrombin of SEQ ID NO:1.

[0031] In some embodiments, a peptide of the invention comprises at least amino acids R245, K247, K248 and K252 of the thrombin of SEQ ID NO:1.

[0032] The internal covalent bond may be as described herein below in the section "Internal Covalent Bonds."

[0033] Preferably, the peptides have one or more of the advantageous properties described herein above in the "Overview" section or below in the "Peptide Function" section.

[0034] The peptides are useful in the treatment of inflammation and / or infection, for example as described in the Methods of Treatment section.

[0035] The sequence of prothrombin is provided herein as SEQ ID NO: 16. Prothrombin contains the amino acid sequence Arg 271 This cleavage generates two fragments, known as fragments 1 and 2, which contain the first 271 residues of prothrombin and the intermediate prethrombin 2, which consists of residues 272 to 579. Fragment 1 and 2 is released as the activated peptide, and prethrombin 2 is released at the Arg 320 to generate active thrombin. The sequence of active thrombin is provided herein as SEQ ID NO:1.

[0036] internal covalent bond One feature of the peptides of the present invention is that they contain a covalent bond between the side chains of two non-adjacent internal amino acids. Thus, the covalent bond is either a direct covalent bond between the side chains of the amino acids, or the side chains are covalently linked to each other via a linker. In other words, a covalent bond in the peptide backbone is not considered as a "covalent bond between the side chains of two non-adjacent internal amino acids" according to the present invention.

[0037] Shorter peptides Although it is possible for a peptide to contain more than one such covalent bond, it is preferred that the peptide contains only one covalent bond between two non-adjacent internal amino acids when the peptide is between 10 and 23 amino acids. Amino acids having an internal bond between their side chains are also designated herein as X1 and X2.

[0038] In the peptides of the present invention, X1 and X2 are linked to each other by a covalent bond, but X1 and X2 may be described herein in their free, unlinked form. Those skilled in the art will understand that even if X1 and X2 are described in their unlinked form, they form a valid covalent bond in the final peptide of the present invention. As an example, X1 and X2 may each be described as "(S)-2-(4'-pentenyl)-alanine", but in the peptides of the present invention, the pentenyl group has reacted, typically by ring-closing metathesis, to form an 8-carbon long alkenyl linker with only one double bond.

[0039] Peptides that contain a covalent bond between two non-adjacent internal amino acids are also known as "stapled" peptides.

[0040] There are many different methods of forming stapled peptides known to those skilled in the art, and the peptides of the present invention can include any type of covalent bond between two non-adjacent internal amino acids useful for peptide stapling.For example, the peptides can include any of the staples described in Li et al., 2020 or International Patent Application No. WO2019018499, both of which are incorporated herein by reference in their entirety.

[0041] When amino acid X1 is located at position n, amino acid X2 is preferably located at position n+3, or position n+4, or position n+5, or position n+6, or position n+7, or position n+8, or position n+9, or position n+10, or position n+11, where n is an integer. More preferably, when amino acid X1 is located at position n, amino acid X2 is located at position n+3, or position n+4, or position n+7, or position n+11, where n is an integer. Even more preferably, when amino acid X1 is located at position n, amino acid X2 is located at position n+4, or position n+7, where n is an integer. The latter arrangement pattern is particularly useful for supporting the α-helical structure of the peptide. Typically, n is an integer in the range of 2 to 18, but n must be selected so that X1 is not located at the most N-terminus, and more preferably, neither X1 nor X2 is located at the most N-terminus or the most C-terminus.

[0042] In principle, X1 can be located at any position in the peptide except the most N-terminus. However, there may be cases where a particular position in the peptide is more advantageous than another position. The peptide of the present invention comprises a continuous sequence of amino acids from thrombin of SEQ ID NO: 1 or GKY25 of SEQ ID NO: 12. In the following, the position of the amino acid is given in relation to the amino acid numbering of GKY25 of SEQ ID NO: 12. Thus, after alignment, any amino acid having the same position as a particular amino acid in GKY25 of SEQ ID NO: 12 is said to "match" that amino acid in GKY25.

[0043] In one embodiment, after aligning the sequence of the peptide of the present invention with the sequence of GKY25 of SEQ ID NO: 12, a. X2 does not match Lys in GKY25 of SEQ ID NO: 12, and b. If X1 matches Lys, then it is preferred that X2 does not match Gln.

[0044] In one embodiment, after aligning the sequence of the peptide of the present invention with the sequence of GKY25 of SEQ ID NO: 12, i) X1 does not match Arg11 in GKY25 of SEQ ID NO: 12; ii) X1 does not match Lys14 in GKY25 of SEQ ID NO: 12; iii) X2 does not match Lys14 in GKY25 of SEQ ID NO: 12; iv) X2 does not match Lys18 in GKY25 of SEQ ID NO: 12; and v) If X1 matches Lys18 of SEQ ID NO: 12, then it is preferred that X2 does not match Gln22.

[0045] In one embodiment, the amino acid X1 is located at position n and the amino acid X2 is located at position n+3, where n is an integer ranging from 2 to 18, and after aligning the sequence of the peptide of the present invention with the sequence of GKY25 of SEQ ID NO: 12, i) X1 does not match Arg11 in GKY25 of SEQ ID NO: 12; ii) X1 does not match Lys14 in GKY25 of SEQ ID NO: 12; and iii) X2 does not match Lys14 in GKY25 of SEQ ID NO:12.

[0046] In one embodiment, the amino acid X1 is located at position n and the amino acid X2 is located at position n+4, where n is an integer ranging from 2 to 18, and after aligning the sequence of the peptide of the present invention with the sequence of GKY25 of SEQ ID NO: 12, i) X1 does not match Arg11 in GKY25 of SEQ ID NO: 12; ii) X1 does not match Leu12 in GKY25 of SEQ ID NO: 12; iii) X1 does not match Lys14 in GKY25 of SEQ ID NO: 12; iv) X2 does not match Lys14 in GKY25 of SEQ ID NO: 12; v) X1 does not match Lys18 in GKY25 of SEQ ID NO: 12; and vi) X2 does not match Lys18 in GKY25 of SEQ ID NO:12.

[0047] In a preferred embodiment, the amino acid X1 is located at position n, and the amino acid X2 is located at position n+3, where n is an integer ranging from 2 to 18. When aligning the sequence of the peptide of the present invention with the sequence of GKY25 of SEQ ID NO: 12, X1 and X2 are located at position n+3, where n is an integer ranging from 2 to 18. Val9 and Leu12; or Phe10 and Lys13; or Leu12 and Trp15; or Lys13 and Ile16; or Ile16 and Val19; or Gln17 and Ile20; or Lys18 and Asp21; or Val19 and Gln22; or Ile20 and Phe23; or Asp21 and Gly24; or Corresponding to Gln22 and Glu25.

[0048] In another preferred embodiment, the amino acid X1 is located at position n, and the amino acid X2 is located at position n+4, where n is an integer ranging from 2 to 18. When aligning the sequence of the peptide of the present invention with the sequence of GKY25 of SEQ ID NO: 12, X1 and X2 are located at position n+4, where n is an integer ranging from 2 to 18. Val9 and Lys13; or Lys13 and Gln17; or Trp15 and Val19; or Ile16 and Ile20; or Gln17 and Asp21; or Val19 and Phe23; or Ile20 and Gly24; or Corresponding to Asp21 and Glu18.

[0049] In a highly preferred embodiment, amino acid X1 is located at position n and amino acid X2 is located at position n+4, where n is an integer ranging from 2 to 18, and when the sequence of the peptide of the present invention is aligned with the sequence of GKY25 of SEQ ID NO: 12, X1 and X2 correspond to Gln17 and Asp21, respectively.

[0050] In one embodiment, X1 and X2 are standard amino acids prior to reaction to form a covalent bond. For example, X1 and X2 may be selected from the group consisting of the following prior to reaction to form a covalent bond: i) X1 is Lys and X2 is selected from the group consisting of Asp, Glu, Lys, Cys and Tyr; ii) X1 is Cys and X2 is selected from the group consisting of Cys, Lys and Met; iii) X1 is Asp and X2 is Lys; iv) X1 is Glu and X2 is selected from the group consisting of Lys and Glu; v) X1 is Tyr and X2 is selected from the group consisting of Lys, Phe and Trp; vi) X1 is Met and X2 is selected from the group consisting of Met and Cys; vii) X1 is His and X2 is His; viii) X1 is Phe and X2 is selected from the group consisting of Phe, Tyr, Ala and Trp; ix) X1 is Ala and X2 is Phe or Tyr; x) X1 is Trp and X2 is selected from the group consisting of Trp, Phe and Tyr.

[0051] In one embodiment, X1 and X2, prior to reaction to form the covalent bond, can be as follows: X1 is Lys and X2 is Asp, Glu, Cys or Lys, or vice versa; X1 and X2 are Cys.

[0052] The covalent bond may be formed by direct reaction between the side chains of standard amino acids or through a cross-linking agent. When X1 and X2 are Cys, the covalent bond may be a disulfide bridge or may be formed through a cross-linking agent, for example a bis-alkylating agent such as a linker containing at least two (bromomethyl) substituents.

[0053] In a preferred embodiment, X1 and X2 are derivatized standard amino acids. Prior to reaction to form the covalent bond, X1 and / or X2 can be selected from the group consisting of, for example, Ser derivatives and Ala derivatives.

[0054] In one embodiment, the covalent bond is formed by linking two non-standard amino acids. For example, the covalent bond can be formed by linking two non-standard amino acids that replace two natural amino acids in a consecutive sequence derived from thrombin.

[0055] In one embodiment, the covalent bond is a hydrocarbon staple.

[0056] In a preferred embodiment, X1 and X2 prior to reaction to form a covalent bond are alkenylated amino acids, such as two α-substituted alkenyl amino acids and / or two C-alkenylated amino acids, such as α,α-disubstituted alkenyl amino acids, and the covalent bond is an olefinic tether formed between said alkenyl residues.

[0057] The alkenylated amino acid can be an amino acid native to alkenylated thrombin, or alternatively, the alkenylated amino acid can be an amino acid that substitutes for an amino acid native to thrombin.

[0058] In a preferred embodiment, X1 and X2, prior to reaction to form a covalent bond, may be individually selected from the group consisting of alkenyl Ala, alkenyl Leu, alkenyl Met, alkenyl Ser, alkenyl Tyr, alkenyl Lys, alkenyl Arg, and alkenyl Phe. In such cases, the covalent bond is an olefin tether formed between said alkenyl residues.

[0059] In a preferred embodiment, one of X1 and X2 prior to reaction to form a covalent bond can be an alkenylated Ala and the other can be independently selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe. In such a case, the covalent bond is an olefinic tether formed between said alkenyl residues.

[0060] In a preferred embodiment, X1 and X2 are α-alkenyl olefin terminal amino acids and / or α,α-disubstituted alkenyl olefin terminal amino acids.

[0061] In a preferred embodiment, X1 and X2 prior to reaction to form a covalent bond can be an alkenylated alanine, preferably an α-substituted alkenyl or an α,α-disubstituted alkenylated alanine, in which case the covalent bond is an olefin tether formed between the alkenyl residues.

[0062] In a preferred embodiment, X1 and X2 prior to reaction to form a covalent bond can be an alkenylated Ser, such as an O-alkenylated Ser.

[0063] The alkenylated amino acid comprises 2-10 carbons in the alkenyl chain, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons, preferably 4, 5 or 6 carbons. The alkenylated amino acid may comprise one or more double bonds, but preferably comprises only one double bond. It is further preferred that the double bond is located at the free end of the alkenyl. Two double bonds located at the free ends of two alkenyl residues can be reacted by RCM to form an olefin tether.

[0064] Thus, X1 and X2 are preferably amino acids linked by an olefin tether formed between the alkenyl residues. 8~14 Alkenyl etc. 6~16 Alkenyl, for example, C or C 14 It may be an alkenyl tether. The tether may contain one or more double bonds, preferably one double bond.

[0065] In a preferred embodiment, X and X prior to reaction to form a covalent bond. 2は , α,α-disubstituted S- or R-pentenylalanine (S5 or R5), or S- or R-octenylalanine (S8 or R8) alanine. Thus, an internal carbohydrate staple can be formed by joining two α,α-disubstituted S- or R-pentenylalanine (S5 or R5), or S- or R-octenylalanine (S8 or R8) alanines.

[0066] In a preferred embodiment, X1 and X2 prior to reaction to form a covalent bond can be (S)-2-(4'-pentenyl)-alanine. Thus, an internal carbohydrate staple can be formed by linking two (S)-2-(4'-pentenyl)-alanines.

[0067] In one embodiment, the covalent bond is established through a ring closure, such as ring closing metathesis (RCM).

[0068] In a preferred embodiment, one of X1 and X2 prior to reaction to form a covalent bond can be a non-standard azido-terminal amino acid and the other can be a non-standard in-terminal amino acid.

[0069] Longer peptides Peptides of the invention that are 24 amino acids or more preferably comprise at least two staples, in other words, peptides of the invention that are 24 amino acids or more, such as 24-40 amino acids, preferably comprise at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids, the first internal covalent bond amino acids being designated X1 and X2 and as described above, and the second internal covalent bond amino acids being designated X3 and X4.

[0070] It is also preferred that the peptide comprising the four most N-terminal amino acids of GKY25 of SEQ ID NO: 12 also comprises at least two staples, the first internal covalently bonded amino acids being designated X1 and X2 and as described above, and the second internal covalently bonded amino acids being designated X3 and X4.

[0071] X3 and X4 may be as described above in the section "shorter peptide". Preferably, X3 and X4 are closer to the N-terminus than X1 and X2. In particular, X3 and X4 may be positioned such that X3 is located at one of the first four amino acids. When the peptide comprises the most N-terminal four amino acids of GKY25 of SEQ ID NO: 12, it is preferred that X3 is located at a position corresponding to one of the most N-terminal four amino acids of GKY25 of SEQ ID NO: 12.

[0072] In some embodiments, amino acid X3 is located at position n and amino acid X4 is located at position n+3, or position n+4, or position n+5, where n is an integer. In preferred embodiments, amino acid X3 is located at position n and amino acid X4 is located at position n+4, where n is an integer in the range of 1 to 18, preferably in the range of 1 to 10, more preferably in the range of 1 to 5. Thus, while X1 is preferably not located at the most N-terminus, X3 may be located at the most N-terminus.

[0073] When X3 is located at the extreme N-terminus, a staple may be formed between the N-terminal -NH2 group and the side chain of an amino acid, preferably the side chain of Glu or Asp, more preferably the side chain of Glu.

[0074] In a particular embodiment, amino acid X3 is located at the most N-terminus of the peptide. In a preferred embodiment, amino acid X3 is located at the most N-terminus, and amino acid X4 is located at the n+4 position. In a highly preferred embodiment, when the sequence of the peptide of the present invention is aligned with the sequence of GKY25 of SEQ ID NO: 12, X3 and X4 correspond to Gly1 and Phe5 of GKY25 of SEQ ID NO: 12, and Phe5 is replaced with either Glu or Asp, preferably Glu. The latter position pattern of X3 and X4 is particularly useful for protecting or masking the protease site of the peptide.

[0075] The covalent bond between X3 and X4 can be, for example, an amide bond formed between a carboxylic acid moiety and an amine. In some embodiments, the carboxylic acid moiety is a glutamic acid side chain. In some embodiments, the amine is an amino acid side chain. In other embodiments, the amine is the N-terminal amine group of the peptide backbone. In some embodiments, the covalent bond between X3 and X4 is a lactam bridge formed between the N-terminal amine group and the side chain carboxylic acid of the respective amino acid.

[0076] In a preferred embodiment, Phe5 of GKY25 of SEQ ID NO: 12 is replaced by Glu5. In such an embodiment, X3 and X4 correspond to Gly1 and Glu5 of SEQ ID NO: 14.

[0077] Peptide properties The peptides of the invention may have one or more of the following properties.

[0078] The present invention shows that longer stapled thrombin-derived peptides, such as GKY25, which contain a single staple, have some less desirable properties. Thus, GKY25, which contains one staple, has high hemolytic activity in blood and low anti-inflammatory activity, making it unusable in vivo.

[0079] In contrast, stapled shorter thrombin-derived peptides, such as stapled HVF18, have relatively low hemolytic activity in blood and high anti-inflammatory activity in blood.

[0080] Furthermore, longer thrombin-derived peptides with double staples, such as GKY25 with two staples, have lower hemolytic activity in blood compared to single-stapled GKY25 and higher stability compared to non-stapled GKY25.

[0081] Shorter peptides Therefore, the peptide of the present invention preferably has a suitable length, and preferably has a length of 10 to 23 amino acids, such as 13 to 18 amino acids, such as 12 to 22 amino acids, such as 14 to 22 amino acids, such as 15 to 21 amino acids, such as 16 to 20 amino acids, such as 17 to 20 amino acids.

[0082] In particular, the peptide may have a length of 18 to 20 amino acids, such as 18 or 19 amino acids.

[0083] The length of a peptide indicates the total length of the peptide. Thus, even though the peptide may be linked to one or more additional moieties, it is preferred that the peptide does not contain more than the number of amino acids indicated.

[0084] Furthermore, it is also preferred that the peptide of the present invention comprises a consecutive amino acid sequence having an appropriate length derived from thrombin of SEQ ID NO: 1. Thus, preferably, the peptide of the present invention comprises or consists of a consecutive amino acid sequence of a range of 10 to 23 amino acids, for example, 13 to 23 amino acids, for example, 13 to 18 amino acids, for example, 14 to 22 amino acids, for example, 15 to 21 amino acids, for example, 16 to 20 amino acids, for example, 17 to 20 amino acids, preferably 17 to 18 amino acids, more preferably 18 to 19 amino acids, derived from thrombin of SEQ ID NO: 1.

[0085] The peptide according to the invention comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1, where up to 6 amino acids may be exchanged. In other words, the peptide may comprise or consist of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1, but 1 to 6 of the amino acids within said consecutive sequence may be replaced by another amino acid. However, it is important that the peptide comprises at least the amino acids K247, K248, K252 of thrombin of SEQ ID NO: 1. In other words, said amino acids should not be replaced.

[0086] In some embodiments, the peptide comprises at least amino acids R245, K247, K248 and K252 of the thrombin of SEQ ID NO:1.

[0087] The peptide according to the present invention has in particular a total length of 10-23 amino acids and comprises or consists of a consecutive sequence of amino acids from GKY25 of SEQ ID NO: 12, where up to 6 amino acids may be exchanged. In other words, the peptide may comprise or consist of a consecutive sequence of amino acids from GKY25 of SEQ ID NO: 12, but 1 to 6 of the amino acids within said consecutive sequence may be replaced by another amino acid. However, it is important that the peptide at least comprises amino acids K13, K14 and K18 of GKY25 of SEQ ID NO: 12. In other words, said amino acids should not be replaced.

