SI23181A - Inhibitory peptides of MyD88-dependent signal pathway - Google Patents

Abstract

The field of invention is a new inhibitory peptide containing a segment which inhibits the activation of MyD88. The invention comprehends the inhibitory peptide and its application for treatment of disease states connected with excessive activation of the innate immune response.

Description

Inhibitory peptides of MyD88 dependent signaling pathways

FIELD OF THE INVENTION

FIELD OF THE INVENTION An inhibitory peptide that inhibits the activation of the innate immune response via the adapter protein MyD88. The invention relates to peptides and their use in the treatment of conditions associated with the overactivation of the innate immune response.

The state of the art

For their survival, multicellular organisms need an immune system to recognize the presence of molecules that are characteristic of pathogenic microorganisms and pathologically altered cells of their own. An important component of the innate immune response is receptors that recognize characteristic molecules of pathogenic microorganisms. Among the receptors, Toll-like or Toll-like receptors are of particular importance for an effective immune response.

Activation of TLR receptors occurs upon binding of molecules that activate the ectodomain TLR (agonists). Agonist binding (pathogen-associated molecule patterns or PAMPs) in most TLR receptors results in the dimerization of ectodomains and the consequent aggregation of the TLR receptor cytosolic domains TIR (Toll / Interleukin-1 receptor). Upon dimerization of the TIR receptor domains, adapter proteins such as MyD88, Mal / TIRAP, TRAM, or TRIF bind to the cytosol. Adapter proteins, through activation of protein kinases, trigger a signaling cascade that leads to activation of transcription factors and transcription of genes involved in the immune response - activation of TLR receptors triggers the production of cytokines such as IL-12, IL-6, IL-8. IL-1 and type I and II interferons.

The characteristic of all adapter proteins (adapters) of TLR signaling pathways is that they contain the TIR domain. The MyD88 adapter is required for activation of MyD88-dependent signaling pathways characteristic of TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 receptors as well as stimulation of interleukin receptors with IL-1, IL-18, and IL-33. Mal / TIRAP is involved in the MyD88-dependent signaling pathway when TLR4 and TLR2 receptors are used. The MyR88-independent signaling pathway is characteristic of the TLR4 and TLR3 receptors, where the TRIF protein plays a major role. The TRAM protein is characteristic of the MyD88-independent signaling pathway only in the case of the receptor

-2TLR4 (Akira S., Takeda K. 2004. Toll-like receptor signaling. Nature Reviews: Immunology, 4: 499-511).

MyD88 and TRIF play an important role in downstream signaling and lead to the synthesis of inflammatory cytokines, whereas Mal / TIRAP and TRAM serve only as a link between MyD88 or TRIF and the corresponding TLR receptor, although their increased expression leads to cell activation (Nishiya T ., Kajita E., Horinouchi T., Nishimoto A., Miwa S. 2007. Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88. FEBS Letters, 581: 3223-3229).

MyD88 is an adapter built from DD (death domain) and TIR domain, and between them there are intermediates INT (intermediate domain). The vertebrate MyD88 molecules are homologous and can be functionally interchangeable. MyD88 represents the central signaling molecule at TLR receptors (with the exception of the TLR3 receptor) and at the interleukin receptor 1 (IL-1R). The TIR MyD88 domain at the C-terminus is responsible for binding to the TLR domain of TIR receptors, and the DD domain at the N-terminus is responsible for binding to the IL-1R-associated kinase 4 kinase and subsequent signal transduction in signaling paths. The role of the INT domain has not yet been studied in detail, and the absence of an INT domain is associated with an inability to signal MyD88. Janssens et al. have reported a form of MyD88 without an INT domain, called MyD88s (MyD88 short), and acts as an inhibitor (Janssens S., Burns K., Tschopp J., Beyaert R. 2002. Regulation of Interleukin-1- and lipopolysaccharide -induced NF-κΒ activation by alternative splicing of MyD88. Current Biology, 12: 467-471). MyD88s can bind to the TIR receptor domain but not to the DD kinase domain IRAK4, since both the DD domain and the INT domain are required for binding to IRAK4. The exact mechanism of activation of MyD88 and downstream kinases is not yet known, since most studies related to MyD88 have focused on the TIR domain.

Inadequate regulation of the immune system can trigger serious disease states, e.g. sepsis. Sepsis is a pathological condition with signs of a severe infection and a systemic response to it, resulting from the over-amplification of an otherwise appropriate host initial response to microorganism infection. Sepsis can go into septic shock, which is characterized by a severe inflammation of the bloodstream, a decrease in blood pressure and organ failure, in addition to a systemic inflammatory response. In the case of chronic inflammation, the innate immune system is often activated via multiple TLR receptors, so inhibition of multiple TLR receptors simultaneously is desirable, for example, through a common MyD88 adapter (US patent 7122656 B2).

-3 Inadequate regulation of the immune system is also involved in chronic inflammatory diseases such as rheumatoid arthritis, putitis, systemic lupus erythematosus and colitis, in the development of cancer and in the development of certain autoimmune diseases. In these diseases, inhibition of signaling may be a useful therapy.

Inhibitors of the innate immune system signaling pathways can be divided into several groups:

- DNA constructs containing a record for a portion of a protein capable of inhibiting the signaling pathway. Thus, in the case of TLR-dependent signaling, MyD88 is predominantly negative. This is a form of MyD88 that contains only the TIR domain and binds to the TIR domain of the TLR receptor but lacks the DD domain and cannot trigger the signaling cascade. Inhibitors in the form of DNA constructs face problems specific to gene therapy, such as problems of target uptake, controlling the level of expression of the inserted gene, the problem of the safety and efficacy of vectors - and, as a consequence, less desirable treatments (Traister RS, Hirsch R. 2008. Gene therapy for arthritis. Mod Rheumatol (2008) 18: 2-14);

- Recombinant proteins are a therapeutically more interesting type of inhibitor. This cluster includes individual protein domains capable of inhibiting signaling pathways. One example is recombinantly obtained dominant negative MyD88 and an inhibitor of IL-1R, which is commercially known as anakinra and is used to treat rheumatoid arthritis. However, recombinant proteins also have their limitations in therapeutic use due to their low stability, mode of administration, pharmacological properties, production costs. Inhibitory peptides and peptidomimetics represent a therapeutically appropriate type of inhibitors since they can be obtained by chemical synthesis. Not only are they of adequate quality and purity, but also commercially available in larger quantities. The TLR4 receptor-dependent signaling pathway is currently known to be inhibitory peptides whose sequence corresponds to the sequence of BB loops of the MyD88, Mal / TIRAP, TRIF and TRAM adapters (patent no. W02006113530 (A2)). Peptides of BBzank adapters are not specifically targeted at a specific target, as they can also bind to other proteins that contain the TIR domain.

