WO2014097126A1 - Chemerin peptides, pharmaceutical composition comprising these peptides and use thereof - Google Patents

Abstract

The invention relates to chemerin peptides, pharmaceutical compositions containing these peptides and use of these peptides for treating pathological epithelium, in particular for use against bacterial and fungal infections and for treating diseases associated with injury or inflammation of the skin, lungs or gastrointestinal tract.

Description

Chemerin peptides, pharmaceutical composition comprising these peptides and use thereof

Technical field

The invention relates to chemerin peptides, pharmaceutical composition containing these peptides and use of these peptides for the manufacture of drug with protective properties with reference to epithelial cells, in particular for use against bacterial and fungal infections and for the treatment of diseases related to inflammation of the skin, lungs or gastrointestinal tract. State of the art

Peptides are well known for use as pharmaceuticals. For example, U.S. Patent No. 6191113 discloses a peptide having an activity of inhibiting growth of smooth muscle cells, and therefore useful in the prevention and treatment of pathological conditions associated with growth of smooth muscle cells such as arteriosclerosis, restenosis after angioplasty, stenosis after transplantation of blood vessel and smooth muscle sarcoma. U.S. Patent No. 6184208 discloses another peptide that have been found to modulate physiological processes suc a gain weight of the epithelial growth zone and hair growth.

Chemerin is the protein product of a gene called TIG2 (Tazarotene-lnduced Gene 2). The name of TIG2 is a result of induction of gene for chemerin by tazarotene (a derivative of retinoic acid), which is a drug used for the treatment of skin diseases such as psoriasis or squamous cell carcinoma. Chemerin is a natural ligand for the metabotropic receptor CMKLR1 and as chemoattractant is involved in regulation of the migration of certain immune system cells to inflammation sites (Wittamer 2003, Zabel 2005). Furthermore, chemerin plays important role in regulation of metabolism, either by influencing the differentiation of fat cells (Bozaoglu 2007, Goralski 2007) as well as by control of production and secretion of insulin by pancreatic cells (Takahashi 2011).

The main site of chemerin formation is likely liver and adipose tissue, which may be responsible for the relatively high concentration of this protein in the blood (nM). Although chemerin is synthesized also by epithelial cells, in pathological conditions of the skin such as e.g. psoriasis, severe inhibition of the expression of this protein occurs (Nagpal, 1997; Albanesi, 2007). Chemerin is synthesized as an inactive precursor (prochemerin), which circulates in the bloodstream. Prochemerin activation occurs through the activity of proteolytic enzymes that modify the carboxyl terminus of the protein (Zabel 2006). The sequence of the chemerin C-terminus is crucial for chemotactic activity. Enzymes that activate chemerin include cysteine and serine proteases involved in the blood coagulation cascade and inflammatory processes (Zabel 2006).

Proteolysis of the C-termius of prochemerin is required for the chemotactic activity , but it can also reduce or terminate chemotactic activity of chemerin (Zabel 2006). These results, on the one hand indicate that the proteolytic modification may be the mechanism of control of chemerin chemotactic activity, but on the other hand indicate chemerin susceptibility to proteolysis.

Antimicrobial peptides (AMP) are a group of low molecular weight (from 20 to 60 amino acids) compounds commonly found in nature (from bacteria to multicellular organisms). AMP characteristic feature is their cationicity (positive electric charge related to the presence of amino acids such as lysine and arginine) and amphipathicity (polar structure, one of the poles of the molecule has a hydrophilic character and the second hydrophobic). The best known AMP produced by human cells include defensins and cathelicidins (Zasloff 2002). Defensins are antimicrobial peptides of compact structure that is stabilized by three disulfide bonds. On the other hand, the only known representative of human cathelicidins - LL37 molecule - is considered linear peptide which adopts the structure of alpha-helix in contact with the cell membrane of bacteria. The consequence of this process is the loss of the integrity of the bacterial membrane, leading to the death of microorganisms (Zasloff 2007).

Due to the increasing number of bacteria strains resistant to antibiotics, AMP compounds are considered attractive for medical purposes as an alternative to antibiotics. This is related to the mode of action of AMP's to the bacteria which relies heavily on immediate perforating of bacteria cell membrane, thus the ability of bacteria to generate resistance to these compounds is limited (Zasloff 2007). An additional argument indicating the absence of bacterial resistance to AMP is example of nisin and a penicillin antibiotic which were discovered almost at the same time (in the twenties of the twentieth century). Nisin, which is commonly used in the preservation of food kept the antibacterial activity until today, while the penicillin is ineffective in the treatment of many infections (Hsu, 2004).