[0088] In some embodiments, the peptide comprises at least amino acids R11, K13, K14, and K18 of GKY25 of SEQ ID NO:12.

[0089] The peptide according to the present invention preferably comprises or consists of a continuous sequence of amino acids derived from thrombin of SEQ ID NO: 1 in the range of 16 to 21 amino acids, more preferably in the range of 17 to 18 amino acids.

[0090] The consecutive sequence may comprise up to 6 amino acid substitutions.For example, the peptide may comprise at least 2 amino acid substitutions, for example 3 amino acid substitutions, for example 4 amino acid substitutions, for example 5 amino acid substitutions, compared with the consecutive sequence of thrombin.Preferably, the consecutive sequence may comprise up to 4 amino acid substitutions, and even more preferably, the consecutive sequence may comprise up to 2 amino acid substitutions.One or more of the substitutions may be conservative substitutions, for example 1, 2, 3 or 4 amino acid substitutions may be conservative substitutions.

[0091] In a highly preferred embodiment, the peptide comprises or consists of a consecutive sequence of amino acids from thrombin according to SEQ ID NO: 1, in which one amino acid is substituted with an amino acid X1 (e.g. any of the X1s described in the "Internal Covalent Bond" section) and another amino acid is substituted with an X2 (e.g. any of the X2s described in the "Internal Covalent Bond" section), said substituted amino acids being preferably positioned relative to each other as described for amino acids X1 and X2 in the "Internal Covalent Bond" section.

[0092] Furthermore, in addition to the continuous sequence of SEQ ID NO: 1, the peptides of the invention may comprise one or more additional amino acids. Preferably, the peptides may comprise up to four additional amino acids, e.g., up to three additional amino acids, such as two additional amino acids. The additional amino acids may, for example, be any of the amino acids described below under "Positively charged amino acids."

[0093] In one embodiment, a peptide according to the invention comprises or consists of a contiguous sequence of amino acids from thrombin of SEQ ID NO: 1, ranging from 17 to 18 amino acids, including up to two amino acid substitutions, and the peptide may include up to four additional amino acids.

[0094] In one embodiment, the peptide according to the invention comprises or consists of a contiguous sequence of amino acids from thrombin of SEQ ID NO: 1 ranging from 17 to 18 amino acids, including one amino acid substitution for amino acid X1 and one amino acid substitution for amino acid X2, and the peptide may comprise up to four additional amino acids.

[0095] Thus, in one embodiment, the peptide of the invention consists of 15-20 contiguous amino acids of thrombin of SEQ ID NO: 1, with two amino acids substituted with X1 and X2, where X1 and X2 are alkenylated amino acids forming an internal carbohydrate staple, in the range of 2-5, preferably 2 additional N-terminal amino acids.

[0096] In one embodiment, the peptide of the invention consists of 16-18 contiguous amino acids of thrombin of SEQ ID NO:1, with two amino acids replaced with X1 and X2, where X1 and X2 are alkenylated amino acids forming an internal carbohydrate staple, and in the range of 2-5 additional N-terminal amino acids, preferably 2 additional N-terminal amino acids.

[0097] In one embodiment, the peptide of the invention consists of 17 consecutive amino acids of thrombin of SEQ ID NO: 1, with two amino acids replaced with X1 and X2, where X1 and X2 are alkenylated amino acids that form an internal carbohydrate staple, and in the range of 2-3 additional N-terminal amino acids.

[0098] The additional N-terminal amino acid is preferably a positively charged amino acid, such as Lys or Arg.

[0099] The peptides of the present invention may further comprise one or more moieties attached to the peptide. The moieties may optionally be linked to the peptide via a linker. The one or more attached moieties may, for example, be selected from the group consisting of alkyl, aryl, heteroaryl, olefin, fatty acid, polyethylene glycol (PEG), saccharide, and polysaccharide.

[0100] Longer peptides However, the peptides of the invention may be longer, in some embodiments the peptides of the invention have a length of 24 to 40 amino acids, such as 25 to 35 amino acids, such as 25 to 30 amino acids, for example 28 to 34 amino acids.

[0101] In particular, the peptide may have a length of 24 to 28 amino acids, such as 25 or 26 amino acids.

[0102] Peptides of the invention that are 24 amino acids or longer contain at least two staples.

[0103] The peptide of the present invention having a length of 24 to 40 amino acids comprises a consecutive sequence of amino acids derived from thrombin of SEQ ID NO: 1. Thus, preferably, the longer peptide of the present invention comprises or consists of a consecutive sequence of amino acids derived from thrombin of SEQ ID NO: 1 in the range of 24 to 40 amino acids, such as 25 to 35 amino acids, for example 25 to 30 amino acids, for example 28 to 34 amino acids, preferably 24 to 28 amino acids, even more preferably 25 and 26 amino acids.

[0104] Longer peptides of the invention may be as defined above in the section "Peptide Characteristics - Shorter Peptides".

[0105] Thus, the longer peptides according to the invention comprise or consist of a continuous sequence of amino acids from thrombin of SEQ ID NO: 1, with up to six amino acids being exchanged. However, it is important that the peptide comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1. In other words, said amino acids should not be substituted. In some embodiments, the longer peptides according to the invention comprise R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0106] In a highly preferred embodiment, the peptide comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1, in which one amino acid is substituted with amino acid X1 (e.g., any of the X1s described in the "Internal Covalent Bond" section), another amino acid is substituted with X2 (e.g., any of the X2s described in the "Internal Covalent Bond" section), another amino acid is substituted with X3 (e.g., any of the X3s described in the "Internal Covalent Bond" section), and another amino acid is substituted with X4 (e.g., any of the X4s described in the "Internal Covalent Bond" section). The substituted amino acids are preferably positioned relative to each other as described for amino acids X1, and X2, and X3, and X4 in the "Internal Covalent Bond" section.

[0107] Positively charged amino acids Interestingly, the present invention shows that the anti-inflammatory effect of a peptide can be significantly enhanced by the insertion of one or more positively charged amino acids.

[0108] Thus, in a preferred embodiment, the peptide according to the invention comprises 1 to 5 (such as 1 to 4), for example 1 to 3, for example 1 to 2 (such as 2), positively charged amino acids inserted at or near the termini of the peptide.

[0109] In particular, the positively charged amino acid may be inserted at or near the N-terminus, such as at a position selected from positions 1, 2, 3, 4 and / or 5 relative to the N-terminus of the peptide.

[0110] In a preferred embodiment, a positively charged amino acid is inserted at the N-terminus.

[0111] In one embodiment, the peptide of the invention comprises two positively charged amino acids inserted at or near the N-terminus, such as at positions selected from 1, 2, and / or 3 relative to the N-terminus of the peptide.

[0112] The positively charged amino acid may preferably be selected from the group consisting of arginine, lysine and histidine. Preferably, the positively charged amino acid is arginine and / or lysine, and even more preferably, the positively charged amino acid is lysine.

[0113] Peptide sequence In some embodiments of the invention, the peptide may have one of the peptide sequences described in this section of the specification. In addition to peptides having the sequences described in this section, it is also preferred that the peptide is: Contains a continuous sequence of 10 to 23 amino acids, preferably 13 to 23 amino acids, derived from thrombin of SEQ ID NO: 1 -Having a total length of 10 to 40 amino acids containing at least the amino acids K247, K248 and K252 of the thrombin of SEQ ID NO: 1, more preferably containing at least the amino acids R245, K247, K248 and K252 of the thrombin of SEQ ID NO: 1 provided, however, that if the total length of the peptide is between 24 and 40 amino acids, it contains at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids.

[0114] Shorter peptides In some embodiments of the invention, the peptide may have one of the peptide sequences described in this section of the specification. In addition to peptides having the sequences described in this section, it is also preferred that the peptide is: Contains a continuous sequence of 10 to 23 amino acids, preferably 13 to 23 amino acids, derived from thrombin of SEQ ID NO: 1 having a total length of 10 to 23 amino acids, preferably 13 to 23 amino acids comprising at least the amino acids K247, K248 and K252 of the thrombin of SEQ ID NO:1, more preferably comprising at least the amino acids R245, K247, K248 and K252 of the thrombin of SEQ ID NO:1.

[0115] In some embodiments of the invention, the peptide may have one of the peptide sequences described in this section of the specification. In addition to peptides having the sequences described in this section, it is also preferred that the peptide is: -Containing a continuous sequence of 10 to 23 amino acids, preferably 13 to 23 amino acids, derived from GKY25 of SEQ ID NO: 12 having a total length of 10 to 23 amino acids, preferably 13 to 23 amino acids - comprising at least amino acids K13, K14, and K18 of GKY25 of SEQ ID NO:12, more preferably comprising at least amino acids R11, K13, K14, and K18 of GKY25 of SEQ ID NO:12.

[0116] X1 and X2 in the sequences in this section can be, for example, as described herein above in the "Internal Covalent Bonds" section.

[0117] U in the sequences in this section can be, for example, His, Arg or Lys, preferably Arg or Lys.

[0118] The Z in the sequences in this section can individually be any standard amino acid. In a preferred embodiment, most or all of the Z in the sequence are selected to correspond to the amino acids of GKY25 in SEQ ID NO:12.

[0119] In one embodiment, the peptide according to the invention has the sequence: -KKZZ-X1-KZZ-X2-Z wherein Z is any standard amino acid, X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0120] In one embodiment, the peptide according to the invention has the amino acid sequence: -UU-(Z) n -IQKVIDQ-(Z) m - comprising or consisting of: The peptide has a total length of 10 to 23 amino acids, each Z is individually any standard amino acid; U is His, Lys or Arg; n is an integer ranging from 0 to 10; m is an integer ranging from 0 to 5; Two of the amino acids are replaced with alkenylated amino acids, the side chains of which are covalently attached.

[0121] In one embodiment, the peptide according to the invention has the sequence: UU-(Z) n -KKZZ-X1-KZZ-X2-Z Or the sequence: UUZZRZKKZZ-X1-KZZ-X2-Z comprising or consisting of: Z is any standard amino acid, U is His, Lys or Arg; n is an integer ranging from 0 to 10; X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0122] In one embodiment, the peptide according to the invention has the sequence: UVFRLKKWI-X1-KVI-X2-ZFGZ comprising or consisting of: Z is any standard amino acid, U is His, Lys or Arg; X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0123] In one embodiment, the peptide according to the invention has the sequence: VFRLKKWI-X1-KVI-X2-QFGE comprising or consisting of: X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0124] In one embodiment, the peptide has the sequence: UUVFRLKKWI-X1-KVI-X2-ZFGZ comprising or consisting of: Z is any standard amino acid, U is His, Lys or Arg; X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0125] In one embodiment, the peptide has the sequence: UUVFRLKKWI-X1-KVI-X2-QFGE comprising or consisting of: U is His, Lys or Arg; X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0126] In one embodiment of the invention, the peptide comprises i) SEQ ID NO:3, ii) SEQ ID NO:4, iii) SEQ ID NO:5, iv) SEQ ID NO:6, v) SEQ ID NO: 7, vi) SEQ ID NO:8, vii) SEQ ID NO: 9, viii) SEQ ID NO: 10, ix) SEQ ID NO: 11, x) SEQ ID NO: 17, xi) SEQ ID NO: 18, xii) SEQ ID NO: 19, xiii) SEQ ID NO: 20, xiv) SEQ ID NO: 21, xv) SEQ ID NO: 22, xvi) SEQ ID NO: 23, xvii) SEQ ID NO: 24, or xviii) SEQ ID NO: 25 wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0127] In one embodiment of the invention, the peptide comprises i) SEQ ID NO:3, ii) SEQ ID NO:4, iii) SEQ ID NO:5, iv) SEQ ID NO:8, or v) SEQ ID NO: 9 wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0128] In one embodiment, the peptide is i) SEQ ID NO:3, ii) SEQ ID NO:4, iii) SEQ ID NO:5, iv) SEQ ID NO:8, or v) SEQ ID NO: 9 wherein X1 and X2 are (S)-2-(4'-pentenyl)-alanine, the side chains of which have reacted with each other to form an alkenyl tether.

[0129] In one embodiment, the peptide is i) SEQ ID NO:3, ii) SEQ ID NO:4, iii) SEQ ID NO:5, iv) SEQ ID NO:8, or v) SEQ ID NO: 9 wherein X1 and X2 are Ala and the side chains are covalently linked to each other by a C8 alkenyl tether containing one double bond.

[0130] In one embodiment, the peptide is i) SEQ ID NO:3, ii) SEQ ID NO:5 iii) SEQ ID NO:6, or iv) SEQ ID NO: 7 wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0131] In one embodiment, the peptide is v) SEQ ID NO:3, vi) SEQ ID NO:5, vii) SEQ ID NO:6, or viii) SEQ ID NO: 7 wherein X1 and X2 are (S)-2-(4'-pentenyl)-alanine, the side chains of which have reacted with each other to form an alkenyl tether.

[0132] In one embodiment, the peptide is ix) SEQ ID NO:3, x) SEQ ID NO:5, xi) SEQ ID NO:6, or xii) SEQ ID NO: 7 wherein X1 and X2 are Ala and the side chains are covalently linked to each other by a C8 alkenyl tether containing one double bond.

[0133] In one embodiment, the peptide is i) SEQ ID NO: 3, or ii) SEQ ID NO:5 wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0134] In one embodiment, the peptide is i) SEQ ID NO: 3, or ii) SEQ ID NO:5 wherein X1 and X2 are (S)-2-(4'-pentenyl)-alanine, the side chains of which have reacted with each other to form an alkenyl tether.

[0135] In one embodiment, the peptide is i) SEQ ID NO: 3, or ii) SEQ ID NO:5 wherein X1 and X2 are Ala and the side chains are covalently linked to each other by a C8 alkenyl tether containing one double bond.

[0136] Longer peptides In some embodiments of the invention, the peptides of the invention are longer peptides containing at least two staples and 24-40 amino acids from thrombin of SEQ ID NO: 1 and may have one of the peptide sequences described in this section of the specification. In addition to peptides having the sequences described in this section, it is also preferred that the peptides are: Contains a continuous sequence of 24 to 40 amino acids, preferably 25 to 30 amino acids, derived from thrombin of SEQ ID NO: 1 having a total length of 24 to 40 amino acids, preferably 25 to 30 amino acids comprising at least the amino acids K247, K248 and K252 of SEQ ID NO:1, preferably comprising at least the amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO:1.

[0137] In some embodiments of the invention, the peptides of the invention are longer peptides that include at least two staples. In addition to peptides having the sequences described in this section, it is also preferred that the peptides are: -Containing a continuous sequence of 24 to 25 amino acids derived from GKY25 of SEQ ID NO:12 having a total length of 24 to 40 amino acids, preferably 25 to 30 amino acids - comprising at least amino acids K13, K14, and K18 of GKY25 of SEQ ID NO:12, preferably comprising at least amino acids R11, K13, K14, and K18 of GKY25 of SEQ ID NO:12.

[0138] In a preferred embodiment, the longer peptides of the invention include i) SEQ ID NO: 14, or ii) SEQ ID NO: 26 wherein X1 and X2 are amino acids whose side chains are covalently linked, and further wherein X3 and X4 are amino acids whose side chains are covalently linked. X1, X2, X3 and X4 in the sequences of this section can be, for example, as described herein above in the "Internal Covalent Bond" section. Preferably, X1 and X2 are (S)-2-(4'-pentenyl)-alanine whose side chains have reacted with each other to form an alkenyl tether, and X3 and X4 are Gly and Glu, respectively, which have reacted with each other to form a lactam bridge.

[0139] Peptide Function As described herein above, a peptide may have one or more of the following functions:

[0140] High in vivo and / or in vitro stability Preferably, the peptide has high in vivo stability. Thus, the peptide according to the invention preferably has increased stability in vivo and / or in vitro compared to a peptide having the same sequence except that the amino acids X1 and X2 are replaced by other amino acids lacking a covalent bond, when said peptide is tested under the same conditions.

[0141] In vivo stability can be determined, for example, by determining anti-inflammatory activity in mice after administration of the peptide over time. If significant anti-inflammatory activity is maintained, for example, after 24 hours, the peptide has high in vivo stability.

[0142] In particular, the anti-inflammatory effect of the peptides may be preferably maintained in vivo for 24 hours following systemic administration of the peptides.

[0143] In vivo stability can be determined, for example, as described in Example 1 herein below.

[0144] Increased protease resistance Preferably, the peptide of the present invention has increased protease resistance, and thus may have increased resistance to one or more proteases compared to a peptide having the same sequence except that amino acids X1 and X2 have been replaced by other amino acids lacking a covalent bond.

[0145] Preferably, the peptides of the invention have increased stability in the presence of proteases, such as human neutrophil elastase (HNE), Pseudomonas elastase (PE), and / or trypsin, when compared to peptides of the same sequence except that amino acids X1 and X2 are replaced by other amino acids lacking a covalent bond, when said peptides are tested under the same conditions.

[0146] Said resistance to proteases can be determined, for example, as described in Example 1 below.

[0147] High anti-inflammatory activity Preferably, the peptides of the present invention have high anti-inflammatory activity. Even more preferably, the peptides of the present invention have both antibacterial and anti-inflammatory activity.

[0148] Anti-inflammatory activity can be determined in a variety of ways, particularly by inducing inflammation in a controlled manner and determining whether inflammation is reduced. Inflammation can be induced, for example, by contacting reporter cells or blood samples with LPS in vitro, or by administering LPS to animals. Inflammation can be determined, for example, by measuring the level of one or more proinflammatory cytokines, or by determining NF-κB activity.

[0149] Thus, the peptides according to the invention preferably reduce the secretion of proinflammatory cytokines in the presence of one or more endotoxins, such as LPS, and in particular preferably reduce the secretion of proinflammatory cytokines in blood containing one or more endotoxins, such as LPS, in vivo.

[0150] The proinflammatory cytokine may, for example, be selected from the group consisting of tumor necrosis factor alpha (TNF-α), interleukin beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interferon (IFN-γ) and / or monocyte chemotactic protein-1 (MCP-1). Preferably, the proinflammatory cytokine is TNF-α and / or IL-1β.

[0151] Preferably, the peptides according to the invention reduce NF-κB activity in the presence of toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), Staphylococcus aureus peptidoglycan (SA-PGN) and / or zymosan.