Stopping within the cell signaling pathway is possible only upon introduction of the inhibitory peptide into the cell. For translocation of peptides into a cell, short sequences derived from segments of cell-capable proteins such as, for example, HIV-1 TAT protein sequence and antennabody transcription factor homeodomain sequence in wine fly (Sebbage V. Cell-penetrating peptides) are commonly used and their therapeutic applications. 2009. Bioscience Horizons. Vol 2 (1): 64-72). Such a sequence was also applied to the case of BB loop peptides. Less known and for the purpose of inhibiting TLR receptor signaling is still • · • ·

The -4-unused approach for the transport of peptides into the cell uses the addition of an acyl chain (Nelson A.R., Borland L., Allbritton N.L., Sims C.E. 2007. Myristoyl-based transport of peptides into living cells. Biochemistry 46: 14771-14781)

The inventors have discovered a new type of peptidin inhibitors that inhibit activation of the innate immune system, specifically MyD88-dependent signaling. These peptides (peptides designated by SEQ ID NO: 4 up to and including 11) comprise a sequence corresponding to the INT domain sequence of the MyD88 INT protein and act via inhibition of signaling pathways.

Summary of the Invention

The invention relates to inhibitory peptides of the sequence of SEQ ID NO: 4 up to and including SEQ ID NO: 11 or peptides of the sequence of SEQ ID NO: 4 to including SEQ ID NO: 11 optionally linked to other amino acid sequences except domain sequences TIR or DD domains of MyD88 protein such that the fusion protein retains the inhibitory properties of peptide inhibitory agents.

The invention relates to inhibitory peptides that are at least 75% identical to the inhibitory peptides described above and include inhibitory peptides with point-modified amino acids and peptides with the amino acid sequence of the vertebrate INT MyD88 domain. The invention relates to inhibitory peptides containing the inhibitory polypeptide and the peptide transfer segment into the cell, the transmit segment being translocation antennapedia sequences from SEQ ID: 13 or TAT peptide from SEQ ID NO: 12. In the second part of the invention, the transfer segment peptides into the cell are represented by acyl chains comprising 8-22 carbon atoms, preferably lauryl or myristoyl chains.

The invention relates to DNA constructs that contain a record for inhibitory peptides and are associated with regulatory elements, such as a promoter and terminator, that allow expression of the inhibitory peptide in the host organism.

The invention relates to small organic molecules, peptidomimetics, which mimic the properties and function of peptide inhibitors.

The invention relates to the preparation of peptide inhibitors for the treatment and treatment or prevention of human and animal diseases when the disease is characterized by activation of the innate immune response and includes in the pharmaceutical composition: (a) inhibitory peptides of the invention, and (b) pharmaceutically acceptable carriers.

The invention relates to methods for the selective inhibition of MyD88-dependent signaling pathways that occur due to the introduction of any combination of inhibitory peptides in an amount sufficient to inhibit.

• ·

-5The invention relates to methods of preventing an inflammatory response involving MyD88-dependent activation in a host with inhibitory peptides, in case the inflammatory response is associated with at least one of the conditions such as allergy, asthma, contact dermatitis, delayed hypersensitivity, wound healing , allergic rhinitis, food hypersensitivity, atopic dermatitis, chronic bowel inflammation, immune diseases of the lungs, autoimmune or immune-mediated skin diseases, psoriasis, enteropathy associated with gluten sensitivity, rheumatoid arthritis, graft rejections, sepsis, septic shock, cancer , systemic lupus erythematosus, colitis, neuropathic pain, neurodegenerative diseases, ischemia, hypoxia, transplants.

According to the invention, the methods include prophylactic or therapeutic administration of inhibitory peptides, and the inhibitory peptide is administered by one or more of the following routes: intravenous, intradermal, subcutaneous, oral, inhalation, deep pulmonary inhalation, nasal, transdermal, transmucosal, vaginal, rectally, intrathecally, intra-articularly, intraperitoneally, or by gene therapy methods and inhibitors, the peptide may be administered in combination with compounds used to treat or prevent inflammation and inflammation-related conditions.

The invention also relates to a kit containing at least one of the inhibitory peptides and a control peptide having no inhibitory properties.

Description of the pictures

Figure 1: INT peptides of the MyD88 INT adapter inhibit signaling via TLR4 / MD-2. HEK293 cells that do not express TLR receptors were transfected with plasmids for TLR4, its MD-2 coreceptor, and reporter plasmids. Peptides were added 24 h after transfection and 6 h later, HEK293 cells were stimulated with sLPS (final concentration 25 ng / ml). After 16 h, cells were lysed and luciferase activity was determined. p71, p72, p73, p74, p92, p93 represent different inhibitory peptides, and p75 the control peptide.

Figure 2: The peptides of the INT domain of the MyD88 adapter inhibit signaling via the interleukin receptor 1. HEK293 cells expressing interleukin receptor 1 were transfected with reporter plasmids. 24 h after transfection, peptides were added and 6 h later, HEK293 cells were stimulated with IL-1 beta (final concentration 5 ng / ml). After 16 h, cells were lysed and luciferase activity was determined. p71, p72, p73, p74, p92 represent different inhibitory peptides, and p75 the control peptide.

Figure 3: The peptides of the INT domain of the MyD88 adapter do not inhibit signaling via the MyD88 independent TLR3 receptor. HEK293 cells that do not express TLR receptors were transfected with plasmids for TLR3 and reporter plasmids. 24h after transfection was added

-6peptides, 6 h later HEK293 cells were stimulated with poly I: C (final concentration 10 μg / ml).

