WO2014095977A1

WO2014095977A1 - Novel pellino peptide

Info

Publication number: WO2014095977A1
Application number: PCT/EP2013/077059
Authority: WO
Inventors: Paul Moynagh
Original assignee: National University Of Ireland, Maynooth
Priority date: 2012-12-21
Filing date: 2013-12-18
Publication date: 2014-06-26
Also published as: GB201223114D0

Abstract

This invention concerns an isolated peptide having the partial sequence of human full-length Pellino-3 protein comprising amino acid residues 37–51 of said Pellino-3 protein, or an analogue thereof having anti-inflammatory activity, pharmaceutical compositions containing it and its use in the treatment or prophylaxis of inflammatory diseases and disorders.

Description

NOVEL PELLINO PEPTIDE

This invention relates to a novel peptide. In particular, it relates to a peptide having antiinflammatory activity, pharmaceutical compositions containing it and its use in the treatment or prophylaxis of inflammatory-based diseases and disorders.

Inflammatory-based diseases and disorders are rarely mediated by a single molecule but instead are due to a number of mediators acting in a complex interacting network. Most of the prior art has focused on targeting a single mediator and yet this may be insufficient to effectively treat the inflammatory-based disease.

The classical treatment of inflammatory-based diseases involves the use of steroids from the glucocorticoid family. Such drugs are very effective in blocking the inflammatory response but suffer from a wide range of severe side effects. Consequently more specific strategies have been explored, including the use of neutralising antibodies to lipopolysaccharide (LPS) (also known as endotoxin) and inhibitors of LPS-induced pro-inflammatory cytokines (e.g. IL-1 and TNF). These approaches have been used with varying degrees of success.

The use of antibodies can lead to problems with neutralising immune responses. The targeting of IL- 1 and TNF is entirely logical but the similar biological activities of these cytokines may dictate that the activities of both cytokines need to be blocked. Furthermore, these products are proteins and their production is associated with high costs.

It is an object of the invention to avoid or minimise the disadvantages of the prior art.

According to the invention there is provided an isolated peptide having the partial sequence of human full-length Pellino-3 protein comprising amino acid residues 37 - 51 of said Pellino-3 protein, or an analogue thereof having anti-inflammatory activity, in which the natural amino acid sequence other than the tyrosine residue at position 44, has been modified by substitution, insertion or deletion of amino acids, or a pharmaceutically acceptable salt thereof.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, including

selenomethionine, as well as those amino acids that are modified after incorporation into a polypeptide, e.g., hydroxyproline, O-phosphoserine, O-phosphotyrosine, gamma-carboxyglutamate and cysteine. Amino acid analogues refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which function in a manner similar to a naturally occurring amino acid. The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired antiinflammatory activity is retained by the peptide. The amino-terminal NH₂ group and carboxy- terminal COOH group of free peptides are typically not set forth in a formula. A hyphen at the amino- or carboxy-terminus of a sequence typically indicates the presence of a further sequence of amino acid residues or a respective NH₂ or COOH terminal group. Amino acids may be referred to herein by either their commonly known three-letter symbols or by their one-letter symbols, as shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

All amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Preferably, a peptide of the invention comprises the following sequence of amino acid residues: B-Xn- PGEEPIKYGELIVLG -, wherein:

B is an amino-terminal group;

X_n is a membrane-translocating peptide, wherein n is 0 or 1 , i.e., the membrane- translocating peptide may be absent (n = 0) or present (n = 1 );

or a pharmaceutically acceptable salt thereof.

Preferably, a peptide of the invention consists of the following sequence of amino acid residues:

B-Xn- PGEEPIKYGELIVLG - Z, wherein:

B is an amino-terminal group;

X_n is a membrane-translocating peptide, wherein n is 0 or 1 , i.e., the membrane- translocating peptide may be absent (n = 0) or present (n = 1 ); and

Z is a carboxy-terminal group;

or a pharmaceutically acceptable salt thereof.

The membrane-translocating peptide, when present, may be any suitable membrane-translocating peptide. Particularly preferred is a 16-amino acid sequence from the Drosophila protein,

Antennapedia, known as penetratin and consisting of the following sequence of amino acid residues: RQIKIWFQNRRMKWKK (SEQ ID NO: 3).