[0152] Preferably, a peptide of the invention at a concentration of 10 μM reduces the secretion of TNF-α and / or IL-1β after incubation in fresh blood in the presence of LPS by at least 50%, such as at least 60%, for example by at least 70%, compared to a peptide of the same sequence except that amino acids X1 and X2 are replaced by other amino acids lacking a covalent bond, when said peptide is tested under the same conditions.

[0153] In one embodiment, the peptide of the present invention can reduce LPS-stimulated blood TNF-α release by 50% at a concentration of less than 10 μM (such as less than 7 μM), for example, in the range of 1 to 10 μM (such as a concentration in the range of 1 to 7 μM).

[0154] Anti-inflammatory activity may be tested by one of the methods described in Examples 1 and 2 herein below.

[0155] Antibacterial activity The peptides of the invention preferably have antibacterial activity. Although the peptides may not have improved antibacterial activity compared to other thrombin-derived peptides, it is preferred that they have at least some antibacterial activity in combination with increased anti-inflammatory activity.

[0156] Therefore, the peptide of the present invention may have bactericidal activity against gram-negative bacteria and / or gram-positive bacteria. Interestingly, the peptide of the present invention has been shown to be effective against gram-negative bacteria. Without being bound by theory, it is believed that this effect is brought about by the binding of the peptide to LPS and / or other membrane structures. The peptide of the present invention has also been shown to be effective against gram-positive bacteria. Without being bound by theory, it is believed that this effect is brought about by the binding of LTA and / or other membrane structures.

[0157] Thus, the peptides may have bactericidal activity mediated through the peptide that can kill bacteria by damaging the bacterial membrane.

[0158] In particular, the peptide of the present invention preferably has a bactericidal effect against one or more bacteria selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Preferably, the peptide has a bactericidal effect against all of the aforementioned bacteria.

[0159] Antibacterial or bactericidal activity can be determined, for example, by radial diffusion assay or viable count assay, which can be performed, for example, as described in Examples 1 and 2 herein below.

[0160] Low hemolytic activity in blood It is highly preferred that the peptides of the invention have low hemolytic activity in blood. Peptides with high hemolytic activity may be toxic and therefore less suitable for systemic administration.

[0161] Generally, a maximum hemolytic activity of 10% is acceptable. Therefore, the hemolytic activity of the peptide of the present invention is preferably less than 10%, and in particular, the hemolytic activity of the peptide of the present invention in fresh whole blood is preferably less than 10%.

[0162] More preferably, the hemolytic activity of the peptide of the present invention is less than 10% at a peptide concentration that has effective anti-inflammatory activity. In a preferred embodiment, the peptide of the present invention has a hemolytic activity of less than 10% at a peptide concentration that reduces the anti-inflammatory activity by 50%. The anti-inflammatory activity is preferably determined as the release of TNF-α in blood stimulated with LPS.

[0163] Therefore, the hemolytic activity of the peptide of the present invention is preferably less than 10% at a peptide concentration capable of reducing the release of TNF-α in LPS-stimulated blood by 50%.

[0164] In one embodiment, the hemolytic activity of the peptides of the invention is preferably less than 5% at peptide concentrations capable of reducing LPS-stimulated blood TNF-α release by 50%.

[0165] The release of TNF-α in blood stimulated with LPS may preferably be determined as described in Example 1 or 2 herein below.

[0166] In some embodiments, when 50 μM of the peptide is incubated in fresh whole blood, the hemolytic activity is at most 5%, such as at most 4%, for example at most 3%, such as at most 2%.

[0167] In some embodiments, when 20 μM of the peptide is incubated in 25% fresh whole blood, the hemolytic activity is up to 5%, such as up to 4%.

[0168] Hemolytic activity is preferably determined by incubating fresh blood with the peptides of the invention, determining hemolysis, and comparing it to the hemolysis of fresh blood in a control prepared by incubating fresh blood with a detergent such as Tween-20. Preferably, hemolytic activity is provided as % hemolysis compared to the control.

[0169] Hemolytic activity may in particular be determined as described in Examples 1 and 2 herein below.

[0170] low toxicity The peptides of the invention also preferably have low toxicity, allowing for systemic administration of the peptides.

[0171] structure The peptides of the invention preferably have a substantially alpha-helical secondary structure in aqueous solution, which is preferably maintained upon binding to a target molecule, such as cluster differentiation 14 (CD14).

[0172] The alpha-helical secondary structure in aqueous solution is preferably determined by circular dichroism spectroscopy.

[0173] Furthermore, it is preferred that the peptides have a low degree of oligomerization. Hydrodynamic radius is an indicator of oligomerization, and therefore it is preferred that the peptides of the present invention have a low hydrodynamic radius. Preferably, the peptides of the present invention have a hydrodynamic radius of less than 150 nm at pH 7.4 and / or less than 100 nm at pH 5. In some embodiments, it is preferred that the peptides of the present invention have a hydrodynamic radius of less than 100 nm at pH 7.4 and / or less than 80 nm at pH 5.

[0174] The hydrodynamic radius is preferably determined by dynamic light scattering, as described in Example 4 below.

[0175] anticoagulant activity The peptides of the present invention may also have anticoagulant activity. Thus, preferably, the peptides of the present invention have the ability to increase the time for blood to form a clot. In particular, it is preferred that the peptides can extend the clotting time of the intrinsic coagulation pathway. Said clotting time can preferably be determined by determining the "activated partial thromboplastin clotting time" (aPTT). Preferably, the peptides of the present invention increase the clotting time determined by aPPT by at least 100% at a concentration of 60 μM. In some embodiments, the peptides of the present invention increase the clotting time determined by aPPT by at least 90% at a concentration of 40 μM peptide. Said increase in clotting time is compared to the clotting time in the absence of the peptide.

[0176] Said clotting time may in particular be determined by aPTT, as described in Example 3 herein below.

[0177] Reduced hemolytic activity against purified RBCs The peptides of the present invention preferably have low hemolytic activity against purified RBCs at peptide concentrations that have effective anti-inflammatory activity. Thus, the peptides of the present invention have IC 50Preferably, the hemolytic activity against purified RBCs is less than 75%, more preferably less than 65%, for example less than 60% at a concentration corresponding to the hemolytic activity against purified RBCs. Said hemolytic activity against purified RBCs may preferably be determined as described in Example 1 under the hemolytic assay section.

[0178] Treatment method The peptides of the present invention are useful in the treatment of a variety of clinical conditions. Accordingly, the present invention provides the peptides disclosed herein for use as a medicament.

[0179] In particular, the peptides of the invention may be for use in a method for the treatment and / or prevention of inflammation in an individual in need thereof, said method typically comprising administering to said individual a therapeutically effective amount of the peptide.

[0180] The peptides of the invention may also be for use in a method for the treatment and / or prevention of an infectious disease in an individual in need thereof.

[0181] In particular, the peptides of the invention are useful for the combined treatment or prevention of inflammation and infection, for example, for the treatment of inflammation associated with an infection in an individual in need thereof.

[0182] The peptides can be administered by any useful method, but they are particularly useful for systemic administration, such as parenteral administration. For example, the peptides can be for subcutaneous or intravenous administration. The peptides can be administered pulmonary, for example, by inhalation. Other routes of administration include intratracheal and intraperitoneal administration.

[0183] The individual receiving the treatment may be any individual in need thereof, for example a human being.

[0184] The infection treated with the peptides of the invention may be a microbial infection, such as an infection with a microorganism selected from the group consisting of bacteria, fungi, viruses and protozoa. Thus, for example, the peptides may be for use in treating a bacterial, fungal or viral infection, or for treating symptoms associated with such infections.

[0185] Thus, the individual may suffer from bacterial infection. The bacterial infection may be acute or chronic bacterial infection. Non-limiting examples of conditions treated with the peptide of the present invention include acute respiratory distress syndrome (ARDS), pneumonia or sepsis.

[0186] In one embodiment, the peptide of the invention is for use in the treatment of an inflammatory disease, which may for example be selected from the group consisting of acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), severe acute respiratory syndrome (SARS), gastroenteritis, and pulmonary inflammation, such as pneumonia or inflammation of the lung tissue.

[0187] The infections treated with the peptides of the present invention may be any infectious bacterial infection. For example, the bacteria may be gram-negative or gram-positive bacteria. Thus, the bacteria may be, for example, of a genus selected from the group consisting of Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Escherichia, Klebsiella, Stenotrophomonas, Shigella, Moraxella, Acinetobacter, Haemophilus, Pseudomonas and Citrobacter.

[0188] In one embodiment, the individual to be treated has an increased level of endotoxin, such as an increased level of LPS, LTA, zymosan and / or SA-PGN. The individual may have an increased level of endotoxin in one or more body fluids. The body fluid may, for example, be selected from the group consisting of blood, serum, saliva, a nasopharyngeal swab sample and a bronchoalveolar lavage (BAL) sample. The endotoxin may in particular be LPS. The increased LPS level may be at a level of at least 50 pg / ml, for example a serum level of LPS of at least 50 pg / ml.

[0189] The individual may also suffer from a viral infection, e.g., an infection with a spike glycoprotein-containing virus (also referred to herein as an "S protein virus"), e.g., a virus of the Coronaviridae family, e.g., the virus is selected from the group consisting of: PorCov-HKU15, SARS-CoV, HCoV NL63 HKU1, MERS-CoV SARS-CoV2, and MERS-CoV.

[0190] The peptides of the invention may be administered alone or in combination with other therapeutic agents, such as antibiotics, anti-inflammatory agents, or antiseptics (such as antibacterial, antifungal, antiviral and antiparasitic agents).

[0191] The present invention relates to both humans and other mammals, particularly horses, dogs, cats, cows, pigs, camels, etc. Thus, the peptides of the present invention are intended for use in both human therapy and veterinary applications. Subjects suitable for such therapy may be identified by established characteristics of an infection, such as fever, pulse, microbial culture, etc. Infections that may be treated with the molecules include infections caused by microorganisms. Examples of microorganisms include bacteria (e.g., gram-positive or gram-negative), fungi (e.g., yeasts and molds), parasites (e.g., protozoa, nematodes, cestodes and flukes), viruses, and prions and mixtures thereof. Certain microorganisms of these classes are well known (see, for example, Davis et al., Microbiology, 3rd edition, Harper & Row, 1980).

[0192] In one embodiment of the present invention, the peptides disclosed herein are for use in the treatment or prevention of a disease, illness or condition, which may be, for example, any of the diseases, illnesses or conditions described below.

[0193] Acute systemic inflammatory diseases with or without an infectious component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, urosepsis, septic shock, etc. Other invasive infectious and inflammatory diseases, including meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, pneumonia, urinary tract infections, and peritonitis.

[0194] Chronic inflammatory and / or infectious diseases, including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal diseases including chronic stomach ulcers.

[0195] Inflammatory and coagulopathy, including thrombosis and disseminated intravascular coagulation (DIC), as well as vasculitis-related inflammatory diseases and allergies, including allergic rhinitis and asthma.

[0196] These include, but are not limited to, inflammation associated with stroke, extracorporeal circulation procedures such as ECMO, cardiopulmonary bypass, or ex vivo lung perfusion processes.

[0197] Excessive contact activity and / or coagulation associated with, but not limited to, stroke, extracorporeal circulation procedures such as ECMO, cardiopulmonary bypass, or ex vivo lung perfusion processes.

[0198] Excessive inflammation due to antibacterial treatment For example, the peptides of the invention may be for use in the treatment or prevention of acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic rhinitis, other types of rhinitis, vasculitis, thrombosis, and / or disseminated intravascular coagulation (DIC).

[0199] In one embodiment, the peptides of the invention exhibit both anti-inflammatory and anticoagulant activity and may be used for the combined treatment or prevention of inflammation and coagulation. Such peptides may be particularly suitable for the treatment and prevention of conditions in which it is desirable to inhibit both inflammatory and coagulation processes in combination, such as ARDS, sepsis, chronic obstructive pulmonary disease (COPD), thrombosis, DIC, acute respiratory distress syndrome (ARDS). Furthermore, other diseases associated with excessive inflammation and coagulation changes, such as cystic fibrosis, asthma, allergic and other types of rhinitis, as well as vasculitis, may also benefit from treatment with the peptides.

[0200] Preparation of peptides Methods for producing peptides are well known in the art.

[0201] Peptides may be produced by recombinant methods well known in the art (see, e.g., Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York).

[0202] Alternatively, peptides can be chemically synthesized by linking multiple amino acids together, for example through amide bonds. Typically, peptides are chemically synthesized by a condensation reaction of the carboxyl group of one amino acid with the amino group of another. Protecting group strategies can be used to prevent undesired side reactions with the various amino acid side chains.

[0203] Well-known liquid or solid phase peptide synthesis techniques are well known to those of skill in the art (such as standard f-Boc or Fmoc solid phase peptide synthesis).

[0204] The covalent bond between the side chains of two non-adjacent internal amino acids can be introduced by any method known to those skilled in the art, for example, any of the methods described in Li et al., 2020.

[0205] When X1 and X2 are alkenylated amino acids, such as olefin-terminated amino acids, the hydrocarbon staples can be introduced by ring-closing metathesis (RCM), for example, ruthenium-catalyzed ring-closing metathesis or RCM using Grubbs first generation catalyst in 1,2-dichloroethane. RCM can be carried out in solution or on a solid support, and several methods are described in Li et al., 2020 and Example 1.

[0206] Similarly, when X3 and X4 are alkenylated amino acids, such as olefin-terminated amino acids, the hydrocarbon staples can be introduced by ring-closing metathesis (RCM), for example, ruthenium-catalyzed ring-closing metathesis or RCM using Grubbs first generation catalyst in 1,2-dichloroethane. RCM can be carried out in solution or on a solid support, and several methods are described in Li et al., 2020 and Example 1.

[0207] When X3 is an N-terminal amino acid and / or an amino acid containing an amine group and X4 is an amino acid containing a carboxylic acid side chain, the covalent bond can be an amide bond between the amine and the carboxylic acid. In other words, the covalent bond can be an amide bond between the amine and the carboxylic acid, i.e., a lactam bridge. Such cyclization can be carried out on a solid phase.

[0208] Peptides according to the invention can also be ordered from companies that specialize in the production of custom-made peptides, for example AmbioPharm Inc. (US). EXAMPLES

[0209] Example 1 Materials and Methods peptide Peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) (SEQ ID NO: 12), HVF18 (HVFRLKKWIQKVIDQFGE) (SEQ ID NO: 2), and their respective stapled versions designated sGKY25 (GKYGFYTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 13), 2sGKY25 (cyclo[GKYGE]YTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 14) and sHVF18 (HVFRLKKWIXKVIXQFGE) (SEQ ID NO: 3) were synthesized by AmbioPharm, Inc. (USA). Briefly, standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPSS) was used. To obtain the hydrocarbon-stapled peptides, the olefin-containing (S)-2-(4'pentenyl)-alanine was inserted at a specific position (indicated as X) in each of the peptide sequences of SEQ ID NOs: 3 and 13. The olefin metathesis reaction was carried out on a solid support using Grubbs first generation catalyst in 1,2-dichloroethane. The resulting peptides were cleaved from the resin and further purified by RP-HPLC. The peptides were obtained as acetate salts and the purity was confirmed by MALDI-TOF MS (>95%). The cyclization of 2sGKY25 was carried out by forming an amide bond, i.e., a lactam bridge, between the N-terminal amine of Gly1 and the carboxylic acid side chain of Glu5 of the peptide using solid-phase conditions.

[0210] Biomaterials Venous blood was collected from healthy donors after written informed consent was obtained. After collection, whole blood or its fractions (e.g., plasma and serum) were either used immediately or stored at -80°C. The use of blood was approved by the ethical committee of Lund University, Lund, Sweden (permit number: DNR2015 / 801).

[0211] Circular dichroism spectroscopy The secondary structures of GKY25, HVF18, and their respective stapled versions, with or without lipopolysaccharide from Escherichia coli O111:B4 (LPS, Sigma-Aldrich, USA), were assessed by circular dichroism (CD). Peptides were diluted to 10 μM in 10 mM Tris at pH 7.4 and diluted at 100 μg mL -1 Incubation with LPS was performed for 30 min at 37 °C. Measurements were performed using a Jasco J-810 spectropolarimeter (Jasco, USA) equipped with a Jasco CDF-426S Peltier set at 25 °C. Spectra were recorded from 190 to 260 nm (scan speed: 20 nm min ) as the average of five measurements in a 0.2 cm quartz cuvette (Hellma, GmbH & Co, Germany). -1 ) were recorded during baseline (10 mM Tris pH 7.4 ± 100 μg mL ) as reported by Morrissette et al. -1 LPS) was subtracted from each spectrum, and the final signal was calculated as the mean residue ellipticity θ (mdeg cm 2 dmol -1 ) was converted to

[0212] In another set of experiments, linear and stapled versions of HVF18 were mixed with 25 or 50% TFE or at 100 μg mL -1 They were mixed in water containing LPS, after which spectra were acquired as indicated above.

[0213] RP-HPLC HVF18 and its stapled version (2.5 μg) were injected onto a reversed-phase C18 column (Phenomenex Kinetex 50 × 2.1 mm 2.6 μM, pore size 100 Å, California, USA) using an Agilent 1260 Infinity System according to the protocol reported by Petruk et al. Biomolecules (2020). Briefly, the column was equilibrated using 95% buffer A containing 0.25% TFA in MilliQ and 5% buffer B containing 0.25% TFA in acetonitrile. The peptides were premixed with buffer A (1:3) 5 min before loading onto the column.

[0214] Peptides were digested with different proteases for different times as described below and then injected onto a reversed-phase C18 column. Analysis was performed as described above. Samples from two different digestions were analyzed.

[0215] In vitro proteolysis of peptides Peptides were resuspended in endotoxin-free water at a concentration of 1 mM. Then, 20 μg of GKY25 and sGKY25 or 14.7 μg of HVF18 and sHVF18 were incubated with 0.2 μg of human neutrophil elastase (HNE, Calbiochem®, Merck KGaA, Darmstadt, Germany), P. aeruginosa elastase (PE, Calbiochem®, Merck KGaA, Darmstadt, Germany), glutamyl-C endopeptidase from S. aureus V8 (EC 3.4.2.11.9) (BioCol GmbH, Michendorf, Germany) or trypsin (Try, Promega, Madison, WI, USA) for various lengths of time (0–18 h) in a final volume of 20 μL. At the end of the incubation, digestion was assessed by Tricine SDS-PAGE and mass spectrometry (for GKY25, sGKY25, HVF18, and sHVF18), and RP-HPLC (for HVF18 and sHVF18). All digestions were performed in three independent experiments.