After 16 h, cells were lysed and luciferase activity was determined. p71, p72, p73, p74, p92, p93 represent different inhibitory peptides, and p75 the control peptide.

Figure 4: INT peptides of the MyD88 INT adapter domain inhibit mRNA synthesis for TNF-alpha in MMF cells stimulated with sFPS. Peptides (final concentration 50 µM) were added to the human monocyte MM6 line and stimulated with sFPS 2 hours later (final concentration 0.5 µg / ml). 1.5 h later, RNA was isolated, RT-PCR and qPCR were performed. p71, p74, p93 represent different inhibitory peptides, and p75 represent the control peptide. *** p <0.005.

Figure 5: Peptides of the INT domain of the MyD88 adapter inhibit mRNA synthesis for TNF-alpha in flagellin-stimulated MM6 cells. Peptides (final concentration 50 μM) were added to the human monocyte MM6 line and stimulated with flagellin (final concentration 50 ng / ml) 2 h later. 1.5 h later, RNA was isolated, RT-PCR and qPCR were performed. p71, p74 represent different peptide inhibitors and p52 represent the control peptide. *** p <0.005.

Figure 6: The peptides of the INT domain of the MyD88 adapter inhibit mRNA synthesis for TNF-alpha in MM8 cells stimulated with R848. Peptides (final concentration 50 μM) were added to the human monocyte MM6 line and stimulated with R848 2 hours later (final concentration 10 μg / ml). 1.5 h later, RNA was isolated, RT-PCR and qPCR were performed. p71, p74 represent different peptide inhibitors and p52 represent the control peptide. * p <0.05, ** p <0.01.

Figure 7: INT peptides of the MyD88 INT adapter domain inhibit phosphorylation of ERK kinases in RAW264.7 cells. The RAW264.7 macrophage murine cell line was added peptides (final concentration 50 μM) and stimulated with sLPS 2 h later (final concentration 0.5 µg / ml). 15 min later, the cells were lysed and the phosphorylation level of the p42 / p44 kinases was determined by antibodies. We showed uniform protein deposition via the detection of a / b tubulin. p71, p73, p92, p93 represent different inhibitory peptides, and p75 the control peptide.

Figure 8: Intemalisation of the peptide with the antennapedia translocation segment (p93) labeled with Alexa Flour 555. The peptide labeled with Alexa Flour 555 was added to the HeLa cell line for 20 min, then the cells were washed and stained with Dio membrane dye and Hoechst core dye. . The left image shows nuclei, the central membrane, and the right shows an intemalised fluorescently labeled peptide.

Figure 9: Intimalisation of peptide with added myristate as translocation signal (p71) labeled with Alexa Flour 555. The peptide labeled with Alexa Flour 555 was added to the HeLa cell line for 20 min, then the cells were washed and stained with Dio membrane dye and • ·

-7th Hoechst dye. The left image shows nuclei, the central membrane, and the right shows an intemalised fluorescently labeled peptide.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly known to those skilled in the art. The terminology used to describe the invention is intended to explain a particular segment of the invention and is not intended to limit the invention. All publications mentioned in the description of the invention are cited as references. In the description of the invention and the claims, the description is in the singular but also includes the plural, which is not specifically emphasized in the description for ease of understanding.

The basis of the invention is the surprising discovery that MyD88-dependent signaling of the innate immune system can be interrupted by peptides with a sequence of the INT MyD88 domain located between the DD domain and the TIR domain in MyD88, and containing the amino acid sequence 110 to 158 in SEQ ID: 1. The present invention describes a novel type of peptide inhibitory composition, the composition of which is based on the inventors' recent insights into innate immunity. The invention is based on the discovery that peptides of the INT MyD88 domain sequence are capable of inhibiting activation of the innate immune system and thus represent a novel tool for the treatment of conditions associated with overactivation of the innate immune system.

The term &quot; peptide &quot; refers to peptides or proteins containing at least two amino acids linked to one another by a peptide bond. The term "peptide" refers to both short chains and longer amino acid chains, commonly referred to as proteins. The peptide may also contain non-genome-encoded amino acids. These amino acids can be modified by natural processes (such as posttranslational modifications) or chemically. Modifications can occur anywhere in the peptide, on the amino acid side chains, at the N-terminal or C-terminal ends. Each peptide may contain several different modifications at different sites. Peptides can be linear, branched or cyclic. Peptide modifications include: actetylation, acylation, ADP-ribosylation, amidation, covalent binding of lipids or lipid derivatives, covalent binding of phosphatidylinositols, cyclization, disulfide bridge formation, covalent bonds, glycosylation, hydroxylation, phosphorylation, phosphorylation, phosphorylation.

The term &quot; inhibitory peptide &quot; refers to peptides with a sequence capable of inhibiting or arresting the signaling pathway in a cell. The term inhibition means any measurable

-8 reducing the expression of TLR receptor activation products, TLR receptor signaling, or TLR-mediated gene expression, for example, cytokine expression.

The term "innate immune response or innate immunity" refers to a cellular response upon activation of Tollu-like and other cellular receptors, where the sequence of receptors is defined in germ cells and does not change in the body.

The term "adapter protein" or "adapter" refers to cytosolic proteins that mediate the transmission of the dimerization signal of TIR TLR domains. These adapter proteins contain TIR domains that can bind to TIR TLR domains and interleukin receptors and other TIR domain containing proteins. Adapters include MyD88, TRIF, Mal / TIRAP, TRAM and SARM.

The term "cell activation" refers to the activation of an immune response via Tollu-like receptors, the activation of innate immunity or the release of cytokines.