The aim of the invention is to provide novel AMP with antibacterial and antifungal properties, which may find application in medicine.

Subject of the Invention

The present invention is a chemerin peptide or its pharmaceutically acceptable salt, characterized in that it comprises the amino acid sequence consisting of at least 11 consecutive amino acids included in general formula: X-X-A1-A2-A3-X-X-A1-A3-X-X- X-X-X-C-X-A3-A3-X-A1 -A3-X-X-X-C-X-X-A3-X-X-X-A3-A3-A3-A3-C-X, where:

A1 represents an amino acid selected from the group of: W, Y and F,

A2 is an amino acid selected from the group: L, I and V,

A3 is an amino acid selected from the group of K, R and H,

X is any amino acid residue, C is cysteine,

wherein preferably its amino acid sequence is chosen among sequences presented as Seq Id. No.: 1 - 15.

Preferably, the peptide according to the invention has a length of 11 to 37 amino acids.

The invention also provides a pharmaceutical composition comprising one or more peptides according to the invention described above.

The subject of the invention is also the peptide according to the invention defined above for use in the manufacture of a medicine for the treatment or prevention of bacterial and fungal infections, particularly related to the epithelial cells.

Preferably, the medicine is designed for administration to the surface of the skin. Detailed description of the Invention

The invention relates to the peptide of length at least of 11 amino acids, chosen from group of peptides derived from chemerin protein, which sequence consists of general formula: X-X-A1 -A2-A3-X-X-A1 -A3-X-X-X-X-X-C-X-A3-A3-X-A1 -A3-X-X-X- C-X-X-A3-X-X-X-A3-A3-A3-A3-C-X, where:

A1 represents an amino acid selected from the group of: W, Y and F,

A2 is an amino acid selected from the group: L, I and V,

A3 is an amino acid selected from the group of K, R and H,

X is any amino acid residue,

C is cysteine,

and their pharmaceutically acceptable salts.

The general formula indicated above is a consensus sequence and was prepared based on the sequences of peptides derived from human chemerin with experimentally confirmed antibacterial activity, which were subsequently subjected to further bioinformatic analysis as described in details below in the specific examples. As a result, it is expected that it does not include random peptides that do not have the desired antimicrobial activity. Based on disclosed general formula an expert will be able to suggest peptides according to the invention having sequences other than the exemplary Seq Id No.: 1-15.

The peptide according to the invention may coincide with the 37 amino acid peptide consistent with the above mentioned consensus sequence, but can also be its shorter fragment having from 11 to 37 amino acids.

A particular embodiment of the invention is a fragment of chemerin protein of the sequence: GIFVRLEFKL QQTSCRKRDW KKPECKVRPN GRKRKCL.

Another particular embodiment of the invention is one of the following peptides: Name of peptide Sequence p4 VRLEFKLQQTSCRKRDWKKP p5 DWKKPECKVRPNGRKRKCLA c1 VRLEFKLQQTSCRKR c2 DWKKPECKVRPNGRKRK c3 GIFVRLEFKLQQTSCRKR c4 RKRDWKKPECKVRPNGR c5 QTSCRKRDWKKPECKVRPNGRKRK c6 VRLEFKLQQTSCRKRDWKKPECK c7 KLQQTSCRKRDWKK s1 LKIDWRINNPTCKHKQFRHS s2 LKFDLRIHDATCKHKQFRHT

S3 FRWNFKWRNPSCRHRHYKQS s4 VRLEFKLAATSCRKRDWKKP s5 VRLEFRLQQTSCRRRDWRRP

The subject of the invention is also pharmaceutical composition that comprises one or more peptides according to the invention.

In another embodiment, the subject of the invention is also application of the peptide for the manufacture of a medicine to increase the protective properties of the epithelial cells.

Preferably, when an increase in the protective properties of the epithelial cells is obtained by the prevention and/or treatment of bacterial and fungal diseases. Equally preferably, when the increase in the protective properties of the epithelial cells is achieved by preventing damage or inflammation of the skin, lungs or gastrointestinal tract.