[0216] SDS-PAGE Two micrograms of peptide from each condition were loaded onto 10-20% Novex Tricine precast gels (Invitrogen, USA). Runs were performed at 100 V for 100 min. Gels were stained with Coomassie Brilliant Blue (Invitrogen, USA). Images were acquired using a Gel Doc Imager (Bio-Rad Laboratories, USA). Samples from three independent digestions were analyzed.

[0217] NMR spectroscopy NMR experiments were performed on a 700 MHz Bruker Avance III HD spectrometer (Swedish NMR Centre, Gothenburg, Sweden) equipped with a QCI cryoprobe and magnetic field gradient pulses. Samples were prepared by dissolving 1.6 mM sHVF18 in 50% 2,2,2-trifluoroethanol (TFE) at pH 4.5 supplemented with 10% DO, 200 μM DSS, and 0.02 vol.% NaN3. 1 H spectra were acquired from the samples at 298 K. 1 Based on the H spectrum and TOCSY spectrum (mixing time 80 min), a series of TOCSY (mixing times 40 and 80 min), NOESY (mixing times 100 and 150 min), ROESY (mixing times 100 and 150 min), DQF-COSY, 13 C-edited HSQC, 15 N-SOFAST-HMQC, 13 C HSQCTOCSY, and 13 C-HMBC spectra were acquired. Spectra were processed using nmrPipe with squared cosine apodization and zero-filling in both dimensions. Spectra were analyzed and assigned using CCPNMR v2.4. Spin systems were identified using a combination of NOESY and TOCSY spectra, and bonds between residues were assigned using NOESY crosspeaks.

[0218] NF-κB activation assay The anti-inflammatory activity of the four peptides was tested in THP1-XBlue-CD14 reporter cells (InvivoGen, San Diego, USA). Briefly, 180,000 cell wells were cultured. -1 , 10%(vv -1 ) heat-inactivated FBS and 1% (vv -1 ) in 96-well plates in phenol red RPMI medium supplemented with antibiotic-antimycotic solution (AA). Then, 100 ng ml -1100 ng ml LPS (Sigma, USA) was added. NF-κB activity was determined after 20 h of incubation, i.e. by mixing 20 μL of supernatant with 180 μL of SEAP detection reagent (Quanti-Blue™, Invivo-Gen) and subsequent measurement of absorbance at 600 nm, according to the manufacturer's instructions (InvivoGen, San Diego, USA). Data shown are means ± SEM from at least four independent experiments, all performed in triplicate. In another set of experiments, cells were incubated with 100 ng ml LPS in the presence or absence of 10 μM linear and stapled HVF18. -1 of E. coli LPS, 1 μg mL -1 S. aureus LTA, 1 μg mL -1 E. coli PGN, 1 μg mLl -1 of S. aureus PGN, 10 μg mL -1 1000 mg of S. cerevisiae zymosan. Experiments were performed as described above. NF-κB activity was assessed as previously described.

[0219] Hemolysis assay Fresh venous blood from healthy donors was collected into lepirudin tubes (50 μg mL -1 50 μL of blood was then transferred to a round-bottom 96-well plate containing 150 μL of peptide pre-diluted in RPMI-1640-GlutaMAX-I without phenol red (Gibco). Blood diluted 1:4 was used as a control. 50 μL of blood mixed with 150 μl of 5% Tween-20 was used as a positive control. Incubation was performed at 37°C and 5% CO 2で After 1 h of incubation, the plates were centrifuged at 800 g and 150 μL of each sample was transferred to a flat-bottom 96-well plate and the absorbance at 450 nm was measured. The percentage of hemolysis was calculated according to the formula reported below:

number

[0220] To evaluate the hemolytic effect of the peptides on red blood cells, blood was collected as reported above and centrifuged at 250g for 10 min. The plasma was then discarded and the red blood cells were washed three times with 150 mM NaCl in 10 mM Tris pH 7.4. The pellet was then diluted 100-fold with saline Tris buffer. 100 μL of this solution was added to a round-bottom 96-well plate that contained 100 μL of peptides prediluted in saline Tris buffer. After 1 h of incubation at 37° C. and 5% CO2, the plate was centrifuged and the absorbance at 450 nm was measured and the percentage of red blood cell lysis was determined using the formula reported above.

[0221] CD14 expression and purification Human His-tagged CD14 (hCD14-his) was produced in insect cells and purified as previously reported. 23 .

[0222] Microscale Thermophoresis Microscale thermophoresis (MST) was performed using a NanoTemper Monolith NT.115 instrument (Nano Temper Technologies, Germany). The Monolith NT Protein Labeling Kit RED-NHS (Nano Temper Technologies, Germany) was used to label 687 μL (20 μM) of recombinant hCD14 according to the manufacturer's protocol. hCD14 (5 μL of 21 nM) was incubated with increasing concentrations of GKY25, HVF18 and their stapled versions (0.03-1000 μM) in 10 mM Tris, pH 7.4, in a 1:1 ratio, with or without 150 mM NaCl. Samples were then loaded into standard glass capillaries (Monolith NT Capillaries, Nano Temper Technologies) and MST analysis was performed (light-emitting diode and infrared laser settings at 80%). Results shown are the mean ± SD of six measurements.

[0223] Whole Blood Assay Lepirudin (50 μg mL -1Fresh venous blood was drawn from healthy donors in the presence of 100 ng ml 2 . Blood was diluted 1:4 in RPMI-1640-GlutaMAX-I (Gibco) and 1 mL of this solution was transferred to a 24-well plate and immediately added increasing concentrations of GKY25, HVF18 and their stapled versions. -1 After 24 h of incubation at 37 °C in 5% CO2, the plates were centrifuged at 1000 g for 5 min, after which the supernatants were collected and stored at 80 °C until analysis. Experiments were performed at least four times, using blood from different donors each time. To evaluate the healing properties of the peptides, blood was diluted with 100 ng ml -1 After stimulation with LPS and incubation at 37°C for 30 min, the mice were treated with increasing doses of the four peptides. In the final set of experiments, the prophylactic anti-inflammatory activity of the peptides was evaluated by exposing blood to increasing concentrations of GKY25, HVF18, and their stapled versions for 30 min. Blood was then diluted with 100 ng ml -1 The cells were stimulated with LPS.

[0224] Cytokine assays Plasma from blood experiments was used to assess cytokine release. Human Inflammation DuoSet® ELISA kits (R&D Systems), specific for TNF-α and IL-1β, were used according to the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm. Data shown are the mean ± SEM from at least four independent experiments, all performed in duplicate.

[0225] Levels of TNF-α, IFN-γ, MCP-1, IL-10, and IL-6 in murine plasma were assessed using a mouse inflammation kit (Becton Dickinson AB) according to the manufacturer's instructions.

[0226] Mouse inflammation model The immunomodulatory effects of HVF18 and sHVF18 were studied in BALB / c tg(NF-κB-RE-Luc)-Xen reporter mice (Taconic, 10-12 weeks old). -1) in a final volume of 200 μL, and E. coli LPS (25 μg mouse -1 ) were injected subcutaneously at the same time as the NF-κB activation assay. The dorsum of the mice (8 mice per treatment group) was carefully shaved and cleaned. After injection, the animals were immediately transferred to individual ventilated cages and images were taken 3 hours later. An In Vivo Imaging System (IVIS Spectrum, Perkin Elmer Life Sciences) was used to measure NF-κB activation. Bioluminescence from the mice was detected and quantified using Living Image 4.0 software (Perkin Elmer Life Sciences). Mice were intraperitoneally administered 100 μL of D-luciferin (150 mg / kg body weight) 15 min before IVIS imaging.

[0227] In vivo LPS model E. coli 0111:B4 LPS was resuspended in 10 mM Tris pH 7.4. A sublethal dose (6 mg per kg body weight) was then injected intraperitoneally (ip) into male C57BL / 6 mice (11-12 weeks, 22 + / - 5 g). Thirty minutes later, mice were injected i.p. with 10, 20, 50, 100 or 500 μg sHVF18 (in 10 mM NaOAc pH 5) per mouse. Eight and 20 hours after LPS administration, mice were deeply anesthetized with isoflurane and blood was collected by cardiac puncture and stored at -80 °C until further analysis.

[0228] bacterial cell culture One colony of E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa O1, as well as eight clinical isolates of S. aureus and seven P. aeruginosa were inoculated into 5 mL of Todd-Hewitt (TH) medium overnight with shaking at 37 °C. The next day, the bacterial cell cultures were diluted 1:50 in fresh TH medium and grown to mid-logarithmic phase. The bacteria were then centrifuged at 3500 g for 10 min, washed, and then cultured at 2 × 10 9 Colony forming units (CFU) mL -1 The pellet was resuspended in 10 mM Tris pH 7.4 to a final concentration of 0.01 mM.

[0229] Radial Diffusion Assay (RDA) Bacteria were grown and prepared as described above. Then, the microorganisms (4 × 10 6 CFU) were added to 15 mL of underlay agarose gel consisting of 0.03% (w / v) TSB, 1% (w / v) low electroosmotic agarose (Sigma-Aldrich) and 0.02% (v / v) Tween 20 (Sigma-Aldrich). The underlay gel was poured into a 144 mm diameter Petri dish. After the agarose solidified, 4 mm diameter wells were punched and 6 μL of the required concentration of peptide solution was added to each well. The plate was incubated at 37 °C for 3 h to allow diffusion of the peptides. The underlay gel was then covered with 15 mL of molten overlay gel (6% TSB and 1% low electroosmotic agarose in distilled H2O). Peptide activity is expressed as the diameter from a clear zone to the well (except for the 4 mm wells). All experiments were performed at least four times.

[0230] Viable Count Assay (VCA) Bacteria were grown and prepared as above. The bacterial suspension was then diluted to 2×10 6 CFUmL -1 The bacteria (50 μL) were incubated with different concentrations of GKY25, sGKY25, HVF18 and sHVF18 (1-20 μM) in 10 mM Tris pH 7.4 in the presence or absence of 150 mM NaCl or 25% human citrated plasma for 2 h at 37 °C. At the end of the incubation, serial dilutions of the samples were plated on TH agar plates, incubated overnight at 37 °C and CFUs were calculated. Bacteria treated with the respective buffer were used as controls. All experiments were performed at least in quadruplicate. Data shown are mean ± SEM.

[0231] Live / dead assay Bacterial membrane permeabilization was previously described. 24The viability of the bacteria was evaluated by the LIVE / DEAD BacLight™ Bacterial Viability kit (Invitrogen, Molecular Probes, Carlsbad, CA, USA) as described in. Briefly, bacterial suspensions were prepared similarly to VCA. S. aureus and P. aeruginosa O1 suspensions (50 μL) were then treated with 1 μM or 5 μM HVF18, respectively, and its stapled version in 10 mM Tris, pH 7.4. Buffer was used as a negative control. After 2 h, samples were mixed with 1 μL of dye mixture per mL of bacterial suspension as indicated in the manufacturer's protocol and incubated for 15 min at room temperature in the dark. At the end of the incubation, 5 μL of the stained bacterial suspension was sandwiched between a slide and an 18 mm square coverslip. Ten fields of view (1 × 1 mm) of mounted samples from three independent sample preparations were examined using a Zeiss AxioScope A.1 fluorescence microscope (objective: Zeiss EC Plan-Neofluar100 / 1.3 oil; camera: Zeiss AxioCam MRm, acquisition software: Zeiss Zen 2.6 (blue edition)).

[0232] Transmission electron microscopy The effect of peptides on S. aureus and P. aeruginosa O1 membranes was further evaluated by transmission electron microscopy (TEM) combined with negative staining (Jeol Jem 1230; Jeol, Japan). In particular, 5 μL of bacterial suspension from VCA was adsorbed onto carbon-coated grids (copper mesh, 400) for 60 s and stained with 7 μL of 2% uranyl acetate for 30 s. The grids were made hydrophilic by glow discharge at low pressure. The analysis was performed on 10 fields of view (magnification '4200) of samples mounted on grids (pitch 62 μm) from three independent experiments.

[0233] MIC and MBC assays The minimum inhibitory concentration (MIC) was determined according to the protocol reported by Wiegand et al. Bacteria were grown and diluted as described above. Bacteria were then further diluted 1:1000 in 2x BBL™ Mueller Hinton II (MH), cation-adjusted broth (Becton, Dickinson and Company, Sparks, USA). Bacteria (50 μL) were added to 96-well round-bottom polystyrene plates (Corning INC, Kennebunk, USA) containing 50 μL of 2x MH broth (control) or 2x MH broth containing peptides (HVF18 or sHVF18) at concentrations ranging from 2.5 to 320 μM. -The plates were then incubated at 37 °C for 24 h. The MIC was verified as the lowest concentration at which no visible bacterial growth was observed. After analysis of the MIC, the same plates were used to determine the minimum bactericidal concentration (MBC). For this purpose, samples from each condition were resuspended using a pipette tip and 10 μL droplets were plated onto THA plates and incubated overnight at 37 °C. The MBC was determined at the concentration at which no bacterial colony formation was observed.

[0234] result Design of dual-acting peptides with improved proteolytic stability The thrombin C-terminal peptide GKY25 has been demonstrated to have dual action targeting both bacterial infection and inflammation via associated TLRs. One approach to enhance peptide stability is peptide hydrocarbon stapling, a modification that uses side chain covalently bonded hydrocarbon bridges to stabilize the secondary structure of a peptide. Stapling has been applied to other peptides, but the effect of stapling is difficult to predict. A hydrocarbon staple moiety was introduced into the sequence of GKY25 (GYGFYTHVFRLKKWIQKVIDQFGE) by replacing specific amino acids with (S)-2-(4'pentenyl)-alanine. The position of stapling was determined based on the information of GKY25 summarized in Table 1. Table 1 [Table 1]

[0235] In addition, the centrally located lysine residues (K13, K14, and K18 - numbers in this section refer to the GKY25 sequence (SEQ ID NO: 12)) may be important for the antibacterial activity of GKY25. Furthermore, protonation of H8 at pH 5.5 increases the antibacterial activity of GKY25 against Gram-negative Escherichia coli by membrane disruption. GKY25 binds to LPS and the LPS-binding hydrophobic pocket of CD14. The residues involved in the interaction of LPS with CD14 are shown in Table 1. Studies have demonstrated that K14 crosslinks to K87 in CD14, and in silico docking studies show that the C-terminal residues of Q17, K18, D21, Q22, and E25 are solvent exposed. NMR studies determined the LPS-bound conformation (PDB code 5z5x) in which the C-terminal α-helix begins at I16. The interaction with LPS is mediated by hydrophobic and positively charged residues H8, R11, K13, and K14. Ignoring the amino acids of GKY25 that may have any of the aforementioned activities, amino acids Q17, D21, Q22, and E25 remained. Stapling was introduced at positions Q17 and D21 because Q22 and E25 are close to the termini.

[0236] The helicity of stapled GKY25 (herein denoted as sGKY25) compared to its native version was analyzed using circular dichroism (CD). The spectrum of GKY25 in the presence of LPS was used as a positive control. sGKY25 exhibited an α-helical structure and the helicity content when bound to LPS was comparable to that of GKY25. In the case of sGKY25 with LPS, there was no change in the α-helical content. To confirm that stapling enhances the proteolytic stability of the peptides, the peptides were exposed to various proteases for different times and analyzed using SDS-PAGE. As shown in Figure 1a, stapling improved the stability of GKY25 in the presence of HNE for up to 6 h. Furthermore, the stability against trypsin was also improved. Indeed, it was possible to detect intact sGKY25 even after 6 h of digestion. Interestingly, none of the peptides was affected by V8 digestion, despite its cleavage site being present in the sequence. To understand which regions were released from digested sGKY25 compared to the native form, we used LC-MS / MS. As expected, the linear peptide showed a wide variety of fragments released already after 30 min of digestion. On the other hand, stapling conferred partial protection to sGKY25. At the time points analyzed, we found few fragments corresponding to the C-terminal part of sGKY25 after digestion with PE and none at all with HNE. This is not surprising, since the N-terminal part of GKY25 is known to be more flexible and susceptible to proteolysis. Although stapling GKY25 resulted in more hemolytic constructs, especially for red blood cells (RBCs) (Figure 4a), sGKY25 also showed significant hemolytic activity in whole blood (Figure 4b). Even at a concentration of 50 μM, sGKY25 showed more than 10% hemolytic activity in whole blood, which is usually the upper limit of acceptable hemolytic activity.

[0237] When tested for anti-inflammatory activity in LPS-stimulated THP-I cells, sGKY25 showed a marked improvement over the linear peptide, but also showed high toxicity at higher concentrations (Fig. 2a).Under a more physiological condition, i.e., in blood, the stapled sGKY25 peptide was completely unable to block LPS-induced inflammation (Fig. 2b).

[0238] The helicity of stapled HVF18 (sHVF18) was confirmed by its hydrophobic environment, such as CD and RP-HPLC characteristics. sHVF18 showed a longer retention time (9.42 min) when compared to linear HVF18 (8.03 min) because the functional binding surface was increased by rocking. Subsequently, the resistance to protease cleavage was tested by SDS-PAGE (Fig. 1b), HPLC, and LC-MS / MS. Stapling improved the stability of HVF18 for up to 18 h when HNE and PE were used. Furthermore, the stability against trypsin was also improved. Indeed, it was possible to detect intact sHVF18 even after 18 h. Similar to linear and stapled GKY25, HVF18 mutants were not affected by V8 digestion. LC-MS / MS results confirmed that stapling of HVF18 conferred complete protection to the peptide against proteolysis. The helical stability of sHVF18 was further tested by exposing the peptide to high temperature and analyzing the secondary structure by CD. It was found that even after exposure to 80°C, sHVF18 still exhibited two characteristic minima in the α-helical spectrum, namely at 208 and 222 nm. Notably, sHVF18 exhibited lower hemolytic activity when compared to sGKY25 (compare Fig. 4b with Fig. 4a). Notably, sHVF18 exhibited significantly lower hemolytic activity in whole blood compared to sGKY25.

[0239] The insertion of the staples into the correct positions was confirmed by nuclear magnetic resonance (NMR). sHVF18 was dissolved in 50% TFE, which is known to increase secondary structure, and TOCSY, NOESY, ROESY, and 13 C-HSQC and 15N-SOFAST-HMQC spectra were collected, and the secondary structure was predicted using the DANGLE dihedral angle and chemical shift index (CSI) module of the CCP NMR suite, which suggested that sHVF18 contains an α-helix consisting of 7–14 residues.