The term "Toll-like receptors" or "TLR" refers to TLR receptors of man or animal. TLR designates members of a type I transmembrane receptor group consisting of leucine-rich repeats on the aminoterminal side, a transmembrane domain, and a cytosolic domain containing a structurally conserved TIR domain. The term refers to native receptors that have the ability to activate cells or the immune response by adding appropriate ligands (agonists). TLR receptors recognize various molecules whose structure and localization are characterized by infection with germs or pathological conditions such as chronic diseases such as cancer, atherosclerosis or autoimmune diseases. Toll-like receptors are localized either on the cell surface (TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, TLR11) or within cell vesicles (TLR3, TLR7, TLR8, TLR9). TLR activation occurs upon binding of an activating molecule (agonist) to the TLR ectodomain consisting of leucine repeats (LRR). Agonist binding results in the dimerization of the ectodomain and through it the dimerization of the cytosolic TIR domains. Upon dimerization of TIR domains, cytosolic adapters such as MyD88 or TRIF bind to the cytosol, triggering a signaling cascade through the activation of protein kinases, which leads to activation of transcription factors and transcription of genes involved in the immune response.

Different TLRs trigger a different response that takes place either via the MyD88-dependent or TRIF-dependent pathway, triggering the production of inflammatory cytokines such as IL-1, IL-6, TNF-alpha or other interferons in the body.

-9The inventors have been able to demonstrate that inhibitory peptides with an INT MyD88 domain sequence can specifically inhibit activation of MyD88-dependent signaling pathways.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly known to those skilled in the art. The terminology used in the description of the invention is intended to explain a particular segment of the invention and is not intended to limit the invention. All publications mentioned in the description of the invention are cited as references. In the description of the invention and the claims, the description is in the singular but also includes the plural, which is not specifically emphasized in the description for ease of understanding.

Inhibitory peptides

The present invention is based on the unexpected discovery that peptides with the INT sequence domain of the adapter protein MyD88 inhibit the MyD88-dependent signaling pathway of innate immunity.

The invention relates to inhibitory peptides (SEQ ID: 4 to and including SEQ ID: 11) containing an inhibitory sequence: that is, the sequence of the INT MyD88 domain (sequence 110 to 130 of the amino acids of MyD88 in SEQ ID: 1 and shorter sequences, that are longer than 10 amino acids) or a shortened sequence, as long as the sequence still retains inhibitory function, as well as point changes (changes in individual amino acids) in the sequence that do not abrogate the inhibitory function of the sequence.

The inhibitory activity of the peptide of the INT domain containing 9 amino acid residues (SEQ ID: 11) was significantly lower than the inhibitory activity of the peptide containing 13 (SEQ ID: 5) or 21 (SEQ ID: 4) of the amino acid residues. Peptides containing a sequence with a length between these two limit values, such as SEQ ID: 6 containing 17 residues, also have inhibitory activity. It also follows from the invention that the inhibitory peptide can be fused as a fusion protein with other proteins or other molecules (with the exception of sequences of the TIR domain or DD MyD88 domain) as long as it retains inhibitory function. It is also understood that the invention also includes multimers of inhibitory peptides of either the same or different lengths.

An additional aspect of the invention is that, in addition to the inhibitory segment that inhibits activation via MyD88, the inhibitory peptides may also have an additional translocation sequence that is involved in cell entry but is not limited to acyl chains or known peptide sequences for cell entry and located on either the N-terminal or C-terminal portion of the inhibitory sequence. The term "translocation sequence" refers to short polypeptide sequences derived from

-10segments of protein capable of entering cells, such as the HIV-1 TAT protein sequence and the antennapedia transcription factor homeodomain sequence in the wine fly. Any translocation sequence that can be used to introduce an inhibitory sequence into a cell can be used in accordance with the present invention.

Some other methods can also be used to introduce an inhibitory peptide into a cell, including non-covalent or covalent linkages with translocation sequences, viral proteins, proteins that allow endocytosis or allow receptor entry, membrane translocation, as well as physical entry methods, such as microinjection, electroporation, through pore formation and others.

A lesser known and inappropriate approach to inhibit TLR receptor signaling, an unused approach for transporting peptides into the cell utilizes the addition of hydrophobic groups such as fatty acid chains, in particular acyl chains of 6 to 28 carbon atoms, preferably between 8 and 22 carbon atoms. Acyl chains may be straight, branched, cyclic, saturated or unsaturated, and may contain heteroatoms such as oxygen, sulfur, nitrogen, phosphorus and others.

Organic compounds with appropriate structural similarities to the inhibitory peptides described above, usually with a molecular weight below 2 kDa, can also be used as inhibitors of TLR and IL-1R activation. Often, for example, small molecules such as peptidomimetics are used. Peptidomimetics, as known to those skilled in the art, are chemically synthesized compounds that provide the appropriate spatial structure necessary for proper interaction with the appropriate protein so that they can inhibit or elicit a response. Therefore, the invention also includes the use of small peptidomimetics that mimic the activity of peptides from the INT MyD88 domain to inhibit MyD88-dependent signaling.

Inhibitome peptides of the present invention can be prepared by methods known in the art, such as e.g. chemical synthesis. Inhibitory peptides can also be prepared in host organisms such as viruses, bacteria, yeasts, mammals and the like, using recombinant DNA technology via a recombinant expression vector that contains a record for the peptides described above or endogenously in the host, as appropriate. to gene therapy methods. It is possible to prepare a DNA construct consisting of a suitable vector and sequence encoding the inhibitory sequence of the INT MyD88 domain (described above). These DNA constructs can be introduced into cells by gene therapy methods and thus have the advantage of intracellular localization, which does not require additional segments for translocation.

• ·

The invention further provides methods for treating conditions associated with TLR receptor overactivation. One method involves administering one or more inhibitory peptides or peptidomimetics described above to an organism in need of treatment, optionally in combination with a pharmaceutically acceptable carrier, in an amount that completely inhibits or diminishes MyD88 signaling.

The biologically active molecules of the present invention can be targeted to any selected cell as long as there is a way to contact these molecules (inhibitory peptides, peptidomimetics) with the selected cell. The cells may be inside the tissue or organ, but are related to the blood vessels into which the peptide is inserted. Further, peptides can be targeted to specific cells via the addition of molecules that bind to a specific receptor, such as the integrin receptor. Additionally, peptides can be introduced directly into the tissue.