In a particularly preferred embodiment, the peptide of the invention consists of the following sequence of amino residues corresponding to amino acid residues 37 - 51 of human Pellino-3: PGEEPIKYGELIVLG (SEQ ID NO: 1 ); or the peptide is tandemly linked to a membrane-translocating peptide and consists of the following sequence of amino acid residues corresponding to amino acid residues 37 - 51 of human Pellino-3 contiguous with the 16-amino acid sequence from the

Drosophila protein, Antennapedia, known as penetratin, referred to above:

RQIKIWFQNRRMKWKK-PGEEPIKYGELIVLG (SEQ ID NO: 2). The peptide of the invention or a pharmaceutically acceptable salt thereof may be used in the treatment or prophylaxis of inflammatory-based diseases and disorders.

Inflammatory-based diseases and disorders that may be treated or prevented using a peptide of the invention include inflammatory bowel disease, including Crohn's colitis, sepsis, multiple sclerosis, lupus and sarcoidosis.

The invention also provides a method for the treatment or prevention of an inflammatory-based disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a peptide of the invention or a pharmaceutically acceptable salt thereof. The invention also provides the use of a peptide of the invention or a pharmaceutically acceptable salt thereof in the treatment or prophylaxis of an inflammatory-based disease or disorder in a subject.

The peptides of the invention may be formulated in any vehicle suitable for in vivo, in vitro or therapeutic use. The peptides may be formulated for oral or parenteral administration or they may be formulated in a cream or ointment for topical application.

The invention also provides a pharmaceutical composition comprising a peptide according to the invention or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable carrier, diluent or excipient therefor.

The pharmaceutical composition may be in any form suitable for administration, e.g., oral, topical or parenteral administration. The pharmaceutical composition of the invention may be used in the treatment or prophylaxis of inflammatory-based diseases and disorders.

A peptide of the invention may be obtained by chemical synthesis using any appropriate

methodology well known to those skilled in the art, such as Fmoc (fluoroenylmethoxy carbonyl) solid phase methodologies. The peptide may be obtained using a peptide synthesizer and a suitable resin such as 2-chlorotrityl chloride resin and suitable scavengers such as thioanisole, DTT, and phenol, or phenol, water and triisopropylsilane, followed by purification using, e.g., HPLC.

Infection by Gram-negative bacteria can frequently lead to a fatal syndrome known as septic shock (sepsis). There is currently no cure for this condition. Sepsis arises due to an exaggerated inflammatory response to LPS in the cell wall of the bacteria. The peptide of the invention inhibits the ability of LPS to trigger the intracellular signalling pathways that promote the inflammatory response and thus may be used in the treatment of sepsis. The peptide of the invention specifically inhibits the ability of LPS to activate intracellular signalling pathways that normally induce pro-inflammatory proteins. When combined contiguously with a membrane-translocating peptide such as penetratin (a 16 amino acid sequence from the Drosophila protein Antennapedia), the application of such a peptide to cells inhibits the LPS activation of intracellular signalling pathways and the induction of pro-inflammatory protein. Of particular note the peptide specifically inhibits LPS signalling and not other agents that employ signalling pathways similar to LPS. The peptide of the invention has the advantage of simultaneously targeting a number of the mediators, a feature that makes it an extremely effective anti-inflammatory agent.

Nucleotide-binding oligomerisation domain-2 (NOD2) is an intracellular protein that recognises breakdown products of bacterial peptidoglycan and responds by inducing pro-inflammatory cytokines. Mutations and polymorphisms in NOD2 are associated with diseases such as Crohn's disease. Applicant has found that NOD2 signalling is defective in the absence of Pellino-3 protein, suggesting that the peptide of the invention may be used as an anti-inflammatory agent in the treatment of colitis, including Crohn's colitis. The invention is illustrated in the following non-limiting Examples.

Example 1

A peptide of the invention having the amino acid sequence PGEEPIKYGELIVLG (corresponding to amino acid residues 37 - 51 of human full length Pellino-3), hereinafter referred to as "Pellino3 peptide", was obtained by chemical synthesis. The peptide was dissolved in phosphate-buffered saline (PBS) to a stock concentration of 5mM.