[0240] A detailed nuclear magnetic resonance (NMR) study of sHVF18 was carried out. The peptide was dissolved in 50% TFE. TOCSY, NOESY, ROESY, 13 C-HSQC, and 15 N-SOFAST-HMQC spectra were collected in sHVF18 in 50% TFE. The TOCSY, NOESY, and ROESY spectra show well-dispersed peaks, and the amino acid types can be easily identified in the TOCSY spectrum. The NOESY and ROESY spectra show many HN-Hα and HN-HN cross peaks, facilitating the sequential assignment of peptides. 13 The C HSQC spectrum shows well-dispersed peaks, with 16 cross-peaks corresponding to the amide backbone atoms. 15 N was detected in the SOFAST-HMQC spectrum, and further side chain cross peaks of 8Trp and 15Gln were also detected. 15 N HMQC and 13 The C HSQC spectrum shows that the sHVF18 sample has well-defined conformational states under these conditions. The presence of multiple HN-HN (I,i+2) and HN-Hα (I,i+2 / 3) structures indicates the presence of well-defined secondary structures. 1We were able to identify 97% of the H resonances. The secondary structure was estimated using the DANGLE dihedral angle estimation and chemical shift index (CSI) module of the CCP NMR suite, and we estimated that sHVF18 contains an α-helix consisting of 7–14 residues, which is also consistent with the NOE pattern. The stapled linker can be easily identified in the NOESY spectrum due to the aromaticity of the staple, and the presence of the staple can be confirmed based on the TOCSY spectrum. The structural ensemble forms an L-shaped structure with two α-helices consisting of residues Val2-Leu5 and Lys11-Gly17. The N- and C-terminal α-helices are structurally well-defined, but there is a relatively large variability in the orientation of the two α-helices relative to each other. When the structure of sHVF18 in TFE is compared to HVF18 in the presence of LPS, a similar L-shaped tertiary structure can be observed with a backbone RMSD of 2.2 Å. The main difference is seen in the N-terminal part of HVF18, where the α-helix seen in sHVF18 is not observed. However, this may be due to TFE inducing sHVF18 into a more helical structure than LPS induces HVF18 into a helical structure, as shown by CD analysis. In sHVF18, the N-terminal part of sHVF18 appears to be more regular than HVF18. The C-terminal α-helix of HVF18 spans residues Ile9-Gly17, which is two amino acids longer than sHVF18. From the sHVF18 structural ensemble, it appears that the staples disrupt the α-helix. When the NOEs obtained for sHVF18 were examined in more detail and compared with the distances in the HVF18 ensemble structure, the NOE distances for residues 5Leu, 7Lys, 8Trp, 9Ile, and 11Lys did not match the HVF18 structural ensemble, indicating structural differences between sHVF18 and HVF18.

[0241] Effect of stapling on the anti-inflammatory activity of peptides Using Microscale Thermophoresis (MST) d The K of sHVF18 against CD14 was determined. dwas significantly decreased even in the presence of salt (Fig. 5). THP1-XBlue-CD14 reporter cells were stimulated with LPS in the presence of increasing concentrations of linear and stapled HVF18 and NF-κB activation was assessed. Figure 3 clearly shows that the anti-inflammatory activity of sHVF18 was significantly improved. Moreover, this was also true for other TLR agonists such as LTA and PGN from S. aureus, PGN from E. coli, and zymosan from S. cerevisiae (Fig. 6a). In THP-1 cells, a stronger inhibition of LPS-induced NF-κB / AP-1 activation by sHVF18 was observed compared to its linear form (Fig. 3). 25% fresh human venous blood was incubated with stapled or linear HVF18 and simultaneously stimulated with LPS for 24 h. We found that sHVF18 efficiently and dose-dependently reduced TNF-α and IL-1β secretion, especially when peptides were added before or together with LPS stimulation (Figures 3 and 7a). When blood was first stimulated with LPS for 30 min and then treated with increasing doses of peptide, the inhibition by sHVF18 was lower but still significant (Figure 7b).

[0242] Effect of stapled peptides on endotoxin responses in experimental mouse models. In the first set of experiments, we investigated whether sHVF18 could suppress LPS-induced local inflammation in NF-κB reporter mice. The same amount of stapled or linear peptide was injected subcutaneously, LPS was added at the same time, and NF-κB activation was then measured over time (Figures 8a and 9). In agreement with the in vitro data, sHVF18 showed potent anti-inflammatory activity already at 50 μg, whereas its linear counterpart was ineffective (Figures 8a and 9). Indeed, higher concentrations of HVF18 were required (200 μg). Moreover, the activity of the linear peptide became unstable over time, with a diminished inhibition of the proinflammatory effects induced by LPS. In the second set of experiments, we evaluated the activity of sHVF18 in a mouse model of endotoxin-induced shock. C57BL / 6 mice were injected ip with a sublethal dose of LPS and treated 30 min later with increasing doses of sHVF18. After 20 hours, mice were sacrificed and cytokine levels in blood samples were analyzed (Figure 9b). Significant reductions in proinflammatory cytokines such as TNF-α, IL-6, IFN-γ, and MCP-1 were observed at 20, 50, and 100 μg of sHVF18 (Figure 9b). Interestingly, 500 μg of sHVF18 failed to reduce cytokine levels at all. Next, the effect of 50 and 100 μg of sHVF18 was investigated using a shorter time point of 8 hours (Figure 8b). A low but significant reduction in cytokine levels was observed at both concentrations of sHVF18.

[0243] Effect of stapling on the antibacterial activity of peptides By using the radial diffusion assay (RDA), it was noted that sHVF18 was similarly active regardless of the conditions, i.e., with or without NaCl, whereas in salt-free experiments, the linear peptide showed better bacterial killing. Indeed, in the presence of NaCl, a decrease in activity was observed (Figure 10a). When testing the antibacterial activity in solution, sHVF18 was found to be more bactericidal than HVF18 in all strains evaluated (Figure 10b, left panel). Moreover, it showed a remarkable affinity for S. aureus, killing it at concentrations below 1 μM (Figure 10c). As one goal of this study was to use sHVF18 as a systemic drug, the activity of the peptide was also evaluated in more complex situations, such as the presence of salt or human plasma (Figure 10b, center and right panels). The antibacterial activity of both peptides decreased with increasing medium complexity, but nevertheless, sHVF18 showed a stronger killing effect than its linear form (Figure 10b, center and right panels).

[0244] The antibacterial activity of sHVF18 was further confirmed by standard minimum inhibitory concentration (MIC) assays (Figure 10d, e). Similar to VCA, sHVF18 showed stronger activity against Gram-positive bacteria.

[0245] Taken together, these results indicate that sHVF18 retains antibacterial activity against Gram-negative bacteria when compared to its linear form, and is more active and therefore more selective against Gram-positive bacteria. Finally, killing is mediated by bacterial membrane permeabilization and disruption.

[0246] Example 2 Materials and Methods peptide Peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) (SEQ ID NO: 12), sGKY25 (GKYGFYTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 13), 2sGKY25 (ciclo[GKYGE]YTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 14), sHVF18 (HVFRLKKWIXKVIXQFGE) (SEQ ID NO: 3), sKVF18 (KVFRLKKWIXKVIXQFGE ) (SEQ ID NO: 4), sKKVF18 (SEQ ID NO: 5), sKKKVF18 (SEQ ID NO: 6), sKKKKVF18 (SEQ ID NO: 7), sRVF18(RVFRLKKWIXKVIXQFGE) (SEQ ID NO: 8), sRRVF18 (SEQ ID NO: 9), sRRRVF18 (SEQ ID NO: 10) and sRRRRVF18 (SEQ ID NO: 11) were synthesized and purified by AmbioPharm, Inc. (USA) as indicated for sHVF18. Peptides were obtained as acetate salts and purity was confirmed by MALDI-TOF MS (>95%).

[0247] Circular dichroism spectroscopy The secondary structure of all stapled peptides was assessed by circular dichroism (CD). Peptides were diluted to 10 μM in 10 mM Tris, pH 7.4, and spectra were measured on a Jasco J-810 spectropolarimeter (Jasco, USA) equipped with a Jasco CDF-426S Peltier set at 25° C. as shown for sHVF18. Data shown are the average values ​​from three independent experiments.

[0248] NF-κB activation assay The anti-inflammatory activity of all stapled peptides was tested in THP1-XBlue-CD14 reporter cells (InvivoGen, San Diego, USA). Experiments were performed as indicated for sHVF18, except that the final concentration of each peptide was 1-10 μM. Data shown are the mean ± SEM from four independent experiments, all performed in triplicate.

[0249] Hemolysis assay The hemolytic effect of stapled peptides on red blood cells or whole blood was tested as indicated for sHVF18. Data shown are the mean ± SEM from at least four independent experiments, all performed in triplicate. Blood from different donors was used in each experiment.

[0250] Whole Blood Assay The anti-inflammatory effect of different stapled peptides in blood was carried out as shown for sHVF18. In each experiment, blood from different donors was used.

[0251] Cytokine assays Plasma from blood experiments was used to assess cytokine release. Human Inflammation DuoSet® ELISA Kits (R&D Systems), specific for TNF-α and IL-1β, were used according to the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm. Data shown are the mean ± SEM from four independent experiments. For 2sGKY25, data shown are the mean ± SEM from two independent experiments.

[0252] Levels of TNF-α, IFN-γ, MCP-1, IL-10, and IL-6 in murine plasma were assessed using a mouse inflammation kit (Becton Dickinson AB) according to the manufacturer's instructions.

[0253] In vivo LPS model E. coli 0111:B4 LPS was resuspended in 10 mM Tris, pH 7.4. A sublethal dose (6 mg per kg body weight) was then injected intraperitoneally (ip) into male C57BL / 6 mice (11–12 weeks, 22 + / - 5 g). Thirty minutes later, mice were injected i.p. with 10 μg per mouse of sHVF18, sKVF18, sKKVF18, sRVF18 or sRRVF18 (in 10 mM NaOAc, pH 5). For untreated mice, 100 μL of 10 mM Tris, pH 7.4 was injected 30 minutes before and 100 μL of 10 mM NaOAc, pH 5 after 30 minutes. Twenty hours after LPS administration, mice were deeply anesthetized with isoflurane and blood was collected by cardiac puncture and stored at -80 °C until further analysis.

[0254] Antibacterial action The antibacterial activity of the various stapled peptides against E. coli ATCC 25922, S. aureus ATCC 29213, and P. aeruginosa O1 was performed similarly to sHVF18 by radial diffusion assay (RDA) or viable count assay (VCA), except that the highest tested concentrations of each peptide were 20 μM and 10 μM for RDA and VCA, respectively.

[0255] result Analysis of the structure and hemolytic activity of stapled peptides As shown in Example 1, stapling of HVF18 increased its stability against proteolysis, significantly improved its anti-inflammatory activity (in vitro and in vivo), and maintained its antibacterial activity, but directed its activity mainly against Gram-positive bacteria, while at the same time having low hemolytic activity. Various mutants of sHVF18 were generated, in which the N-terminal His residue was replaced with one to four Lys or Arg residues. In addition, another mutant of stapled GKY25 (2sGKY25) was generated, which has two staple regions, one mutant in the C-terminal region like sGKY25, but with an additional staple in the N-terminal region. As described in Example 1, sGKY25 was unable to block LPS-induced inflammation in a complex environment such as human blood (see Figure 2b), so the purpose of double stapling was to evaluate whether 2sGKY25 could exhibit any anti-inflammatory activity.

[0256] The secondary structure of sHVF18 and its mutants was evaluated by circular dichroism (CD). In Figure 11, it can be seen that the K and R mutants of sHVF18 have a more defined α-helical structure with two minima, one at 208 nm and one at 222 nm. The hemolytic activity of these peptides was compared in red blood cells (RBCs) and whole blood (Figures 12a and b, respectively). All peptides were found to be hemolytic for RBCs. Different amounts of K and R residues did not make any difference in this respect (Figure 12a). When the same analysis was performed in whole blood, it was observed that the hemolytic activity of the K and R mutants was significantly increased when the number of positive residues was 3 or 4 compared to 1 or 2 (Figure 12b). When analyzing the hemolytic activity of 2sGKY25 in whole blood, it was noted that the hemolytic activity was slightly lower than that of sGKY25, but clearly higher than that of sHVF18 (Figure 12c - see dashed line). The dotted line in Figure 12 indicates 10% hemolytic activity. Generally, it is preferred that the hemolytic activity of a peptide is less than 10%.

[0257] Evaluation of the anti-inflammatory activity of stapled peptides in vitro and in vivo The ability of the stapled peptides to counteract LPS-induced inflammation was first evaluated in THP-1-XBlue-CD14 reporter cells. Data for the K-containing variants of HVF18 are shown in Figure 13a and for the R-containing variants in Figure 13b. When tested at a final concentration of 10 μM, all peptides showed improved activity over the original sHVF18. The variants with three and four K or R showed better activity already at 2 μM and 5 μM, respectively, compared to 10 μM sHVF18. Next, the anti-inflammatory activity was evaluated in a more physiological environment, i.e., in 25% whole blood. Blood was stimulated with LPS in the presence or absence of the different peptides, and then the release of TNF-α and IL-1β in plasma was quantified by ELISA (Figures 14a and b). Surprisingly, here the activity of peptides with three and four K or R was lower than that of peptides with one or two positively charged residues. In particular, peptides with two K or R showed improved efficacy. Notably, these peptides were found to significantly reduce TNF-α release even at very low concentrations of 1-2 μM, and at 5-10 μM peptides, TNF-α release was almost completely abolished. These peptides were also found to significantly reduce IL-1β release at 2-5 μM. We also tested the activity of double-stapled GKY25 with respect to its linear and single-stapled versions in LPS-stimulated blood (Figure 14c). Closing GKY25 at the N-terminal region also proved to better suppress LPS-induced TNF-α and IL-1β release than GKY25, but higher doses were required when compared to the mutants of sHVF18.

[0258] The activity of 10 μg of sHVF18 with one and two K or R mutants was compared in mice stimulated with LPS. We found that all peptides could reduce the levels of various cytokines in mice treated with peptides. The two K and R mutants showed the strongest effect on cytokine reduction (Figure 15).

[0259] Evaluation of the antibacterial activity of stapled peptides in vitro The activity of antimicrobial peptides is strongly related to their specific sequence properties. Therefore, the effect of the addition of K and R in the stapled sHVF18 mutants on the activity against E. coli, P. aeruginosa O1 and S. aureus was measured. The clearance zone was measured in the absence of NaCl (Figure 16a) and in the presence of 150 mM NaCl (Figure 16b). In general, peptides with a lower net positive charge were found to be more active in salt-free medium and vice versa. Furthermore, the ability of sHVF18K and R mutants to eliminate bacteria on plates without NaCl was generally better at lower concentrations when compared to the original sHVF18. In plates with NaCl, which is more similar to the in vivo situation, the mutants with an increased number of Ks in particular showed significantly improved antimicrobial activity. The bactericidal activity of the peptides in solution was again investigated in the absence (Figure 17a) and presence (Figure 17b) of NaCl. Here, sHVF18 with K and R mutations was found to be more active against all bacterial strains compared to the original sHVF18, especially in the presence of salt, and furthermore, the activity was higher as the net charge of the peptide increased.

[0260] conclusion The overall results for the different linear and stapled peptides investigated in Examples 1 and 2 are summarized in Table 2 and Figure 20d. It was demonstrated that the anti-inflammatory activity of the stapled peptides is significantly increased in a complex environment such as human blood. In particular, the mutants of sHVF18 with 2R or K showed excellent results. On the other hand, when His was replaced with 3 and 4R or K, the anti-inflammatory effect was lower than that of the mutants with 1 and 2R or K, probably due to the higher toxicity of these peptides as shown by the hemolysis results. These results indicate that there is an optimal amount of positively charged residues that can be inserted into the peptide structure, which in this case corresponds to two residues of K or R. Both of these mutants showed a higher therapeutic index, i.e. a larger difference between the therapeutic concentration and the dose that causes toxicity. With regard to the retention of antibacterial activity, it was observed that the replacement of His with one or more Lys or Arg residues enhances the bactericidal effect of these mutants, especially in the presence of salt, when compared to the original sHVF18.

[0261] Table 2: Comparison of hemolytic, anti-inflammatory and antibacterial activities of linear and stapled peptides [Table 2] *I C 50 For peptides >10 μM, the exact IC 50 Although the hemolytic activity was unknown, it was selected to exhibit hemolytic activity at 50 μM.

[0262] Example 3 - Anticoagulant properties of peptides Materials and Methods Clotting assays A coagulometer (Amelung, Lemgo, Germany) was used for all clotting time measurements. Freshly collected human citrated plasma was used for all experiments. To measure activated partial thromboplastin time (aPTT), 100 μL of kaolin-containing solution (Dapttin, Technoclone) and plasma-peptide mix were incubated at 37°C for 200 s, and then clot formation was initiated by adding 100 μL of 30 mM fresh CaCl2 solution. Prothrombin clotting time (PT, thromboplastin reagent (Trinity Biotech)) was recorded by adding 100 μL of coagulation reagent to 100 μL of pre-warmed (37°C for 60 s) plasma-peptide mix.

[0263] result Evaluation of the anticoagulant properties of peptides Peptides sHVF18, sKVF18, sKKVF18, sRVF18 and sRRVF18 were tested in the clotting assay. The sequences of these peptides are shown in the sequence summary below. The results are shown in Figure 18.

[0264] Excessive activation of the coagulation cascade via LPS- and bacteria-induced contact activation contributes to the deleterious effects observed during sepsis and septic shock. Therefore, we investigated the possible effect of the peptides of the invention on the coagulation pathway. Analysis of the effect of the peptides on activated partial thromboplastin time (aPTT) and prothrombin time (PT) showed that all peptide variants dose-dependently inhibited the activation of the intrinsic pathway of human plasma coagulation (aPTT) in vitro, but the RR and KK variants sHVF18 were particularly effective (Figure 18, upper panel). In contrast, the extrinsic pathway of coagulation, monitored by measuring the prothrombin time (PT), was not affected at any of the doses tested (Figure 18, lower panel).

[0265] conclusion Activation of coagulation, inhibition of fibrinolysis, and consumption of coagulation inhibitors result in a procoagulant state and fibrin deposition in microvessels, as observed in ARDS and sepsis, diseases that may be complicated by disseminated intravascular coagulation (DIC). As a consequence, microvascular thrombosis contributes to accelerated organ dysfunction. Moreover, excessive contact activation leads to the release of the proinflammatory peptide bradykinin and subsequent induction of an inflammatory response, contributing to severe complications such as hypotension and vascular leakage. Thus, peptides that modulate several pathways, including inflammation and coagulation, in biologically relevant situations, as demonstrated with sHVF18, especially the KK and RR mutants, are of interest for developing future peptide-based therapies for patients that show excessive activation of these pathways, such as those seen in ARDS, sepsis, and other systemic inflammatory diseases. Moreover, since activation of the contact system occurs in several non-infectious diseases, interference with peptides may also be beneficial in other conditions with coagulation dysfunction.