Peptide uptake can be ex vivo or in vivo. Ex vivo administration involves the collection of a sample of immune cells, endothelial cells, mucosal epithelial cells in the lungs or intestines, or any other type of cell in which treatment is beneficial to the organism. The collected cells are then grown in vitro, the selected inhibitory peptides are added, and after exposure of the cells to the inhibitory peptides, the cells can be returned to the body to provide therapeutic or prophylactic treatment. In vivo uptake refers to the uptake of a selected peptide or peptides directly into the body. Selected peptides can be administered by any suitable means, but not limited to injection (eg intravenous, subcutaneous, intraperitoneal, intradermal, intraarticular, intrathecal), inhalation, ingestion ... Oral administration is also possible if the peptides of the invention are prepared in encapsulated or enteric form. Inhibitory peptides of the invention in the form of DNA constructs can be introduced using a viral carrier, lipid complexes, nanoparticles, physical methods such as electroporation, biolistic methods, microinjection and similar methods of gene therapy. In addition, the molecules of the invention may be administered in combination with other compounds or active substances, including immunogenic additives, adjuvants, antiviral drugs, antibiotics, anti-inflammatory drugs, and pharmaceutically acceptable drugs or derivatives thereof. The invention is used to prevent or treat diseases caused by infections of microorganisms including bacteria, viruses, fungi, primates and other microorganisms, to prevent and treat allergies, cancer, neurodegenerative diseases, neuropathic pain, rheumatoid arthritis, putika, systemic lupus erythematosus and other autoimmune diseases and diseases where activation of the immune response requires signaling via MyD88.

-12The term "treatment" refers to a disease state of a subject that is improved or partially improved in at least one of the clinical indicators, and the term also refers to a delayed progression of the disease or disorder. The term also includes the prevention of infection or the onset of a disease state, but it is not intended to be a complete prevention of a disease state but also a delayed development of a subject's disease state. The method of "treatment of a disease state" includes therapeutic methods of treatment and prevention of a disease state. Treatment of the condition can include both preventive (prophylactic) and therapeutic treatment. Preventive treatment refers to treatment that begins before the onset of symptoms or signs of a medical condition. Such preventative treatment generally decreases the likelihood that the organism will develop a disease state and reduces the consequences of the condition already acquired, or both. Therapeutic treatment, however, refers to treatment that begins after the onset of symptoms or signs of a medical condition. Thus, therapeutic treatment is intended to limit and reduce the progression of the disease state. Conditions that can be treated using the methods of the invention include, but are not limited to, viral, bacterial, fungal, parasitic, neoplastic, neurodegenerative diseases, neuropathic pain, cardiovascular disease, autoimmune disease, cancer, sepsis, septic shock ...

Selective promotion of TLR4 via the MyD88-independent pathway, such as using monophosphorylated lipid A (MPLA), has been shown to offer a potential advantage in vaccination as it prevents inflammatory responses induced by MyD88 activation. The peptides and peptidomimetics of the invention can be used in combination with adjuvants that stimulate TLR receptors and other receptors of the innate immune response so as to avoid unwanted inflammation but retain the beneficial properties of the vaccines. This can be achieved by the addition of the peptides or peptidomimetics of the invention to any combination of immunogens, adjuvants, or mixtures thereof, by any route of administration, such as, but not limited to, subcutaneous, intramuscular, nasal, oral administration.

Pharmaceutical composition

The invention further provides a pharmaceutical composition comprising the inhibitory peptides of the invention with pharmaceutically acceptable carriers. The term "pharmaceutically acceptable" refers to a material that is non-toxic to the host organism that are, but not limited to, humans and other vertebrates.

In the invention, the inhibitory peptides and peptidomimetics of the invention are present in the pharmaceutical composition in an "inhibitory effective amount". The term "inhibitomally effective" amount refers to an amount that sufficiently suppresses an immune response (cellular or humoral) in an organism of which

-13Pharmaceutical compound introduced. Optionally, the dosage amount is sufficient when it suppresses the activation of the immune response. The protection obtained is not necessarily complete, it is sufficient that the benefit of administering the pharmaceutical composition outweighs the undesirable effects. The inhibitomally effective amount depends on the route of administration, on the peptide inhibitors and on the organism. Effective amounts, dosages, are determined in a manner known in the art and are preferably in the range of 1 to 1000 micrograms / kg body weight, depending on the type of disease, tissue, route of administration and other factors.

The pharmaceutical composition of the invention may include other medical agents, pharmaceutical agents, stabilizing compounds, buffers, carriers, diluents, salts, wetting agents, osmostabilizers.

The invention also enables the manufacture of a kit which can be used in the field of research of the immune system for carrying out the invention. The kit contains a package containing one or more of the inhibitory peptides described above, a control peptide that has no inhibitory properties and is composed of the amino acid residues of the abovementioned peptide inhibitors, but in an altered order. The kit also contains the appropriate solutions necessary for carrying out the invention and written documentation with instructions for carrying out the invention and analytical information such as the amount of reagent in the packaging and the like.

The following are embodiments intended to illustrate the invention. The description of embodiments is not intended to limit the invention but should be construed as demonstrating the operation of the invention.

Implementation examples

Example 1. Schematic representation of peptides of the INT domain of the MyD88 adapter.

Example 1 shows the inhibitors and control peptides (Table 1) prepared to demonstrate the operation of the invention.

-14Table 1: Schematic representation of the peptides of the INT domain of the MyD88 adapter used in embodiments in order to illustrate the operation of the invention.

peptide name translocation motif peptide sequence inhibitory peptide _P 71 Miristat INT MyD88 - 21 AK domain sequence (EEDCQKYILKQQQEEAEKPLQ) p72 Miristat INT sequence of MyD88 - 13 A A (LKQQQEEAEKPLQ) p73 Miristat INT MyD88 - 17 AA domain sequence (EEDCQKYILKQQQEEAE) p74 Laurat cationic AK GRVRRLS sequence and INT sequence MyD88-21 AA (EEDCQKYILKQQQEEAEKPLQ) p51 Miristat INT sequence of MyD88 - 9 AA (LKQQQEEAE) p92 Miristat cationic AK GRVRRLS sequence and INT sequence MyD88-21 AA (EEDCQKYILKQQEEAEKPLQ) p93 sequence antennapedia (RQIKIWFQNRRM KWKK) INT MyD88 - 21 A A domain sequence (EEDCQKYILKQQEEAEKPLQ) control peptide _P 75 - cationic AK GRVRRLS sequence and INT sequence MyD88-21 AA (EEDCQKYILKQQQEEAEKPLQ) p52 myristate altered sequence 21 of the AK domain INT MyD88

Example 2. Inhibition of innate immunity in the HEK293 cell line at the inhibition level of the NF-κB activation reporter.