The advantage of the use of chemerin peptides as antibacterial agents instead of recombined chemerin is that it makes possible to separate the bactericidal functions of this protein from other functions such as chemotaxis. Other features that are located in other domains of chemerin may cause undesirable or unpredictable effects during treatment.

Another advantage of the chemerin peptides according to the invention is lower susceptibility to degradation by proteolytic enzymes. As mentioned above, chemerin is extremely susceptible to proteolysis, while its smaller fragments are inherently more resistant to this process. Furthermore, these low molecular fragments can more easily penetrate into the sites of infection.

An additional advantage of the use of the peptides according to the invention is competitive cost of their synthesis.

Description of the figures and tables comprises experimental data

Object of the invention is demonstrated in the embodiment in the accompanying drawings, in which:

Figure 1 shows the sequence of human chemerin;

Figure 2 shows the sequence pattern in PROSITE format; motif has a length of 37 amino acid residues and corresponds to the sequence in Figure 1 ;

Figure 3 shows the sequence of chemerin protein, with underscores the position of tested peptides in a protein sequence;

Figure 4 demonstrates that chemerin peptide 4 ( p4 ) inhibits the growth of bacteria E. coli, in microdilution assays ( MBD, micotitre broth dilution assay) and a radial diffusion (RDA, radial diffusion assay);

Figure 5 demonstrates that the chemerin peptide p4 at concentration 100 μΜ is more effective than cathelicidin (LL37) in inhibiting the growth of bacteria E. coli and fungi C. albicans; Figure 6 demonstrates that the chemerin peptide p4 at concentration≥ 40 μΜ is at least equally efficient as well-known antimicrobial agent lysozyme in inhibiting the growth of bacteria E. coli and fungi C. albicans;

Table II shows that the chemerin peptide p4 has broad antimicrobial activity by inhibiting 13 (all tested) micro-organisms which can colonize the skin;

Figure 7 shows that the chemerin peptide p4 causes lysis of the bacteria in pH- and NaCI-dependent manner;

Figure 8 shows the frequency distribution of particular amino acid residues of chemerin formed on the basis of peptide pools endowed with a positive charge and able to accept the structure of the amphipathic alpha-helix or twisted beta-sheet;

Figure 9 shows that the amino acids in the basic sequence of p4 peptide may be replaced by specific amino acids and that the sequence may differ in length;

Figure 10 shows that the peptide p4 inhibits bacterial growth in vivo.

Embodiment of the invention

Synthesis of chemerin peptides

Antimicrobial and antifungal effect of the chemerin peptides has been shown on the basis of studies carried out on 14 synthetic peptides derived from chemerin protein, each about 20 amino acids in length, spanning the entire chemerin sequence. The location of the peptides in the structure of chemerin protein is depicted in Figure 3 where the top panel shows an entire chemerin sequence, underlines indicate the position of tested peptides in a protein sequence. Additionally, in Figure 3, the 20-amino acid signal peptide is shown in Italics. The sequence in bold shows peptide p4 which is the basis of the invention.

The sequence of human chemerin protein was also shown in Figure 1 ( number gi.4506427, an indication of the NCBI Protein database record: NP_002880, the range of amino acid residues from position 63 to 99 with a length of 37 amino acids is shown in red). In turn, in Figure 2 the pattern of the sequence in PROSITE format is shown, where the individual items in the sequence are separated by a hyphen, X is any amino acid residue and the notation [nnn] (e.g., [KRH]) denotes the occurrence of chosen amino acid residues in given position (here: lysine, arginine and histidine). This pattern describes the sequences of length of 37 amino acid residues.

The starting point for the construction of the pattern from Figure 2 was a sequence profile (i.e. position-specific scoring matrix) identified by MEME program for a pool of 500 peptides, whose sequence was generated by computer on the basis of evolutionarily fixed amino acid substitutions described by scoring matrix PAM250. In the final regular expression (Fig. 2), the reduction of information about variability at different motif positions helped to identify these motif positions, which are the most conservative.

Application of the sequential pattern from Fig. 2 for searching the database of amino acid sequences gives the same results as in the case of a sequence profile identified by the MEME program.