[0266] Example 4 - Oligomerization of Peptides Materials and Methods Dynamic Light Scattering (DLS) The hydrodynamic radius of particles in solution was measured using a DynaPro Plate reader (WYATT Technology) equipped with a temperature-controlled chamber (25°C). Peptides (HVF18, sHVF18, sKVF18, sKKVF18, sRVF18, and sRRVF18 - sequences are given in the sequence summary below) were resuspended in 10 mM Tris, pH 7.4 or 10 mM NaOAc, pH 5 at a final concentration of 1 mM immediately before analysis. 30 μL of each sample was used for analysis. Each measurement was performed in triplicate with 10 subruns. The hydrodynamic radius was analyzed using Dynamics 7.19 software. Results are expressed as mean values ​​± DS obtained from three independent experiments.

[0267] result Peptide Oligomerization The results are shown in Figure 19. Peptide oligomerization is considered a disadvantage, as it is often associated with toxicity, immunogenicity, and even reduced activity. TCP-25 and HVF18 oligomerize. The effect of stapling on peptide oligomerization was measured. The size of the hydrodynamic radius (in nm) of the particles in solution was evaluated by dynamic light scattering (DLS) immediately after dissolving the peptides in 10 mM Tris at pH 7.4 or 10 mM NaOAc at pH 5. We have previously reported that at acidic pH, TCP-25 oligomerization is inhibited. Moreover, at this pH, the peptide is completely unstructured. Particles of HVF18 were larger at pH 7.4 than at pH 5. On the other hand, sHVF18 showed particles with significantly smaller size compared to HVF18 at both pHs. Analysis of the hydrodynamic radii of the K and R mutants of sHVF18 showed that the size of the particles was smaller and completely independent of pH, suggesting that both stapling and positive charges make the peptide less prone to oligomerization. Interestingly, no significant differences were observed between the K and R mutants and the number of these positively charged amino acids in the sequence.

[0268] Example 5 Materials and Methods peptide Peptide sHVF18 (HVFRLKKWIXKVIXQFGE) (SEQ ID NO: 3), its short versions (designated as sVFR17, sFRL16, sRLK15, sLKK14, sKKW13), mutants of sKKW13 (designated as sKKK14, sKKK15, sRKK14, sRRK15), and doubly stapled GKY25 (cyclo(GKYGFY)THVFRLKKWIXKVIXQFGE) designated as 2sGKY25 were synthesized by AmbioPharm, Inc. (USA). Briefly, standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPSS) was used. To obtain the hydrocarbon stapled peptides, an olefin-containing (S)-2-(4'pentenyl)-alanine was inserted at a specific position (designated as X) in each of the peptide sequences of SEQ ID NO: 3 and 13. Olefin metathesis reactions were carried out on solid support using Grubbs first generation catalyst in 1,2-dichloroethane. The resulting peptides were cleaved from the resin and further purified by RP-HPLC. The peptides were obtained as acetate salts and the purity was confirmed by MALDI-TOF MS (>95%).

[0269] Hemolysis assay The hemolytic effect of stapled peptides on red blood cells and / or whole blood was tested as indicated for sHVF18. Data shown are the mean ± SEM from at least four independent experiments, all performed in triplicate. Blood from different donors was used in each experiment.

[0270] Whole Blood Assay The anti-inflammatory effect of different stapled peptides in blood was carried out as shown for sHVF18. In each experiment, blood from different donors was used.

[0271] Cytokine assays Plasma from blood experiments was used to assess cytokine release. Human Inflammation DuoSet® ELISA kits specific for TNF-α and IL-1β (R&D Systems) were used according to the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm. Data shown are the mean ± SEM from four independent experiments.

[0272] Porcine ARDS model Animal preparation: Adult sows and boars of wild-type American Yorkshire pigs (Sus scrofa domesticus) bred on farms were included in the study. Animals were stratified into treatment or non-treatment groups. A total of 10 pigs with an average weight of 45 kg were treated with ketamine (Ketaminol® Veterinary 100 mg / mL; Farmaceutici Gellini SpA, Aprilia, Italy; 20 mg kg -1 ) and xylazine (Rompun® Veterinary 20 mg mL -1 ;Bayer AG,Leverkusen,Germany;2mg kg -1) was premedicated. A urinary catheter was inserted into the bladder. A peripheral intravenous (IV) line was placed in an earlobe and general anesthesia was maintained with ketamine (Ketaminol® Veterinary, MSD Animal Health Sweden, Stockholm, Sweden), midazolam (Midazolam Panpharma®, Panpharma Nordics AS, Oslo, Norway) and fentanyl (Leptanal®, Piramal Critical Care BV, Voorschoten, Netherlands). Mechanical ventilation was established using a Siemens-Elema ventilator (Servo900C, Siemens, Solna, Sweden) with a 7.5 size endotracheal tube for intubation. Volume-controlled ventilation (VCV) was used according to the manufacturer's instructions with a flow pattern switch at "constant flow" to reduce peak pressures. To achieve an I:E ratio of 1:2, inspiration time was set at 25% and pause time at 10%, and ventilation was adjusted to maintain carbon dioxide levels (PaCO2) between 33 and 41 mmHg. Tidal volumes (Vt) were 6–8 mL kg -1 The dynamic compliance was determined using equation (a).

number

[0273] In addition, an arterial line (Secalon-T™, Merit Medical Ireland Ltd, Galway, Ireland) was inserted into the right common carotid artery. A pulmonary artery catheter (Swan-Ganz CC Ambo V and Introflex, Edwards Lifesciences Services GmbH, Unterschleissheim, Germany) was placed into the right internal jugular vein.

[0274] ARDS induction by lipopolysaccharide: E. coli LPS (O111:B4, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was used intravenously to induce ARDS according to the Berlin criteria (Force et al.). Before administration, LPS was diluted in saline (2 μg kg -1 minutes -1 LPS administration resulted in hemodynamic instability in all animals, and norepinephrine (40 μg mL -1 , 0.05-2 μg kg -1 minutes -1 ; Pfizer AB, Sollentuna, Sweden) and dobutamine (2 mg mL -1 , 2.5-5 μg kg -1 minutes -1 Continuous inotropic support was required with intravenous infusion of 100 mg / kg of sucrose (Hameln Pharma Plus GmbH, Hameln, Germany). Acetated Ringer's solution (Baxter Medical AB, Kista, Sweden) was used to compensate for fluid losses. PaO2 and FiO2 were measured according to the Berlin definition (Force et al.) -1 Based on the ratio, different ARDS stages were defined: mild ARDS if the ratio was 201–300 mmHg, moderate ARDS if the ratio was 101–200 mmHg, and severe ARDS if the ratio was ≤100 mmHg. ARDS status was defined as the combination of two separate arterial blood gas measurements taken within 15 minutes of each other that exceeded the PaO2FiO2 ratio according to the Berlin definition. -1 It was considered as confirmed if it was within the range.

[0275] sHVF18 Treatment: In the treatment cohort, each animal received two doses of peptide solution (12 mg in 50 mL) intravenously over 30 minutes using a central venous catheter in the superior vena cava.

[0276] Arterial blood gas analysis: Arterial blood was collected every 30 min and analyzed with an ABL 90 FLEX blood gas analyzer (Radiometer Medical ApS, Bronshoj, Denmark). According to clinical standards, measurements were normalized to a blood temperature of 37 °C.

[0277] Hemodynamic measurements: Animals were closely observed and hemodynamic parameters were measured and recorded using a Swan-Ganz catheter and thermodilution via an arterial line before the start of ARDS induction and every 30 min thereafter. Parameters recorded were heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO), systolic pulmonary pressure (SPP), diastolic pulmonary pressure (DPP), mean pulmonary pressure (MPP), pulmonary artery wedge pressure (PAWP), systemic vascular resistance (SVR), and pulmonary vascular resistance (PVR).

[0278] Histopathological Analysis: After ARDS was confirmed, biopsies of the right lower lobe were taken. Biopsies were immediately transferred to 10% neutral buffered formalin solution (Sigma Aldrich, St. Louis, Missouri, USA) and fixed overnight at 4°C. Formalin-fixed tissues were treated with a graded ethanol series (solutions obtained from Histolab Products AB, Gothenburg, Sweden) and clearing solution (Sigma Aldrich) before paraffin embedding (Histolab). 4 μm sections were cut and transferred to SuperFrost Plus microscope slides (Thermo Fisher Scientific, Waltham, Massachusetts, US). Sections were allowed to dry overnight at room temperature. After deparaffinization, sections were stained with hematoxylin and eosin (both Merck Millipore, Darmstadt, Germany) and subsequently dehydrated in successive graded ethanol and xylene solutions (Histolab). Stained sections were finally mounted with Pertex solution (Histolab). Bright-field images were acquired by an Olympus CKX53 microscope (Olympus, Shinjuku, Tokyo, Japan). Images from each animal were independently scored for lung injury by three blinded scorers based on five parameters: the number of immune cells in the alveolar and interstitial spaces, the occurrence of proteinaceous debris, the presence of thickening and architectural changes in the alveolar septa, and finally the presence of hemorrhage, hyaline membranes, or other signs of enhanced injury. Scores were given based on a scale of 0 to 6 for each criterion. Scores are presented as the mean of the sum of the characteristic scores for each sample.

[0279] result Analysis of hemolytic and anti-inflammatory activities of shorter mutants of sHVF18 As shown in Example 1, stapling of HVF18 increased its stability against proteolysis, significantly improved its anti-inflammatory activity (in vitro and in vivo), and directed its activity mainly against Gram-positive bacteria while maintaining its antibacterial activity, with low hemolytic activity. To evaluate how small the active peptide can be made, shorter mutants of sHVF18 were generated. Double-stapled GKY25 (2sGKY25), one at the C-terminal region as in sGKY25 and one at the N-terminal region, were used for comparison.

[0280] A library of different stapled peptides was generated and then screened for their hemolytic effect (Fig. 20a) versus anti-inflammatory activity (Fig. 20b). IC of TNF-α and IL-1β in LPS-stimulated blood 50 When showing the percentage of hemolytic activity of all peptides against erythrocytes as a function of the concentration of the same peptide required for hemolysis, sHVF18 in particular turned out to be promising (Figure 20c).

[0281] Analysis of hemolytic and anti-inflammatory activities of sKKW13 mutants Both sLKK14 and sKKW13 showed low hemolytic activity (Fig. 20a), but sLKK14 tended to oligomerize and lose its immunomodulatory effect when in solution, whereas sKKW13 did not (Fig. 20b). Therefore, we hypothesized that adding one or two K and R residues to sKKW13 might improve its anti-inflammatory activity. We first compared the hemolytic activity of these new peptides with the original peptide and sHVF18 against whole blood (Fig. 21a). The K and R mutants showed much higher hemolytic activity. We then tested their ability to reduce cytokine production in LPS-stimulated blood (Fig. 21b). All K and R mutants were less active than sHVF18 and did not provide significant improvement when compared to sKKW13.

[0282] Effect of sHVF18 in a porcine ARDS model The therapeutic effect of sHVF18 in an established preclinical porcine model of ARDS was further investigated. ARDS was induced by intravenous injection of E. coli LPS (study summary shown in Fig. 22a). All pigs became hemodynamically unstable and required inotropic support with norepinephrine after LPS administration (inotropic support refers to the use of dobutamine and norepinephrine drugs for clinical purposes to maintain hemodynamic stability). This hemodynamic instability as well as differences between treated and untreated pigs over the time course of the experiment are shown by PaO2 / FiO2 ratio (Fig. 22b), cardiac output (Fig. 22c), urine output (Fig. 22d), norepinephrine (NA, Fig. 22e), and lactate levels (Fig. 22f).

[0283] Overall, after intravenous administration of sHVF18, treated animals were hemodynamically stable and required significantly less inotropic support compared to untreated animals. Treated animals did not deteriorate as much as untreated animals in terms of PaO2 / FiO2 ratio, and only one of five treated animals showed mild ARDS (according to the Berlin definition of ARDS) according to blood gases.

[0284] Lung tissue samples were taken from the right lower lobe at the end of the experiment and compared with lung tissue samples from five healthy control pigs. Lung biopsies taken from healthy controls for histological analysis appeared normal without any abnormalities (Figure 22g, left panel). All biopsies taken from both treated and untreated animals at the end of the experiment showed signs of diffuse alveolar damage, such as immune cell infiltration and thickening of the alveolar-capillary barrier due to intra-alveolar hemorrhage, but less in treated pigs (Figure 22g, middle and right panels). In addition to the subjective analysis of lung histology, blinded scoring was performed on all pigs by three independent observers. A significant increase in cumulative lung injury score was seen in the untreated group compared to healthy controls, explaining the multiple signs of lung injury following the onset of acute lung injury by LPS administration. A significant difference was seen between treated and untreated pigs (Figure 22h), with treated pigs showing less signs of lung injury.

[0285] Example 6 Materials and Methods In silico analysis of peptide staple positions The NMR structure of HVF18 (PDB:5Z5X) (Saravanan et al., 2018) was docked into the modeled structure of human CD14 using the ClusPro web server (Kozakov et al., 2017). Similar results were obtained as described in Saravanan et al., 2018, whereby the peptide binds to the N-terminus of CD14. The structure with the highest scoring docking pose was selected as a template to model GKY25. The N-terminal GKYGFYT residues were modeled using Modeller version 9.21, and the model with the lowest individually optimized protein energy score (Shen et al., 2006) was selected. The GKY25-CD14 complex was solvated with TIP3P water and NaCl salt using CHARMM-GUI Solution Builder as described in (Jo et al., 2008). Steepest descent energy minimization and 125 ps equilibration simulations (whereby positional restraints are applied to the protein and peptide backbone atoms) were performed following the standard CHARMM-GUI protocol (Lee et al., 2016). The final snapshots after equilibration were extracted and the binding energy between GKY25 and CD14 was calculated using the molecular mechanics Poisson-Boltzmann surface area (MMPBSA) method (Kumari et al., 2014). Hydrophobic staples were added to the GKY25 peptide by mutating residues at positions i and i+3 to alanines and linking them with two pentene segments using CHARMM-GUI Solution Builder (Jo et al., 2008). Stapled GKY25 bound to CD14 was then subjected to the same solvation, minimization and equilibration procedures as above, after which their binding energies were determined using MMPBSA. A similar protocol was performed to add staples at positions i and i+4. The binding energy difference relative to unstapled GKY25 was then calculated. A similar analysis of peptide staple positions was also performed on the shorter HVF18 peptide.

[0286] result Design of dual-acting peptides with improved proteolytic stability Next, an in silico analysis of peptide staple positions was performed, and a short hydrophobic pentenylalanine staple linking residues i and i+3 or residues i and i+4 was added along the sequence of GKY25. To determine the effect of this staple addition on binding to CD14, the binding energy difference between the non-stapled and stapled versions of the peptide was then calculated. The results are shown in Figure 23. We found that adding staples to most positions of the longer GKY25 peptide, especially to the N-terminal region of the peptide, reduced binding as indicated by the positive binding energy difference (see results in Figures 23A and B). The reduced affinity may have been caused by the staples disrupting the interaction between the peptide and CD14. Some staples improved binding to CD14. In the i-i+3 configuration, these include I16-V19, V19-Q22, I20-D23, and D21-G24, and in the i-i+4 configuration, these include V9-K13 and Q17-D21. With the exception of the Q17-D21 staple, all staples that conferred more favorable binding to CD14 contain hydrophobic residues that may be important for interaction with LPS.

[0287] In contrast, the shorter HVF18 peptide showed improved affinity at most staples (see Figures 23C and D). In the i-i+3 configuration, at least the following staples showed improved affinity: V2-L5, F3-K6, L5-W8, K6-I9, I9-V12, Q10-I13, K11-D14, V12-Q15, I13-F16, D14-G17, and Q15-E18. In the i-i+4 configuration, at least the following staples showed improved affinity: V2-K6, K6-Q10, W8-V12, I9-I13, Q10-D14, V12-F16, I13-G17, and D14-E18. Of the staples that provided improved binding, Q10-D14 (corresponding to Q17-D21 in GKY25) has one of the most negative binding energy differences.

[0288] References Force, A.D.T. et al. Acute respiratory distress syndrome: the Berlin Definition. JAMA 307, 2526 - 2533 (2012). Jo, S., Kim, T., Iyer, V. & Im, W. CHARMM - GUI: A Web - Based Graphical User Interface for CHARMM. J. Comput. Chem. 29, 1859 - 1865 (2008). Kalle, M. et al. Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin - mediated shock and Pseudomonas aeruginosa sepsis. PLoS One 7, e51313 (2012). Kozakov, D. et al. The ClusPro web server for protein - protein docking. Nat. Protoc. 12, 255 - 278 (2017). Kumari, R., Kumar, R. & Lynn, A. G - mmpbsa - A GROMACS tool for high - throughput MM - PBSA calculations. J. Chem. Inf. Model. 54, 1951 - 1962 (2014). Lee, J. et al. CHARMM - GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM / OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput. 12, 405 - 413 (2016). Li,X.,et al.Stapled Helical Peptides Bearing Different Anchoring Residues,Chem.Rev.2020,120,10079-10144 Morrisett,J.D.,et al.Interaction of an apolipoprotein(apoLP-alanine) with phosphatidylcholine.Biochemistry 12,1290-1299(1973). Papareddy,P.et al.Proteolysis of human thrombin generates novel host defense peptides.PLoS Pathog 6,e1000857(2010). Petruk,G.,et al.SARS-CoV-2 Spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity.Journal of Molecular Cell Biology,12(12) 916-932(2020). Petruk,G.,et al.,Concentration-and pH-dependent oligomerization of the thrombin-derived C-terminal peptide TCP-25.Biomolecules,10(11),1572(2020). Puthia,M.et al.A dual-action peptide-containing hydrogel targets wound infection and inflammation.Sci Transl Med 12(2020). Puthia, M., et al. A dual-action peptide-containing hydrogel targets wound infection and inflammation. Science translational medicine, 12(524)(2020). Saravanan, R., et al. Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides. Nature communications, 9(1), 1-14(2018). Shen, M., Devos, D., Melo, F. & Sali, A. A composite score for predicting errors in protein structure models. Protein Sci. 15, 1653-1666(2006). Shi, X.E. et al. Hydrogen exchange-mass spectrometry measures stapled peptide conformational dynamics and predicts pharmacokinetic properties. Anal Chem 85, 11185-11188(2013). Stromdahl, A.C. et al. Peptide-coated polyurethane material reduces wound infection and inflammation. Acta Biomater(2021). [Table 3]

[0289] Abbreviation: AMP, antimicrobial peptides; AMR, antimicrobial resistance; CD, circular dichroism; PAMP, pathogen-associated molecular pattern; TCP-25, 25 amino acid thrombin C-terminal peptide; TEM, transmission electron microscopy; TLR, toll-like receptor.