Example 2

Effects of Pellino3 peptide on LPS - induced NFKB:

HEK293 cells expressing TLR4 (HEK-TLR4 cells) were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 μg ml streptomycin and maintained in a 37°C humidified atmosphere with 5% C0₂. For luciferase reporter assays HEK-TLR4 cells were seeded (1.5 x 10⁵ cells/ml; 200 μΙ in 96-well plates and grown for 24 h. Cells were then transfected using Lipofectamine 2000 transfection reagent, with constructs encoding (A) NFKB-regulated firefly luciferase (80 ng) or (B) pFR firefly luciferase reporter construct (60 ng) and IRF3 (30 ng) (fused downstream of the yeast Gal4 DNA-binding domain) and constitutively expressed TK Renilla- luciferase reporter construct (phRL-TK) (20 ng) (Promega Biosciences). Total DNA was kept constant (200 ng/well) using the appropriate empty vector. Transfected cells were left overnight and then pretreated for 1 h with various concentrations of Pellino3 peptide prepared in Example 1 prior to stimulation with LPS for 6 hours. Cell extracts were generated and assayed for firefly luciferase activity (normalised for transfection efficiency by assaying co-transfected TK Renilla luciferase). The results are illustrated in Figure 1. Data are expressed relative to unstimulated cells. The Pellino3 peptide is especially effective in inhibiting TLR4 signalling that is triggered by LPS / endotoxin.

Human Pellino3 (protein) has been described to negatively regulate activation of NFKB, and these cell-based studies show that the Pellino3 peptide is capable of inhibiting LPS-induced activation of the pro-inflammatory transcription factor NFKB, i.e., it can negatively regulate activation of NFKB, suggesting that the Pellino3 peptide can functionally mimic Pellino3 in this respect.

Example 3

Pellino3 peptide protects against LPS-induced sepsis

Sepsis was induced in mice (10 each) via intraperitoneal (i.p.) injection of ultrapure

lipopolysaccharide (LPS) at a dose of 20mg/kg. Pellino3 peptide prepared in Example 1 was injected i.p 1 hour prior to LPS treatment. Animals were scored at hourly intervals using the following criteria: score 0 - no symptoms; score 1 - piloerrection, huddling; score 2 - piloerrection, huddling, diarrhoea; score 3 - lack of interest in surroundings, severe diarrhoea; score 4 - decreased movement, listless appearance; and score 5 - loss of self-righting reflex. Mice were humanely killed when they reached reach score 5. The results, which are illustrated in Figure 2, show that Pellino3 peptide protects against LPS-induced sepsis.

Example 4

Exacerbation of DSS-induced colitis in Pellino3-deficient mice:

Given the role for Pellino3 in negatively regulating pro-inflammatory signalling, the applicant generated a Pellino3 knockout and assessed the effect of Pellino3 deficiency on inflammatory diseases. Colitis was chosen as an inflammatory disease model. Mice were treated with 2.5% DSS dissolved in the drinking water for 5 d, followed by 5 d on normal water. Body weight, occult blood in faeces, and stool consistency/diarrhoea were recorded daily for each mouse to determine the disease activity index (DAI). The maximum DAI score was 12 based on assigning a 1-4 scoring system for each parameter: score 0, no weight loss, normal stool, and no blood; score 1 , 1-3% weight loss; score 2, 3-6% weight loss, loose stool (a loose stool was defined as the formation of a stool that readily becomes paste upon handling), and blood visible in stool; score 3, 6-9% weight loss; and score 4, <9% weight loss, diarrhoea, and gross bleeding. When mice were killed, the lengths of the colon were measured. The results, which are illustrated in Figure 3 (a)-(e), reveal that PellinoS-deficient mice showed exacerbation of colitis-associated pathology. In Figure 3, the following applies: ":p<0 05; **:p<0.01 ; ***:p<0.001.

Example 5

Defective NOD2 signalling (by MDP) in Pellino3 deficient mice:

Given that NOD2 protects against colitis, the effects of Pellino3 deficiency on NOD2 signalling were examined. Wild type (WT) and Pellino3 deficient (KO) mice received i.p. injection of MDP (100 μg) or PBS and serum samples generated 4h after administration. Serum was assayed for levels of IL-6 by sandwich ELISA. The results, which are illustrated in Figure 4, show that NOD2 signalling is defective in the absence of Pellino3. In Figure 4, the *** applies to the difference (p<0.001 ) between MDP treated wild type and Pellino3 knockout (KO) mice and this comparison is represented by the line underneath *** that spans the two groups.