With the release 2012_10 of database Uniprot/Swissprot containing 214 310 sequences, there are five fragments of chemerin sequences corresponding to this pattern (given amino acid sequence, the species from which the chemerin sequence originates and scope of residues). Lowercase letters indicate the amino acid residues determined as any residues (X) in the presented pattern: giFVRIeFKIqqtsCrKRdWKkpeCkvRpngRKRKCI human 63 - 99 giFVRIeFKIqqtsCrKRdWKkpeCkvRpngRKRKCI orang-utan 63 - 99 gqFVRIeFKIqqtsCrKKdWRkedCkvKpngRKRKCI bullock 63 - 99 gtFVRIeFKIqqtsCfKKdWKnpeCkiKangRKRKCI hamster 65 - 101 gtFVRIeFKIqqtnCpKKdWKkpeCtiKpngRRRKCI mouse 65 - 101

A measure of the uniqueness of the described pattern is predicted probability of obtaining a random match between a sequence and a pattern, which for the release 2012_10 of Uniprot / Swissprot database is of the order 10^"10. This means that at 200 000 times more sequences in the database (i.e., more than 42 billion sequences), one of the sequences have been accidentally matched to the pattern shown in Fig. 2.

Examples of peptide sequences according to the invention are illustrated in Table 1. Table 1

Relative

Peptide Number of Total mean

Sequence

name aminoacids charge hydrophobic moment p4 VRLEFKLQQTSCRKRDWKKP 20 +5 0,375 p5 DWKKPECKVRPNGRKRKCLA 20 +6 0,295 c1 VRLEFKLQQTSCRKR 15 +4 0,625 c2 DWKKPECKVRPNGRKRK 17 +6 0,496 c3 GIFVRLEFKLQQTSCRKR 18 +4 0,476 c4 RKRDWKKPECKVRPNGR 17 +6 0,389 c5 QTSCRKRDWKKPECKVRPNGRKRK 24 +9 0,322 c6 VRLEFKLQQTSCRKRDWKKPECK 23 +5 0,238 c7 KLQQTSCRKRDWKK 14 +5 0,137 s1 LKIDWRINNPTCKHKQFRHS 20 +5 0,511 s2 LKFDLRIHDATCKHKQFRHT 20 +4,5 0,521

S3 FRWNFKWRNPSCRHRHYKQS 20 +7 0,511 s4 VRLEFKLAATSCRKRDWKKP 20 +5 0,363 s5 VRLEFRLQQTSCRRRDWRRP 20 +5 0,375

Table 1 shows a few groups of peptides. Peptides p4 and p5 are derived from human chemerin and are subject to the detailed analysis presented in Figures 3-6. Peptides c1 - c7 are derived from the sequence of human chemerin according of the invention. Peptides s1 - s5 are peptides designed in silico on the basis of simulated amino acid substitutions described with the PAM250 matrix for peptide p4. For each peptide following properties are specified: the amino acid sequence, the length (number of amino acid residues), the total charge at pH = 6.0, which characterizes the environment of the water/bacterial membrane interface, and the relative mean hydrophobic moment determined on the assumption of the spatial structure of the peptide as twisted beta sheet, which corresponds to the periodicity of 160 degrees in the Edmundson projection.

The inhibition of the E. coli bacteria growth by chemerin peptide 4 (p4)

Chemically synthesized chemerin peptides p1 - p14, covering the entire sequence of the chemerin (Fig. 3 ) were tested for antimicrobial activity against two strains of E. coli HB101 (Fig. 4A ) and E. coli ATCC 25922 (Fig. 4B). The antibacterial activity of the chemerin peptides was tested with two standard tests: MBD (Fig. 4A ) and RDA (Fig. 4B , 4C ). In MBD test bacteria were cultured in MBD medium to the logarithmic growth phase, and then incubated in MHB culture medium with indicated peptides at a concentration of 100 μΜ. After 24 hr. incubation, the bacteria were plated on plates with agar and MHB medium, and grown colonies were counted. Bacteria without added peptides was treated as a control group (100 %), and the results are shown as a percentage of the control. The MBD test results show the average of four independent experiments with standard deviation (Fig. 4A). The RDA test measured the zone of inhibition of bacterial growth shown on plates containing TSB medium supplemented with 0.15 M NaCI and agarose (Fig. 4B ), or TSB medium and agarose ( Fig. 4C). The peptides were administered to the cut-wells on plates at a concentration of 100 μΜ. After overnight incubation, the extent of growth inhibition (visible as clear zone around the well with peptide) was measured in millimeters [mm]. RDA results are shown as the Mean ±SD of three independent experiments (Fig. 4B, Fig. 4C).