In this example, we have shown that inhibitory peptides inhibit the activation of natural immunity via the TLR4 / MD-2 receptor complex and IL-1R (MyD88-dependent signaling pathway) but not through the TLR3 receptor (MyD88-independent signaling pathway). This was demonstrated by inhibition of reporter activation by the NF-κΒ promoter characteristic of MyD88-dependent signaling, whereas activation was not inhibited in the case of the reporter by the IFN-beta promoter characteristic of MyD88-independent signaling.

The methods and techniques of cell line cultivation are well known to those of skill in the art and are only explained in the framework herein, in terms of explaining an embodiment. The HEK293 cell line was grown at 37 ° C and 5% CO ₂ . DMEM medium with 10% FBS containing all the necessary nutrients and growth factors was used for its cultivation. With sufficient cell density, we transplanted them • »

- 15 thinned thinner. To be used in the experiments, cells were first counted with a hemocytometer and grafted onto a 96-well microtiter plate appropriate for cell culture cultivation. The grafted plates were incubated in an incubator at 37 ° C and 5% CO ₂ until the cells reached a suitable transfection sprouting. GeneJuice, JetPei or Lipofectamine transfection reagents were used for cell transfection. Transfection was performed according to the manufacturer's instructions following a customized transfection procedure in 96-well microtiter plates.

The following DNA was used for transfection of the HEK293 cell line: pNF-KB-Fluc 50 ng, pRluc 10 ng, pTLR4 2.5 ng, pMD-2 2.5 ng when attempting to activate the TLR4 / MD-2 receptor complex (Fig. 1), pNF-KB-Fluc 50 ng, pRluc 10 ng when attempting to activate IL1-R (Fig. 2), or pIFN-beta-Fluc 50 ng, pRluc 10 ng, pTLR3 20 ng when attempting to activate the TLR3 receptor (Fig. 3).

24 h after transfection, inhibitory peptides were added to the cells in the amount indicated in the figures, 6 h later stimulated with the appropriate agonist - sLPS for TLR4 / MD-2, IL-1 beta for IL1-R, and poly I: C for TLR3 . 16 h later, cells were lysed and luciferase activity was measured. A test with two reporter proteins was used for luciferase activity: (a) godfather luciferase (Fluc) and (b) Renilla luciferase (Rluc). Cresnic luciferase (Fluc), which uses CoA, ATP, and luciferin as the substrate, is functionally linked to a promoter that senses the activation of NFkB or IRF-3 transcription factor. Activation of innate immunity via TLR receptors results in activation of NFkB (in the case of MyD88-dependent signaling) or activation of IRF-3 (in the case of MyD88-independent activation), which can be detected by measuring the activity of godfather luciferase. The second reporter transfected into cells concomitantly with the pNF-KB-Fluc plasmid. pIFN-beta-Fluc and plasmids for TLR receptors, serves as a reporter of transfection efficiency. The reporter plasmid bears a DNA record for Renilla luciferase (Rluc), for which coelenterazine is a suitable substrate. Rluc is expressed in cells independently of the conditions.

For analysis of reporter protein expression, cells were lysed with buffer according to the manufacturer's instructions (Promega). First, we measured activity for firefighter luciferase (NF-kB-F1uc or IFN-beta-Fluc) and then Renilla luciferase activity (Rluc; phRL-TK http://www.promega.com/vectors/prltk.txt). We used the double luciferase method according to the manufacturer's instructions (Promega). Rluc activity therefore tells us the proportion of cells transfected, while Fluc activity shows us the activation of innate immunity. Fluc / Rluc Ratio (RLU • ·

-16ang. relative luciferase units) therefore tells us the normalized value of stimulated cells relative to transfected cells.

The results shown in Figures 1, 2 and 3 show that the inhibitory peptides inhibit MyD88-dependent signaling in the case of TLR4 / MD-2 and IL-1R receptor complex activation, but not MyD88-independent signaling in the case of TLR3 receptor activation , which confirms the inventor's thinking that peptides of the INT MyD88 domain inhibit activation of innate immunity.

Example 3. The peptides of the INT domain of the MyD88 adapter inhibit mRNA synthesis for TNF-alpha in the stimulated human MM6 monocyte line.

Example 3 illustrates the function of peptides of the INT domain of the MyD88 adapter on the MM6 human monocyte model line. The MM6 line was grown at 37 ° C and 5% CO ₂ . For its cultivation we used RPMI medium with 5% FBS and addition of nonessential amino acids. For the experiment, 10 ⁶ cells were taken and peptides were added and stimulated 2 hours later by agonists with different TLR receptors. The MM6 line is a human monocyte line and itself expresses different TLR receptors, which means that we do not need to enter them into cells by transfection. 1.5h after stimulation, cells were lysed with TRIzol reagent and whole RNA was isolated from them with a commercially available RNA isolation kit. In order to remove DNA residues from the RNA isolates, DNase purification was also performed. Subsequent reverse transcription of RNA into cDNA followed with the corresponding reverse starting oligonucleotides (for cytokines and for HPRT as appropriate internal control). The cDNA obtained was then amplified by qPCR with the corresponding starting nucleotides. The qPCR result tells us the amount of mRNA for a given cytokine normalized to the amount of mRNA for appropriate internal control. In Example 3, we wanted to show the efficacy of the inhibitory peptides of the invention in the case of inhibiting mRNA synthesis for the cytokine TNF-alpha, which is known to begin to be produced upon activation of innate immunity. The results shown in Figures 4, 5 and 6 show that the inhibitory peptides inhibit mRNA synthesis for TNF-alpha in the case of stimulation of MM6 cells with TLR4 / MD-2 agonists (Fig. 4), TLR5 (Fig. 5) and TLR8 (Fig. 6) characterized by triggering MyD88-dependent signaling, which confirms the inventor's thinking that peptides of the INT MyD88 domain inhibit activation of innate immunity.