[0290] Overview of Arrays [Table 4] TIFF2024539138000007.tif212159

[0291] In the above sequence listing, X1 and X2 are initially (S)-2-(4'pentenyl)-alanine, respectively, which react with each other by RCM to form an olefin tether. The sequence listing shows the sequence of the unreacted peptide. Those skilled in the art will understand that even if the unreacted sequence is provided, the peptide is generally used in a stapled form, i.e., after the olefin tether is formed by RCM. In addition, X3 and X4 are glycine and glutamic acid, respectively, which react with each other to form a covalent bond in the form of a lactam bridge.

[0292] item The present invention may be further defined by any one of the following: 1. A peptide comprising a continuous sequence of 10 to 23 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, i) having a total length of 10 to 40 amino acids, ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) further comprising at least the amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; provided that, when the peptide has a total length of 24-40 amino acids, the peptide comprises at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids, the amino acids of a first internal covalent bond being designated X1 and X2, and the amino acids of a second internal covalent bond being designated X3 and X4.

[0293] 2. The peptide according to item 1, wherein the peptide comprises a continuous sequence of 10 to 40 amino acids derived from thrombin of SEQ ID NO:1.

[0294] 3. A peptide comprising a continuous sequence of 10 to 23 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, the peptide comprising: i) having a total length of 10 to 23 amino acids; ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) The peptide comprises at least the amino acids K247, K248 and K252 of thrombin of SEQ ID NO:1.

[0295] 4. A peptide comprising a continuous sequence of 10 to 23 amino acids derived from GKY25 of SEQ ID NO: 12 containing up to 6 amino acid substitutions, the peptide comprising: i) having a total length of 10 to 23 amino acids; ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) The peptide comprising at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO:12.

[0296] 5. The peptide according to any one of the preceding items, wherein the peptide comprises a contiguous sequence of 13 to 23 amino acids derived from thrombin of SEQ ID NO: 1 or GKY25 of SEQ ID NO: 12.

[0297] 6. Amino acid sequence: -UU-(Z) n-IQKVIDQ-(Z) m - A peptide comprising or consisting of The peptide has a total length of 10 to 23 amino acids, each Z is individually any standard amino acid; U is His, Lys or Arg; n is an integer ranging from 0 to 10; m is an integer ranging from 0 to 5; The peptide, wherein two of the amino acids are replaced with alkenylated amino acids, the side chains of which are covalently attached.

[0298] 7. The peptide according to any one of the preceding claims, wherein the peptide has an overall length of 13 to 23 amino acids.

[0299] 8. A peptide comprising a continuous sequence of 13 to 23 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, the peptide comprising: i) having a total length of 13 to 23 amino acids; ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) The peptide comprises at least the amino acids R245, K247, K248, and K252 of thrombin of SEQ ID NO:1.

[0300] 9. The peptide according to any one of items 1 and 8, wherein the peptide comprises at least the amino acids R245, K247, K248, K252 of thrombin according to SEQ ID NO:1.

[0301] 10. A peptide comprising a continuous sequence of 13 to 23 amino acids derived from GKY25 of SEQ ID NO: 12 containing up to 6 amino acid substitutions, the peptide comprising: i) having a total length of 13 to 23 amino acids; ii) comprising at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, said amino acids being designated X1 and X2; iii) The peptide comprising at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO:12.

[0302] 11. The peptide according to item 10, wherein the peptide comprises at least the amino acids R11, K13, K14 and K18 of thrombin according to SEQ ID NO: 12.

[0303] 12. The peptide of any one of the preceding claims, wherein the peptide contains only one internal covalent bond between two non-adjacent internal amino acids.

[0304] 13. A peptide according to any one of the preceding items, wherein the amino acid X1 is located at position n and the amino acid X2 is located at position n+3, or at position n+4, or at position n+5, or at position n+6, or at position n+7, or at position n+8, or at position n+9, or at position n+10, or at position n+11, where n is an integer in the range of 2 to 18.

[0305] 14. The peptide according to any one of the preceding items, wherein the amino acid X1 is located at position n and the amino acid X2 is located at position n+3, or at position n+4, or at position n+7, or at position n+11, where n is an integer in the range of 2 to 18.

[0306] 15. The peptide according to any one of the preceding items, wherein the amino acid X1 is located at position n and the amino acid X2 is located at position n+3, or at position n+4, or at position n+7, where n is an integer ranging from 2 to 18.

[0307] 16. The peptide according to any one of the preceding items, wherein the amino acid X1 is located at position n and the amino acid X2 is located at position n+4 or at position n+7, where n is an integer ranging from 2 to 18.

[0308] 17. When the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12, a. X2 does not match Lys in GKY25 of SEQ ID NO: 12; b. A peptide according to any one of the preceding items, provided that if X1 matches Lys, then X2 does not match Gln.

[0309] 18. When the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12, a. X1 does not match Arg11 in GKY25 of SEQ ID NO: 12; b. X1 does not match Lys14 in GKY25 of SEQ ID NO: 12; c. X2 does not match Lys14 in GKY25 of SEQ ID NO: 12; d. X2 does not match Lys18 in GKY25 of SEQ ID NO: 12; and e. A peptide according to any one of the preceding items, provided that if X1 corresponds to Lys18 of SEQ ID NO: 12, then X2 does not correspond to Gln22.

[0310] 19. An amino acid X1 is located at position n and an amino acid X2 is located at position n+3, where n is an integer ranging from 2 to 18, provided that when the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12, a. X1 does not match Arg11 in GKY25 of SEQ ID NO: 12; b. X1 does not match Lys14 in GKY25 of SEQ ID NO: 12; and c. The peptide of any one of the preceding items, provided that X2 does not match Lys14 in GKY25 of SEQ ID NO:12.

[0311] 20. Amino acid X1 is located at position n and amino acid X2 is located at position n+4, where n is an integer ranging from 2 to 18, provided that when the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12, a. X1 does not match Arg11 in GKY25 of SEQ ID NO: 12; b. X1 does not match Leu12 in GKY25 of SEQ ID NO: 12; c. X1 does not match Lys14 in GKY25 of SEQ ID NO: 12; d. X2 does not match Lys14 in GKY25 of SEQ ID NO: 12; e. X1 does not match Lys18 in GKY25 of SEQ ID NO: 12; and f. The peptide of any one of the preceding items, provided that X2 does not match Lys18 in GKY25 of SEQ ID NO:12.

[0312] 21. A peptide according to any one of the preceding items, wherein amino acid X1 is located at position n and amino acid X2 is located at position n+3, where n is an integer in the range of 2 to 18, and when the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12, X1 and X2 correspond to Val9 and Leu12; or Phe10 and Lys13; or Leu12 and Trp15; or Lys13 and Ile16; or Ile16 and Val19; or Gln17 and Ile20; or Lys18 and Asp21; or Val19 and Gln22; or Ile20 and Phe23; or Asp21 and Gly24; or Gln22 and Glu25 of SEQ ID NO: 12.

[0313] 22. A peptide according to any one of the preceding items, wherein amino acid X1 is located at position n and amino acid X2 is located at position n+4, where n is an integer ranging from 2 to 18, and when the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12, X1 and X2 correspond to Val9 and Lys13; or Lys13 and Gln17; or Trp15 and Val19; or Ile16 and Ile20; or Gln17 and Asp21; or Val19 and Phe23; or Ile20 and Gly24; or Asp21 and Glu18 of SEQ ID NO: 12.

[0314] 23. A peptide according to any one of the preceding items, wherein amino acid X1 is located at position n and amino acid X2 is located at position n+4, where n is an integer ranging from 2 to 18, and X1 and X2 correspond to Gln17 and Asp21 of SEQ ID NO: 12 when the peptide sequence is aligned with the sequence of GKY25 of SEQ ID NO: 12.

[0315] 24. The peptide according to any one of the preceding items, wherein X1 and X2 are selected from the group consisting of: i) X1 is Lys and X2 is selected from the group consisting of Asp, Glu, Lys, Cys and Tyr; ii) X1 is Cys and X2 is selected from the group consisting of Cys, Lys and Met; iii) X1 is Asp and X2 is Lys; iv) X1 is Glu and X2 is selected from the group consisting of Lys and Glu; v) X1 is Tyr and X2 is selected from the group consisting of Lys, Phe and Trp; vi) X1 is Met and X2 is selected from the group consisting of Met and Cys; vii) X1 is His and X2 is His; viii) X1 is Phe and X2 is selected from the group consisting of Phe, Tyr, Ala and Trp; ix) X1 is Ala and X2 is Phe or Tyr; x) X1 is Trp and X2 is selected from the group consisting of Trp, Phe and Tyr; xi) X1 is Gly and X2 is Glu.

[0316] 25. A peptide according to any one of the preceding items, wherein X1 is Lys and X2 is Asp, Glu, Cys or Lys, or vice versa.

[0317] 26. The peptide according to any one of the preceding items, wherein X1 and X2 are Cys and the covalent bond is either a direct covalent bond (i.e. a disulfide bridge) or via a cross-linking agent, the cross-linking agent being, for example, a bis-alkylating agent, for example a linker comprising at least two (bromomethyl) substituents.

[0318] 27. A peptide according to any one of the preceding paragraphs, wherein either X1 or X2 is a derivatized standard amino acid, for example selected from the group consisting of Ser derivatives and Ala derivatives.

[0319] 28. The peptide according to any one of the preceding items, wherein the covalent bond is formed by linking two non-standard amino acids, optionally wherein the non-standard amino acids replace two natural amino acids of thrombin.

[0320] 29. The peptide of any one of the preceding claims, wherein the covalent bond is a carbohydrate staple.

[0321] 30. The peptide according to any one of the preceding items, wherein X1 and X2 are alkenylated amino acids, such as two α-substituted alkenyl amino acids and / or two C-alkenylated amino acids, such as α,α-disubstituted alkenyl amino acids, and the covalent bond is an olefin tether formed between the alkenyl residues.

[0322] 31. The peptide according to item 30, wherein the alkenylated amino acid is an alkenylated thrombin-specific amino acid and / or an alkenylated amino acid that substitutes for a thrombin-specific amino acid.

[0323] 32. The peptide of any one of the preceding claims, wherein X1 and X2 are independently selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe.

[0324] 33. A peptide according to any one of the preceding items, wherein one of X1 or X2 is alkenylated Ala, and the other is selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe.

[0325] 34. A peptide according to any one of the preceding items, wherein X1 and / or X2 is an alkenylated alanine.

[0326] 35. A peptide according to any one of the preceding items, wherein X1 and / or X2 is an α,α-disubstituted alkenylated alanine and the covalent bond is an olefinic tether formed between the alkenyl residues.

[0327] 36. The peptide of any one of the preceding items, wherein X1 and / or X2 are linked by a tether, said tether being an alkene chain of 10 carbon atoms counting from the C-alpha carbon.

[0328] 37. The peptide of any one of the preceding claims, wherein X1 and / or X2 are alkenylated alanines linked to each other by ring-closing metathesis to form an alkene tether, said tether being an alkene chain of 10 carbon atoms counting from the C-alpha carbon.

[0329] 38. The peptide according to item 36 or 37, wherein the alkene chain is an unbranched alkene chain.

[0330] 39. A peptide according to any one of the preceding items, wherein X1 and / or X2 is alkenylated Ser, such as O-alkenylated Ser.

[0331] 40. A peptide according to any one of items 30 to 39, wherein the alkenylated amino acid contains 2 to 10 carbons in the alkenyl chain, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons, preferably 4, 5 or 6 carbons.

[0332] 41. The peptide according to any one of items 30 to 40, wherein the alkenylated amino acid is an α-substituted alkenyl olefin terminal amino acid and / or an α,α-disubstituted alkenyl olefin terminal amino acid.

[0333] 42. A peptide according to any one of the preceding items, wherein X1 and / or X2 is an α,α-disubstituted S- or R-pentenylalanine (S5 or R5) and S- or R-octenylalanine (S8 or R8) alanine.

[0334] 43. The peptide of any one of the preceding items, wherein the internal carbohydrate staple is formed by linking two (S)-2-(4'-pentenyl)-alanines.

[0335] 44. The peptide according to any one of the preceding items, wherein the covalent bond is established by ring closure.

[0336] 45. The peptide according to any one of the preceding items, wherein the covalent bond is established by ring-closing metathesis (RCM).

[0337] 46. ​​A peptide according to any one of the preceding items, wherein X1 is a non-standard azido-terminal amino acid and X2 is a non-standard in-terminal amino acid, or vice versa.

[0338] 47. A peptide according to any one of the preceding items, wherein X3 and X4 are closer to the N-terminus than X1 and X2.

[0339] 48. The peptide according to any one of the preceding items, wherein the amino acid X3 is located at position n and the amino acid X4 is located at position n+3, or at position n+4, or at position n+5, where n is an integer. In a preferred embodiment, the amino acid X3 is located at position n and the amino acid X4 is located at position n+4.

[0340] 49. The peptide according to any one of the preceding items, wherein amino acid X3 is located at the extreme N-terminus of the peptide.

[0341] 50. A peptide according to any one of the preceding claims, wherein amino acid X3 is located at the extreme N-terminus of the peptide and amino acid X4 is located at the n+4 position.

[0342] 51. A peptide according to any one of the preceding claims, wherein the internal covalent bond is an amide bond formed by reaction of an amine with a carboxylic acid.

[0343] 52. The peptide according to any one of the preceding claims, wherein the internal covalent bond is a lactam bridge formed by reaction of an amine with a carboxylic acid.

[0344] 53. The peptide of any one of the preceding claims, wherein X3 is an N-terminal amino acid having an N-terminal amine group and X4 is an amino acid having a carboxylic acid side chain, and X3 and X4 are linked by an internal covalent bond formed by reacting the amine with the carboxylic acid.

[0345] 54. The peptide according to any one of the preceding items, wherein when the sequence of the peptide of the present invention is aligned with the sequence of GKY25 of SEQ ID NO:12, X3 and X4 correspond to Gly1 and Phe5 of GKY25 of SEQ ID NO:12.

[0346] 55. The peptide according to any one of the preceding paragraphs, wherein X3 and X4 correspond to Gly1 and Glu5 of SEQ ID NO:14.

[0347] 56. The peptide according to any one of the preceding items, wherein the peptide has a length of 14 to 22 amino acids, such as a length of 15 to 21 amino acids, such as a length of 16 to 20 amino acids, such as a length of 17 to 20 amino acids.

[0348] 57. The peptide according to any one of the preceding items, wherein the peptide has a length of 18 to 20 amino acids, for example 18 or 19 amino acids.

[0349] 58. The peptide according to any one of the preceding items, wherein the peptide comprises a consecutive amino acid sequence of 14 to 22 amino acids, such as 13 to 18 amino acids, such as 15 to 21 amino acids, such as 16 to 20 amino acids, such as 17 to 20 amino acids, preferably 17 to 18 amino acids, derived from thrombin of SEQ ID NO:1.

[0350] 59. The peptide according to any one of the preceding items, wherein the peptide has a length of 24 to 40 amino acids, such as a length of 25 to 35 amino acids, such as a length of 25 to 30 amino acids, such as a length of 28 to 34 amino acids.

[0351] 60. A peptide according to any one of the preceding items, wherein the peptide comprises or consists of a contiguous sequence of amino acids derived from thrombin of SEQ ID NO: 1, ranging from 16 to 21 amino acids, including up to 6 amino acid substitutions.

[0352] 61. The peptide according to any one of the preceding items, wherein the peptide comprises or consists of a contiguous sequence of amino acids from thrombin of SEQ ID NO:1, ranging from 17 to 18 amino acids, including up to two amino acid substitutions, and the peptide may include up to four additional amino acids.

[0353] 62. The peptide of any one of the preceding claims, wherein the peptide comprises or consists of a contiguous sequence of amino acids from thrombin of SEQ ID NO: 1, ranging from 17 to 18 amino acids, including one amino acid substitution for amino acid X1 and one amino acid substitution for amino acid X2, and the peptide may include up to four additional amino acids.

[0354] 63. A peptide according to any one of the preceding items, wherein the peptide comprises at least two amino acid substitutions, such as three amino acid substitutions, such as four amino acid substitutions, such as five amino acid substitutions, compared to the consecutive sequence of thrombin.

[0355] 64. The peptide according to any one of the preceding items, wherein one or more of the substitutions are conservative substitutions, e.g. 1, 2, 3 or 4 amino acid substitutions are conservative substitutions.

[0356] 65. The peptide of any one of the preceding claims, wherein the peptide further comprises one or more moieties attached to the peptide, and optionally the peptide and the one or more moieties are attached to each other by a linker, and the one or more moieties are selected from the group consisting of alkyl, aryl, heteroaryl, olefin, fatty acid, polyethylene glycol (PEG), saccharide, and polysaccharide.

[0357] 66. A peptide according to any one of the preceding items, wherein the peptide further comprises 1 to 5, such as 1 to 4, such as 1 to 3, such as 1 to 2, such as 2 positively charged amino acids inserted at or near the termini of the peptide.

[0358] 67. The peptide according to item 66, wherein the positively charged amino acid is inserted at or near the N-terminus, for example at a position selected from positions 1, 2, 3, 4 and / or 5 relative to the N-terminus of the peptide.

[0359] 68. The peptide according to item 66 or 67, wherein the positively charged amino acid is inserted at the N-terminus.

[0360] 69. A peptide according to any one of the preceding items, wherein the peptide comprises two positively charged amino acids inserted at or near the N-terminus, e.g., at a position selected from positions 1, 2 and / or 3 relative to the N-terminus of the peptide.

[0361] 70. The peptide according to any one of the preceding items, wherein the peptide consists of in the range of 15-20 contiguous amino acids of thrombin of SEQ ID NO:1, in which two amino acids are replaced with alkenylated amino acids forming an internal carbohydrate staple, and in the range of 2-5 additional N-terminal amino acids.

[0362] 71. The peptide according to any one of the preceding items, wherein the peptide consists of in the range of 16-18 contiguous amino acids of thrombin of SEQ ID NO:1, in which two amino acids are replaced with alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2-5 additional N-terminal amino acids.

[0363] 72. The peptide according to any one of the preceding items, wherein the peptide consists of 17 consecutive amino acids of thrombin of SEQ ID NO:1, with two amino acids replaced with alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2-3 additional N-terminal amino acids.