The effectiveness of chemerin peptide In inhibition of bacteria growth

In addition, it has been shown that the chemerin peptide p4 at concentration 100 μΜ is more effective in inhibiting the growth of E. coli and C. albicans than 100 μΜ cathelicidin LL37 (Figure 5) and at least equally effective in >40 μΜ concentration as lysozyme tested in comparable concentration 40 μΜ (Fig. 6).

The RDA test was used to study the zone of growth inhibition of microbes listed above by chemerin peptide p4 and cathelicidin LL37 (both compounds at a concentration of 100 μΜ). In addition, the activity of the chemerin peptide p4 at concentrations 195 μΜ, 130 μΜ, 40 μΜ, 13 μΜ and 4 μΜ was also compared. It was demonstrated that activity of chemerin peptide p4 at 40 μΜ was similar whereas at≥ 130 μΜ was stronger than effect of lysozyme at concentration 40 μΜ. RDA assay was performed as described above. RDA results are shown as the Mean ±SD of three (Fig. 5) or two (Fig. 6) independent experiments. The asterisk indicates a statistically significant result (student's t-test, p <0.005).

It was also shown that the chemerin peptide inhibits 13 strains of bacteria that may cause infections of the skin (Table I). In the minimum dilution assay these bacteria after reaching the logarithmic growth phase were incubated in 10 mM phosphate buffer at pH 7.4 containing 1% TSB for 2 h at 37°C. P4 was added to the medium in concentrations: 100 μΜ, 50 μΜ, 25 μΜ, 12 μΜ, 6 μΜ, 3 μΜ, 1.5 μΜ and 0.8 μΜ. After 24 hr. incubation, the bacteria were plated on agar plates and the colonies grown were counted. MIC (i.e. the lowest concentration of the peptide causing 100 % inhibition of bacterial growth) was determined.

Results shown in Table II illustrate the Mean ±SD of three independent experiments. These results indicate broadantimicrobial activity of peptide p4.

In addition, the MIC results obtained are better or at least comparable with MIC values of other antimicrobial peptides such as defensins (Zasloff 2002 , 2007).

Table II. MIC values for peptide p4 for given bacteria strains tested in minimum dilution assays.

Bacteria strain MIC (μΜ)

E. coli HB101 3-6

E. coli ATCC 11775 3-6

S. aureus ATCC 6538 12

S. capitis ATCC 27840 6

S. hominis ATCC 27844 6-12

S. epidermidis ATCC 12228 12

S. epidermidis ATCC 14990 12

P. aeruginosa ATCC 10145 6

Streptococcus infantis ATCC 700779 6

Streptococcus sanguinis ATCC 10556 12

Streptococcus agalactiae ATCC 13813 12

Dermabacter hominis ATCC 49369 12

C, albicans ATCC 24433 6 Incubation of E. coli with peptide p4 at 10 μΜ resulted in release of cytoplasmic proteins into the environment, which indicates the perforation of the cell membrane of E. coli by the peptide p4. Inhibitory effect of p4 on the growth of E. coli depends on environmental conditions such as pH and salt content (Fig. 6). It was also shown that the chemerin peptide p4 causes lysis of bacteria in the pH-and NaCI-dependent manner (Fig.7A and 7B, respectively).

The peptide p4 at10 μΜ was incubated for 30 min with a strain of E. coli stably transfected with beta-galactosidase enzyme. Experiments were performed in 20 mM citrate-phosphate buffer at the specified pH and phosphate buffer pH = 7 with the addition of the indicated concentrations of NaCI. Control cells were treated with 1% Triton X-100 solution, and considered as lysed in 100%. The activity of beta- galactosidase released into the culture medium was measured calorimetrically after the addition of substrate for beta-galactosidase. The results are expressed as the Mean ±SD of three independent experiments.

The overall conclusion is that the peptide 4 (p4) derived from chemerin and to a lesser extent peptide 5 (p5), which partially overlaps with p4 demonstrated potent inhibitory effects against growth of E. coli bacteria in microdilution assays (MBD) and a radial diffusion (RDA) as shown in Figure 4. Peptide p4 also inhibited the growth of other microorganisms, such as S. aureus, P. aeruginosa and C. albicans (Fig. 5) as well as other several Staphylococcus and Streptococcus strains (Table II). It is worth emphasizing that all tested microorganisms (13 in total), which can colonize the skin, were inhibited by the chemerin peptide p4. In addition, the peptide p4 at 100 μΜ showed stronger antifungal and antibacterial effect than the cathelicidin LL37, widely considered to be one of the most versatile and effective AMP (Fig. 5). Concurrently the peptide p4 in 40 μΜ concentration had an activity similar to lysozyme (Fig. 6), another antibacterial agent with very high potency.