-17Example 4. The peptides of the INT domain of the MyD88 adapter inhibit phosphorylation of ERK kinases in murine RAW264.7 macrophages.

Example 4 illustrates the functioning of peptides of the INT domain of the MyD88 adapter on the RAW264.7 murine macrophage model line. The RAW264.7 line was grown at 37 ° C and 5% CO ₂ . DMEM medium with 10% FBS was used for its cultivation. For the experiment, 10 ⁶ cells were taken and peptides added. Two hours later, cells were stimulated with sLPS and lysed with lysis buffer after 15 min, to which phosphatase inhibitors were added. When TLR receptors are activated on the surface of murine macrophages, phosphorylation of MAP kinases occurs very rapidly, including ERK kinases (p44 / p44). Thus, activation of innate immunity can also be monitored via phosphorylation of ERK kinases. The protein concentration of the cell lysates was determined by protein concentration and 40 μg of protein was applied to the SDS gel. After SDS, the polyacrylamide gel electrophoresis is transferred to a nitrocellulose membrane by Westem transfer by size of the separated protein. Phosphorylated kinases are detected by specific antibodies. From the results shown in Figure 7, the inhibitory peptides inhibit the phosphorylation of ERK kinases, confirming the inventor's thinking that peptides of the INT MyD88 domain inhibit activation of innate immunity.

Example 5. Internalization of peptides of the INT domain of the MyD88 adapter.

Example 5 represents the localization of fluorescently labeled peptides in a HeLa cell line. The localization of fluorescently labeled peptide inhibitors in a cell line was determined by the inventors by microscopy with a confocal microscope. Techniques and methods for working with a confocal microscope and staining cell elements are widely known to those skilled in the art. Fluorescently labeled peptides were added to HeLa cells, which had been inoculated 24 hours beforehand into suitable microscopy dishes. 30 min later, cells were washed with PBS buffer and fresh medium was added. Cell nuclei were stained with Hoechst dye and cell membranes were stained with Dio. The labeled live cells were examined on a Leica TCS SP5 confocal microscope. This microscope is designed for laser scanning of fluorescently labeled live or fixed cells. We used a 63χ oil immersion lens. Images were acquired with LAS AF 1.8.0. Leica Microsystems. The use of lasers was dependent on the wavelengths at which the individual fluorophores (dyes) were to be excited.

Figures 8 and 9 show that inhibitory peptides localize inside the cell, a prerequisite for their successful operation.

LIST SEQUENCE <160> 13 <170> Patent Version 3.4 <210> <211> <212> <213> 1 296 PRT Homo sapiens

<220>

<221> <222> <223> I <400> mature (D .. HyD8 £ 1 Ala peptide (296) > Ala Gly Sir 10 Ala Ala Pro Val Sir 15 Sir Met 1 Ala Gly Gly 5 Pro Gly Thr Sir Sir Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30 Leu Sir Leu Phe Leu Asn Val Arg Thr Gin Val Ala Ala Asp Trp Thr 35 40 45 Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gin Leu 50 55 60 Glu Thr Gin Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gin Gly 65 70 75 80 Arg Pro Gly Ala Sir Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu 85 90 95 Gly Cys Asp Asp Val Leu Leu Glu Leu Gly Pro Sir Ile Glu Glu Asp 100 105 110 Cys Gin Lys Tyr Ile Leu Lys Gin Gin Gin Glu Glu Ala Glu Lys Pro 115 120 125 Leu Gin Val Ala Ala Val Asp Sir Sir Val Pro Arg Thr Ala Glu Leu 130 135 140 Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg 145 150 155 160 Phe Asp Ala Phe Ile Cys Tyr Cys Pro Sir Asp Ile Gin Phe Val Gin 165 170 175 Glu Met Ile Arg Gin Leu Glu Gin Thr Asn Tyr Arg Leu Lys Leu Cys 180 185 190 Val Sir Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Sir Ile Ala 195 200 205

Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser • ·

-19210 215

Asp 225 Asp Tyr Leu Gin Sir 230 Lys Glu Cys Asp Phe 235 Leu Sir Leu Sir Pro 245 Gly Ala His Gin Lys 250 Arg Tyr Lys Ala Met 260 Lys Lys Glu Phe Pro 265 Sir Ile Val Cys Asp 275 Tyr Thr As n Pro Cys 280 Thr Lys Sir Leu Ala Lys Ala Leu Sir Leu Pro

220

Gin Thr Lys Phe Ala 240

Leu Ile Pro Ile Lys 255

Leu Arg Phe Ile Thr 270

Trp Phe Trp Thr Arg 285

290 295 <210> 2 <211> 49 <212> PRT <213> Homo sapiens <220>

<221> mature peptide <222> (1) .. (49) <223> INT MyD88 domain <400> 2

Glu Glu Asp Cys Gin Lys Tyr Ile Leu Lys Gin 1 5 10 Glu Lys Pro Leu 20 Gin Val Ala Ala Val 25 Asp Sir Ala Glu Leu Ala Gly Ile Thr Thr Leu Asp Asp

Pro 35 40 <210> 3 <211> 49 <212> PRT <213> Mus musculus <220> <221> mature peptide <222> (D · . (49) <223> domain INT MyD88 Mus Musculus <400> 3

Gin Gin Glu Glu Ala 15

Ser Val Pro Arg Thr 30

Pro Leu Gly His Met 45

Glu 1 Glu Asp Cys Gin 5 Lys Tyr Leu Gly Lys 10 Gin Gin As n Gin Glu 15 Sir Glu Lys Pro Leu Gin Val Ala Arg Val Glu Sir Sir Val Pro Gin Thr 20 25 30 Lys Glu Leu Gly Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly Gin Thr