[0364] 73. The peptide according to any one of items 66 to 72, wherein the additional N-terminal amino acid is positively charged.

[0365] 74. The peptide according to any one of items 66 to 73, wherein the positively charged amino acid is selected from the group consisting of arginine, lysine and histidine, preferably, the positively charged amino acid is arginine and / or lysine, even more preferably, the positively charged amino acid is lysine.

[0366] 75. A peptide according to any one of the preceding items, wherein the alkenylated amino acid is as defined in any one of items 30 to 43.

[0367] 76. The peptide has the sequence: -KKZZ-X1-KZZ-X2-Z wherein each Z is individually any standard amino acid; Item 11. The peptide of any one of the preceding items, wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0368] 77. The peptide has the sequence: UU-(Z) n -KKZZ-X1-KZZ-X2-Z wherein each Z is individually any standard amino acid; U is His, Lys or Arg; n is an integer ranging from 0 to 10; Item 11. The peptide of any one of the preceding items, wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond.

[0369] 78. The peptide has the sequence: wherein each Z is individually any standard amino acid; Item 11. The peptide of any one of the preceding items, wherein X1 and X2 are amino acids linked by a covalent bond.

[0370] 79. The peptide has the sequence: UUZZRZKKZZ-X1-KZZ-X2-Z comprising or consisting of: each Z is individually any standard amino acid; U is His, Arg or Lys; Item 11. The peptide of any one of the preceding items, wherein X1 and X2 are amino acids linked by a covalent bond.

[0371] 80. The peptide has the sequence: VFRLKKWI-X1-KVI-X2-QFGE comprising or consisting of: Item 11. The peptide of any one of the preceding items, wherein X1 and X2 are amino acids linked by a covalent bond.

[0372] 81. The peptide has the sequence: UUVFRLKKWI-X1-KVI-X2-QFGE comprising or consisting of: U is His, Arg or Lys; Item 11. The peptide of any one of the preceding items, wherein X1 and X2 are amino acids linked by a covalent bond.

[0373] 82. The peptide according to any one of items 76 to 81, wherein U is Lys or Arg.

[0374] 83. The peptide, i) SEQ ID NO:3, ii) SEQ ID NO:4, iii) SEQ ID NO:5, iv) SEQ ID NO:6, v) SEQ ID NO: 7, vi) SEQ ID NO:8, vii) SEQ ID NO: 9, viii) SEQ ID NO: 10, ix) SEQ ID NO: 11, x) SEQ ID NO: 17, xi) SEQ ID NO: 18, xii) SEQ ID NO: 19, xiii) SEQ ID NO: 20, xiv) SEQ ID NO: 21, xv) SEQ ID NO: 22, xvi) SEQ ID NO: 23, xvii) SEQ ID NO: 24, xviii) SEQ ID NO: 25, or xix) SEQ ID NO: 26 wherein X1 and X2 are covalently bonded amino acids and X3 and X4 are covalently bonded amino acids.

[0375] 84. The peptide, iv) SEQ ID NO:3, v) SEQ ID NO: 4, vi) SEQ ID NO:5, vii) SEQ ID NO:8, or viii) SEQ ID NO: 9 3. A peptide according to any one of the preceding claims, comprising or consisting of a sequence as set forth in:

[0376] 85. The peptide, i) SEQ ID NO:3, ii) SEQ ID NO:5, iii) SEQ ID NO:6, or iv) SEQ ID NO: 7 wherein X1 and X2 are amino acids whose side chains are covalently linked together, optionally X1 and X2 are (S)-2-(4'-pentenyl)-alanine whose side chains have reacted with each other to form an alkenyl tether, and further optionally X1 and X2 are Ala whose side chains are covalently linked together by a C8 alkenyl tether comprising one double bond.

[0377] 86. The peptide, i) SEQ ID NO: 3, or ii) SEQ ID NO:5 wherein X1 and X2 are amino acids whose side chains are covalently linked together, optionally X1 and X2 are (S)-2-(4'-pentenyl)-alanine whose side chains have reacted with each other to form an alkenyl tether, and further optionally X1 and X2 are Ala whose side chains are covalently linked together by a C8 alkenyl tether comprising one double bond.

[0378] 87. The peptide, i) SEQ ID NO: 14, or ii) SEQ ID NO: 26 wherein X1 and X2 are amino acids whose side chains are linked by a covalent bond, and further wherein X3 and X4 are amino acids whose side chains are linked by a covalent bond, preferably X1 and X2 are (S)-2-(4'-pentenyl)-alanine whose side chains have reacted with each other to form an alkenyl tether, and X3 and X4 are Gly and Glu, respectively, that have reacted with each other to form a lactam bridge.

[0379] 88. The peptide according to any one of items 49 to 55, wherein X1 and X2 are as defined in any one of items 13 to 47.

[0380] 89. A peptide according to any one of the preceding items, wherein X3 and X4 are as defined in any one of items 13 to 55.

[0381] 90. The peptide according to any one of the preceding items, wherein the peptide comprises at least one alpha-helical secondary structure unit in aqueous solution.

[0382] 91. The peptide according to item 90, wherein the alpha-helical secondary structural unit is maintained upon binding to a target molecule, such as cluster differentiation 14 (CD14).

[0383] 92. The peptide according to any one of the preceding items, wherein the peptide has a hydrodynamic radius of less than 150 nm, such as less than 100 nm at pH 7.4, and / or less than 100 nm, such as less than 80 nm at pH 5.

[0384] 93. The peptide according to any one of the preceding items, wherein the peptide has increased stability in vivo and / or in vitro compared to a peptide having the same sequence except that amino acids X1 and X2 are replaced by other amino acids lacking a covalent bond, when the peptide is tested under the same conditions.

[0385] 94. The peptide according to any one of the preceding items, wherein the peptide has increased stability in the presence of a serine protease, for example, compared to a peptide having the same sequence except that amino acids X1 and X2 have been replaced by other amino acids lacking a covalent bond, when the peptide is tested under the same conditions.

[0386] 95. The peptide of any one of the preceding items, wherein 50 μM of the peptide has a hemolytic activity of up to 5% (such as up to 4%), for example up to 3% (such as up to 2%) in fresh whole blood.

[0387] 96. A peptide according to any one of the preceding items, having a hemolytic activity of up to 10%, preferably up to 5%, in fresh whole blood at a peptide concentration that reduces TNF-α release in LPS-stimulated blood in vitro by 50%.

[0388] 97. The peptide of any one of the preceding items, wherein the peptide has anticoagulant activity.

[0389] 98. The peptide of any one of the preceding items, wherein the peptide increases the clotting time measured by aPPT by at least 100% at a concentration of 60 μM and / or increases the clotting time measured by aPPT by at least 90% at a peptide concentration of 40 μM.

[0390] 99. The peptide of any one of the preceding claims, wherein the peptide reduces inflammation and / or infection.

[0391] 100. The peptide of any one of the preceding claims, wherein the peptide reduces secretion of proinflammatory cytokines in the presence of one or more endotoxins, such as LPS.

[0392] 101. The peptide according to any one of the preceding items, wherein the peptide reduces in vivo secretion of proinflammatory cytokines in blood, including one or more endotoxins, such as LPS.

[0393] 102. The peptide according to item 100 or 102, wherein the proinflammatory cytokine is selected from the group consisting of tumor necrosis factor alpha (TNF-α), interleukin beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interferon (IFN-γ) and / or monocyte chemotactic protein-1 (MCP-1).

[0394] 103. The peptide of any one of the preceding claims, wherein the peptide reduces NF-κB activity in the presence of a toll-like receptor (TLR) agonist, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), Staphylococcus aureus peptidoglycan (SA-PGN) and / or zymosan.

[0395] 104. The peptide according to any one of the preceding paragraphs, wherein the peptide at a concentration of 10 μM reduces the secretion of TNF-α and / or IL-1β after incubation in fresh blood in the presence of LPS by at least 50%, such as at least 60%, for example at least 70%, when compared to a peptide of the same sequence except that the amino acids X1 and X2 are replaced by other amino acids lacking a covalent bond, when the peptide is tested under the same conditions.

[0396] 105. The peptide of any one of the preceding items, wherein the peptide is bactericidal, e.g., the peptide can kill bacteria by damaging bacterial membranes.

[0397] 106. The peptide according to item 105, wherein the bacteria is selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

[0398] 107. The peptide according to any one of the preceding items, wherein the anti-inflammatory effect of the peptide is maintained in vivo for 24 hours after systemic administration of the peptide.

[0399] 108. A peptide according to any one of the preceding items for use as a medicament.

[0400] 109. A peptide according to any one of the preceding items for use in a method for the treatment and / or prevention of inflammation and / or infection in an individual in need thereof.

[0401] 110. The peptide for use according to item 109, wherein the inflammation is associated with an infection.

[0402] 111. A peptide according to any one of items 1 to 108 for use in a method for the treatment or prevention of a disease selected from the group consisting of acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic rhinitis, other types of rhinitis, vasculitis, thrombosis, disseminated intravascular coagulation (DIC), gastroenteritis, and pulmonary inflammation.

[0403] 112. The peptide for use according to item 109 or 110, wherein the inflammation is selected from the group consisting of acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), gastroenteritis, and pulmonary inflammation.

[0404] 113. The peptide for use according to item 109 or 110, wherein the inflammation is ARDS.

[0405] 114. Peptide for use according to any one of items 109 to 113, wherein the treatment is systemic.

[0406] 115. Peptide for use according to any one of items 109 to 114, wherein the method comprises parenteral administration of the peptide, such as subcutaneous administration or intravenous administration.

[0407] 116. The peptide for use according to any one of items 109 to 115, wherein the individual is a human.

[0408] 117. The peptide for use according to any one of items 109 to 116, wherein the infection is an infection caused by a microorganism, such as a bacterial infection or a viral infection.

[0409] 118. The peptide for use according to any one of items 109 to 117, wherein the individual is suffering from a bacterial infection.

[0410] 119. The peptide for use according to item 118, wherein the bacterial infection is an acute or chronic bacterial infection, for example an infection with a gram-negative bacterium.

[0411] 120. The peptide for use according to any one of items 109 to 119, wherein the individual has increased levels of endotoxins, such as increased levels of LPS, LTA and / or SA-PGN.

[0412] 121. The peptide for use according to any one of items 109 to 120, wherein the individual has an increased level of endotoxin in one or more body fluids, optionally wherein the body fluid is selected from the group consisting of blood, serum, saliva, a nasopharyngeal swab sample and a bronchoalveolar lavage (BAL) sample, and further optionally wherein the endotoxin is LPS.

[0413] 122. The peptide for use according to any one of items 109 to 121, wherein the increased level of LPS is at a level of at least 50 pg / ml, such as a serum level of LPS of at least 50 pg / ml.

[0414] 123. The peptide for use according to any one of items 109 to 122, wherein the individual is suffering from a viral infection.

[0415] 124. The peptide for use according to item 123, wherein the viral infection is an infection with an S protein virus, for example a virus of the Coronaviridae family, and the virus is selected from the group consisting of: PorCov-HKU15, SARS-CoV, HCoV NL63, HKU1, MERS-CoV, SARS-CoV 2, and MERS-CoV.

[0416] 125. A method for the treatment and / or prevention of inflammation and / or infection in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a peptide according to any one of items 1 to 107.

[0417] 126. The method according to item 125, wherein the treatment, the inflammation, the infection and / or the individual are defined in any one of items 110 to 124.

[0418] 127. Use of a peptide according to any one of items 1 to 107 for the preparation of a medicament for the treatment and / or prevention of inflammation and / or infection in an individual in need thereof.

[0419] 128. The use according to item 127, wherein the treatment, the inflammation, the infection and / or the individual are defined in any one of items 110 to 124.

[0420] 129. A pharmaceutical composition comprising a peptide according to any one of items 1 to 107.

Claims

1. A peptide comprising a contiguous sequence of 10 to 23 amino acids derived from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, i) has an overall length of 10 to 40 amino acids; ii) comprises at least one internal covalent bond between the side chains of two non-adjacent internal amino acids, wherein said amino acids are １ and X ２ is shown by iii) further comprising at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; provided that, when the peptide has an overall length of 24 to 40 amino acids, it comprises at least two internal covalent bonds between the side chains of two non-adjacent internal amino acids, and the amino acid of the first internal covalent bond is X １ and X ２ and said amino acid of the second internal covalent bond is represented by X ３ and X ４ Denoted by peptide.

2. Amino acid sequence: -U-U-(Z) n -I-Q-K-V-ID-Q-(Z) m - A peptide comprising or consisting of the peptide has an overall length of 10 to 23 amino acids; During the ceremony, each Z is individually any standard amino acid; U is His, Lys or Arg; n is an integer ranging from 0 to 10; m is an integer ranging from 0 to 5; two of the amino acids are replaced with alkenylated amino acids, the side chains of which are covalently bonded; peptide.

3. 2. The peptide of claim 1, wherein the peptide comprises at least amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO:

1.

4. 2. The peptide of claim 1, wherein the peptide has a total length of 10 to 23 amino acids.

5. 2. The peptide of claim 1, wherein the peptide has a total length of 13 to 23 amino acids.

6. Amino Acid X １ is located at position n, and amino acid X ２ is located at position n+3, or position n+4, or position n+5, or position n+6, or position n+7, or position n+8, or position n+9, or position n+10, or position n+11, wherein n is an integer ranging from 2 to 18.

7. When the sequence of the peptide is aligned with the sequence of GKY25 of SEQ ID NO: 12, a) X １ does not match Arg11 in GKY25 of SEQ ID NO: 12; b) X １ does not match Lys14 in GKY25 of SEQ ID NO: 12; c) X ２ does not match Lys14 in GKY25 of SEQ ID NO: 12; d) X ２ does not match Lys18 in GKY25 of SEQ ID NO: 12; and e) X １ matches Lys18 of SEQ ID NO: 12, then X ２ The peptide of claim 1, provided that:

8. 2. The peptide of claim 1, wherein the covalent bond is a hydrocarbon staple.

9. X １ and X ２ are alkenylated amino acids, such as two α-substituted alkenyl amino acids or two C-alkenylated amino acids, such as α,α-disubstituted alkenyl amino acids, and the covalent bond is an olefin tether formed between the alkenyl residues.

10. X ３ and X ４ are covalently linked by a lactam bridge, optionally wherein the lactam bridge is ３ and the N-terminal amine group of X ４ The peptide according to claim 1, wherein the peptide is formed between the side chain carboxylic acid of

11. X １ and / or X ２ are linked by a tether, said tether being an alkene chain of 10 carbon atoms counting from the C-alpha carbon.

12. 9. The peptide of claim 8, wherein the internal hydrocarbon staple is formed by linking two (S)-2-(4'-pentenyl)-alanines.

13. 2. The peptide of claim 1, wherein the peptide has a length of 14 to 22 amino acids.

14. 2. The peptide of claim 1, wherein the peptide further comprises 1 to 5 positively charged amino acids inserted at or near the termini of the peptide.

15. 2. The peptide of claim 1, wherein the peptide comprises two positively charged amino acids inserted at positions selected from positions 1, 2 and 3 relative to the N-terminus of the peptide.

16. 2. The peptide of claim 1, wherein the peptide consists of 15 to 20 consecutive amino acids of thrombin of SEQ ID NO: 1, with two amino acids replaced with alkenylated amino acids forming an internal hydrocarbon staple, and two to five additional N-terminal amino acids.

17. The peptide is i) SEQ ID NO: 3, ii) SEQ ID NO: 4; iii) SEQ ID NO: 5; iv) SEQ ID NO: 6, v) SEQ ID NO: 7, vi) SEQ ID NO: 8, vii) SEQ ID NO: 9; viii) SEQ ID NO: 10; ix) SEQ ID NO: 11, x) SEQ ID NO: 14, xi) SEQ ID NO: 17, xii) SEQ ID NO: 18; xiii) SEQ ID NO: 19, xiv) SEQ ID NO: 20, xv) SEQ ID NO: 21, xvi) SEQ ID NO: 22, xvii) SEQ ID NO: 23, xviii) SEQ ID NO: 24, xix) SEQ ID NO: 25, or xx) SEQ ID NO: 26 wherein X comprises or consists of a sequence as set forth in １ and X ２ are covalently linked amino acids, and X ３ and X ４ is a covalently linked amino acid.

18. The peptide is i) SEQ ID NO: 3, ii) SEQ ID NO: 5; iii) SEQ ID NO: 6, or iv) SEQ ID NO: 7 wherein X comprises or consists of a sequence as set forth in １ and X ２ is an amino acid, the side chain of which is covalently attached, and optionally X １ and X ２ is (S)-2-(4'-pentenyl)-alanine, the side chains of which have reacted with each other to form an alkenyl tether, and optionally, X １ and X ２ is Ala, the side chain of which is C containing one double bond ８ The peptides of claim 1, covalently linked to each other by an alkenyl tether.

19. The peptide is i) SEQ ID NO: 3, or ii) SEQ ID NO: 5 wherein X comprises or consists of a sequence as set forth in １ and X ２ is (S)-2-(4'-pentenyl)-alanine, the side chains of which have reacted with each other to form an alkenyl tether, and optionally X １ and X ２ is Ala, the side chain of which is C containing one double bond ８ The peptides of claim 1, covalently linked to each other by an alkenyl tether.

20. The peptide of claim 1, SEQ ID NO: 3 comprising or consisting of the sequence set forth in the peptide further comprises 1 to 5 positively charged amino acids inserted at the N-terminus; The peptide of claim 1.

21. The peptide of claim 1, SEQ ID NO: 3 comprising or consisting of the sequence set forth in the peptide further comprises two positively charged amino acids inserted at the N-terminus; The peptide of claim 1.

22. The peptide of claim 21, wherein the positively charged amino acid is selected from the group consisting of arginine, lysine and histidine.

23. The peptide of claim 21, wherein the positively charged amino acid is arginine.

24. The peptide of claim 1, wherein the peptide has a maximum hemolytic activity of 10% in fresh whole blood at a peptide concentration that reduces TNF-α release in LPS-stimulated blood by 50% in vitro.

25. A pharmaceutical composition for use in medicine comprising the peptide of claim 1.

26. A pharmaceutical composition for use in the treatment or prevention of inflammation or infection, comprising the peptide of claim 1.

27. 27. The pharmaceutical composition of claim 26, wherein the treatment or prevention is treatment or prevention of a disease selected from acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic rhinitis, other types of rhinitis, vasculitis, thrombosis, disseminated intravascular coagulation (DIC), gastroenteritis, and pulmonary inflammation.

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