In addition, a systematic bioinformatic analysis of the human chemerin sequence was performed. The analysis was based on the determination of the total charge at pH = 6.0 and the relative mean hydrophobic moment (RMHM) for each peptide originating from chemerin and having the length between 11 and 37 residues.. Studies have shown that these two parameters are correlated with the antibacterial activity of tested peptides derived from sequence of human chemerin with p <0.05. Calculations of RMHM were performed with the assumption of one of the two selected peptides structures: the alpha-helix and twisted beta sheet. On the basis of a pool of peptides endowed with a positive charge which are able to form structure of the amphipathic alpha-helix (Fig. 8, green line, peptide charge > +2 and RMHM > 0,2) or twisted beta sheet (Fig. 8, red line, peptide charge > +2 and RMHM > 0,2 and blue line peptide charge > +3 and RMHM > 0,3), the frequency distribution of chemerin peptides which meet criteria mentioned above was created.

On the basis of Figure 8 it can be concluded that the N-terminal fragment of human chemerin exhibits the tendency to adopt a structure of two amphipathic helixes, while the section of the sequence between residues 63 and 99 (which covers whole claimed sequence of 37 amino acids in length) has the ability to form a twisted beta-sheet structure. This is according to results of chemerin secondary structure prediction (Cole, 2008). Because chemerin is a relatively small protein it can be assumed that the described structural tendencies will also characterize its sequence fragments interact with lipid membranes. The performed analysis clearly shows that part of the chemerin with sequence GIFVRLEF KLQQTSCRKR DWKKPECKVR PNGRKRKCL may be the source of positively charged peptides potentially strongly interacting with bacterial membranes, where the surface is of a slightly anionic (pH about 6). The analysis of 3D structure of the transmembrane bacterial protein OmpA (PDB ID: 2GE4) indicates that the peptide in conformation of the twisted beta sheet must be at least 11 amino acid residues in length to extend across the entire width of the lipid membrane (Cierpicki, 2006). As both peptides p4 and p5 most probably adopt a twisted beta sheet structure when bound to a lipid membrane, the minimum length of the chemerin peptide having antimicrobial properties should therefore be 1 amino acid residues.

On the basis of experimental results concerning antimicrobial activity of the peptides p4 and p5, as also results of the bioinformatic analysis described above, the model of antimicrobial active peptide was proposed. This antimicrobial peptide is≥ 11≤ 37 in length and its sequence matches the pattern shown in Fig. 2, where the key amino acids are marked as A1 -A3, and X is any amino acid. The claimed peptide of 37 amino acids covers sequences of peptides p4 and p5.

The methodology of the case is based on the use of antimicrobial peptide pattern shown in Fig. 2 with a length of min. 11 and max 37 amino acids, where in the positions A1 , A2 and A3 are the key amino acids selected on the basis of the original chemerin sequence and the need to maintain a possibly high value of the total charge at pH = 6.0and the relative mean hydrophobic moment (RMHM). The peptide is chemically synthesized and tested for antimicrobial properties in in vitro assays (RDA, MBD) and/or in vivo.

In order to demonstrate that the proposed model accurately anticipates antimicrobial functions, several experiments were performed. Results are shown in Fig. 9. Using the RDA test antimicrobial properties of the chemerin peptides p4, c6, c7, s4, s5 were compared in relation to E. coli. Peptide C6 (23 amino acids) and C7 (14 amino acids) are derived from human chemerin sequence. On the other hand, peptides s4 (QQ substituted by AA) and s5 (all K converted R) are peptides designed in silico based on the simulated amino acid substitutions described with PAM250 matrix for peptide p4. Tested peptides were selected to make substitutions in positions marked as X in the general sequence (peptide s4 - substitution concerns X at position 11 and 12) or in key position, i.e. A3 (peptide s5 - substitution concerns A3 at positions 9, 17, 21 and additionally X at position 22). Obtained results confirm the high antimicrobial activity of all tested analogs of p4.