The data in Figures 3 and 4 strongly indicate a protective role for Pellino3 against colitis-associated pathology. Given that it has been shown above in Example 2 that the Pellino3 peptide can functionally mimic Pellino3, this peptide is likely to show therapeutic efficacy in colitis. Example 6

Effect of Pellino3 peptide on NOD2 signalling

In order to further probe the anti-inflammatory potential of the Pellino3 peptide in Crohn's disease, a clinical subtype of inflammatory bowel disease, the regulatory effects of the peptide on NOD2, a protein in the body that protects against development of Crohn's disease, was assessed and the results are illustrated in Figure 5. The human monocytic cell line THP1 was stimulated for 1 hour in the absence (no peptide) or presence of the Pellino3 peptide (20μΜ) prior to stimulation with the NOD2 ligand muramyl dipeptide (MDP) (50 μg ml) for a further 30 minutes. Cell lysates were generated and probed by Western blotting for phosphorylated IkBa (phospho ΙκΒα), p38 (phospho- p38) and JNK (phospho-JNK). MDP was shown to promote increased phosphorylation of all 3 proteins with further augmentation in the presence of the Pellino3 peptide. The peptide alone also caused some stimulatory effects. Given the protective role of NOD2 signalling in Crohn's disease, this finding further supports a potential protective feature for the Pellino3 in Crohn's disease.

The following abbreviations are used herein:

DMEM: Dulbecco's Modified Eagle's Medium

DSS: Dextran Sodium Sulphate

DTT: Dithiothreitol

HEK: Human Embryonic Kidney

IL-1 : lnterleukin-1

JNK: Jun N-terminal protein kinase

LPS: Lipopolysaccharide

MDP: Muramyl Dipeptide

NFKB: Nuclear-Factor-kappa B

NOD2: Nucleotide-binding Oligomerisation Domain-2

PBS: Phosphate-Buffered Saline

TLR: Toll-Like Receptor

TNF: Tumour Necrosis Factor

Claims

CLAIMS:

1. An isolated peptide having the partial sequence of human full-length Pellino-3 protein

comprising amino acid residues 37 - 51 of said Pellino-3 protein, or an analogue thereof having anti-inflammatory activity, in which the natural amino acid sequence other than the tyrosine residue at position 44, has been modified by substitution, insertion or deletion of amino acids, or a pharmaceutically acceptable salt thereof.

2. An isolated peptide according to claim 1 , comprising the following sequence of amino acid residues:

B-Xn- PGEEPIKYGELIVLG -, wherein:

B is an amino-terminal group;

X_n is a membrane-translocating peptide, wherein n is 0 or 1 ;

or a pharmaceutically acceptable salt thereof.

3. An isolated peptide according to claim 1 or 2, consisting of the following sequence of amino acid residues:

B-Xn- PGEEPIKYGELIVLG - Z, wherein:

B is an amino-terminal group;

X_n is a membrane-translocating peptide, wherein n is 0 or 1 ; and

Z is a carboxy-terminal group;

or a pharmaceutically acceptable salt thereof.

4. An isolated peptide according to claim 2 or 3, wherein n is 0.

5. An isolated peptide according to claim 2 or 3, wherein n is 1.

An isolated peptide according to claim 5, wherein the membrane-translocating peptide consists of the following sequence of amino acid residues: RQIKIWFQNRRMKWKK (SEQ ID NO: 3).

7. An isolated peptide according to claim 1 or 2, consisting of the following sequence of amino acid residues: PGEEPIKYGELIVLG (SEQ ID NO: 1 ).

An isolated peptide according to claim 1 or 2, which is contiguous with a membrane- translocating peptide consisting of the following sequence of amino acid residues:

RQIKIWFQNRRMKWKK-PGEEPIKYGELIVLG (SEQ ID NO: 2).

An isolated peptide according to any preceding claim or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis of inflammatory-based diseases and disorders.

An isolated peptide or a pharmaceutically acceptable salt thereof for use according to claim 9, wherein the inflammatory-based disease or disorder is selected from inflammatory bowel disease, including Crohn's colitis, sepsis, multiple sclerosis, lupus and sarcoidosis.

A pharmaceutical composition comprising a peptide according to any of claims 1 to 7 or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable carrier, diluent or excipient therefor.

A pharmaceutical composition according to claim 1 1 , which is in a form suitable for oral, parenteral or topical administration.

A pharmaceutical composition according to claim 1 1 or 12 for use in the treatment or prophylaxis of inflammatory-based diseases or disorders.

A pharmaceutical composition according to claim 13, wherein the inflammatory-based disease or disorder is selected from inflammatory bowel disease, including Crohn's colitis, sepsis, multiple sclerosis, lupus and sarcoidosis.