The results obtained for peptides c6 and c7 indicate that the change in length of the peptide preserves antimicrobial activity that characterizes the peptide p4. On the other hand, lack of change in length of the peptide with simultaneous point substitutions based on in silico analysis and consistent with claimed sequence pattern (Fig. 2) shows that activity of synthetic peptides s4 and s5 is equal to or even slightly higher than the activity of the peptide p4 (Fig. 9).

Inhibition of bacterial growth by peptide p4 in the skin environment.

To investigate whether the chemerin peptide p4 inhibits bacterial growth in vivo, mice with genetic deficiency of chemerin (KO) and the age- and sex-matched mice C57BL6 (designated as WT) containing the chemerin in the skin were used. Mice deficient in chemerin are convenient as a model as they closely mimic the conditions of significantly inhibited chemerin expression, such as is the case of certain skin diseases in humans, for example psoriasis. On the other hand, in WT mice it was tested how the addition of peptide p4 affects the ability of the epithelium to inhibit bacterial growth in an environment where endogenous chemerin is present. The dorsal skin of KO and WT mice was shaved. A rubber ring with 1 cm was subsequently attached, which formed the incubation chamber, wherein the skin was the bottom of the chamber. Where indicated, p4 (or vehicle) was added into the cavity formed by the ring to a final 100 microM and allowed to dry.

1 x 10⁷ CFU of S. aureus was thereafter injected into the cavity formed by the rubber ring.

Bacteria grown in vitro (that can not be killed by skin-produced AMPs) were used as a control (100%). Bacteria from the cavity were retrived 24h latter, spread on agar plates and counted after over night incubation. Data from one experiment (the mean of two spots) per sample,

representative for two independent experiments are shown. Numbers above bars demonstrate % of killing relative to the control (Fig. 10).

The results show the strong antimicrobial activity of the chemerin peptide p4 in the skin environment, both, devoid of endogenous chemerin (reduction from 86% to 0.36 %) and containing chemerin (down from 0.75% to 0.12%).

SEQUENCE LISTING

<110> Uniwersytet Jagiellonski

Stanford University

<120> Chemerin peptides, pharmaceutical composition comprising these peptides and use thereof

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References

1. Albanesi C et al. (2007). J. Exp. Med. 206: 249-258.

2. Allen SJ et al. (2007). Biomol NMR Assign 1 : 171-173.

3. Bozaoglu K et al. (2007). Endocrinology 148: 4687-4694.

5 4. Cierpicki et al. (2006) J. Am. Chem. Soc. 128:6947-6951.

5. Cole Ch et al. (2008). Nucl. Acids Res. 36 (suppl 2): W197-W201.

6. Goralski KB et al. (2007). J Biol Chem 282: 28175-28188.

7. Hsu et al. (2004). Nature 11 :963-967.

8. Kulig P et al. (2011). J Immunol 187: 1403-1410.

10 9. Nagpal, S et al. (1997). J. Invest. Dermatol 109: 91-95.

10. Takahashi M et al. (2011). Sci Rep 1 : 123.

11. Wittamer V et al. (2003). J Exp Med 198: 977-985.

12. Zabel BA et al. (2005). J Immunol 174: 244-251.

13. Zabel BA et al. (2006). Exp Hematol 34: 1021-1032.

15 14. Zasloff M (2002). Nature 415: 389-395.

15. Zasloff M (2007). J Am Soc Nephrol ^: 2810-2816.

Claims

Claims

1. Chemerin peptide or its pharmaceutically acceptable salt characterized in that it comprises amino acid sequence of at least 11 subsequent amino acids with general formula:

X-X-A1-A2-A3-X-X-A1-A3-X-X-X-X-X-C-X-A3-A3-X-A1-A3-X-X-X- C-X-X-A3-X-X-X-A3-A3-A3-A3-C-X,

where:

A1 means amino acid selected from a group: W, Y i F,

A2 means amino acid selected from a group: L, I i V,

A3 means amino acid selected from a group: K, R i H,

X means any amino acid residue,

C means cysteine,

favourably when its amino acid sequence was chosen from the sequence presented as Seq Id. No.: 1 - 15.

2. Peptide according to claim 1 , characterized in that the peptide is of 11 to 37 amino acids in length.

3. Pharmaceutical composition comprising one or more peptides defined in claims 1 to 2.

4. The peptide according to claims 1 to 2 for use in manufacturing the medicine for treatment and prevention of bacterial and fungal diseases, particularly related to epithelial cells.

5. The peptide for use according to claim 4, wherein manufactured medicine is designed for skin surface application.