• e

Pro 35 40 45

<210> 4 <211> 21 <212> PRT <213> artificial sequence <220> <221> mature peptide <222> d) - (21) <223> p71 <400> 4 Glu Glu . Asp Cys Gin Lys 1 5 Glu Lys Pro Leu Gin 20 <210> 5 <211> 13 <212> PRT <213> artificial sequence <220> <221> mature peptide <222> (1) ·· (13) <223> p72 <400> 5 Leu Lys ; Gin Gin Gin Glu 1 5 <210> 6 <211> 17 <212> PRT <213> artificial sequence <220> <221> mature peptide <222> (D · · (17) <223> p73 <400> 6 Glu Glu Asp Cys Gin Lys 1 5 Glu <210> 7 <211> 28 <212> PRT

<213> artificial sequence

Tyr

Glu Ala

Tyr

Ile Leu Lys Gin 10

Glu Lys 10

Ile Leu Lys 10

Gin Gin Glu

Pro Leu Gin

Gin Gin Gin

Glu

Glu Ala 15

Glu Ala 15

-21 <220>

<221> mature peptide <222> (1) .. (28) <223> ρ74 <400> 7

Gly Arg Val Arg Arg Leu Ser Glu Glu Asp Cys Gin Lys Tyr Ile Leu 15 10 15

Lys Gin Gin Gin Glu Glu Ala Glu Lys Pro Leu Gin 20 25 <210> 8 <211> 28 <212> PRT <213> Artificial Sequence <220>

<221> mature peptide <222> (1) .. (28) <223> p75 <400> 8

Gly Arg Val Arg Arg Leu Ser Glu Glu Asp Cys Gin Lys Tyr Ile Leu 15 10 15

Lys Gin Gin Gin Glu Glu Ala Glu Lys Pro Leu Gin 20 25 <210> 9 <211> 27 <212> PRT <213> artificial sequence <220>

<221> mature peptide <222> (1). . (27) <223> p92 <400> 9

Gly Arg Val Arg Arg Leu Ser Glu Glu Asp Cys Gin Lys Tyr Ile Leu 15 10 15

Lys Gin Gin Glu Glu Ala Glu Lys Pro Leu Gin 20 25

<210> 10 <211> 36 <212> PRT <213> artificial sequence <220> <221> mature peptide <222> (1) · (36) <223> p93 <400> 10

-22Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys

Glu Glu Asp Cys Gin 20 Lys Tyr Ile Leu 25 Lys Pro Leu Gin 35 <210> 11 <211> 9 <212> PRT <213> artificial sequence <220> <221> mature peptide <222> (D .. . (9) <223> p51 <400> 11 Leu Lys Gin Gin Gin Glu Glu Ala Glu

<210> 12 <211> 14 <212> PRT <213> HIV-1 VIRUS <220>

<221> mature peptide <222> (1) .. (14) <223> protein TAT <400> 12

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin Gin 15 10 <210> 13 <211> 16 <212> PRT <213> Drosophila melanogaster <220>

<221> mature peptide <222> (1) .. (16) <223> antennapedia <400> 13

Arg Gin Ile Lys Ile Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys 15 10 15

Claims (16)

Claims 1. The peptide inhibitory sequence of SEQ ID NO: 4 to SEQ ID NO: 11. An inhibitory peptide according to claim 1 characterized in that it is optionally linked to other amino acid sequences other than the TIR or DD MyD88 domain sequences so that the fusion protein retains the inhibitory properties of the inhibitory peptide according to claim 1. Inhibitomes peptide according to claims 1 or 2, characterized in that the inhibitory peptide is 75% identical to the inhibitory peptide according to claim 1, and which includes inhibitory peptides with point amino acid changes or an inhibitory peptide from the INT MyD88 vertebrate domain. An inhibitory peptide according to any one of claims 1 to 3, characterized in that it optionally contains a translocation segment that allows the inhibitory peptide to enter the cell, and wherein the translocation segment consists of translocation sequences that transfer the inhibitory peptide into the cell. An inhibitory peptide according to claim 4, characterized in that the translocation segment is from the peptide antennapedia SEQ ID: 13 or the peptide TAT SEQ ID NO: 12. An inhibitory peptide according to any one of claims 1 to 3, characterized in that it optionally contains a translocation segment, wherein the translocation segment is an acyl chain containing 8 to 22 carbon atoms, preferably a lauryl or myristoyl chain. A peptide inhibitor according to any one of claims 1 to 6 for the preparation of therapists for the treatment or prevention of human and animal diseases when the disease is characterized by activation of an innate immune response or inflammation. The DNA encoding the peptides according to any one of claims 1 to 5 and the DNA is operatively linked to regulatory elements, such as a promoter and terminator, that allow expression of the inhibitory peptide in the host organism. A small organic molecule (peptidomimetic) that mimics the properties and action of peptides according to any one of claims 1 to 6. A pharmaceutical composition comprising: (a) an inhibitory peptide according to any one of claims 1 to 7 and (b) a pharmaceutically acceptable carrier. • * -2411. A method for selectively interfering with the MyD88 dependent pathways, which introduces into the cell any combination of inhibitory peptides according to any one of claims 1 to 7 in an amount sufficient to cause obstruction. A method for preventing an inflammatory response involving the activation of MyD88 in the body by introducing an inhibitory peptide according to any one of claims 1 to 7. 13. The method of claim 12, wherein the inflammatory response is associated with at least one of the allergic conditions, asthma, contact dermatitis, delayed hypersensitivity, wound healing, allergic rhinitis, food hypersensitivity, atopic dermatitis, bowel disease, immunological lung disease, autoimmune or immune-mediated skin disease, psoriasis, enteropathy associated with gluten hypersensitivity, rheumatoid arthritis, graft rejection, sepsis, septic shock, cancer, gout, systemic lupus erythematosus, colitis, neuropathic pain, neurodegenerative diseases, ischemia, hypoxia. 14. The method of claim 12 or 13, wherein the inhibitory peptide is administered prophylactically or therapeutically. A method according to any one of claims 12 to 14, characterized in that the inhibitory peptide or DNA encoding the inhibitory peptide according to claim 8 is introduced in one or more ways intravenously, intradermally, subcutaneously, orally by inhalation, by deep inhalation lungs, nasal, transdermal, transmucosal, vaginal, rectal, intrathecal, intraarticular, intraperitoneal or gene therapy methods. A method according to any of claims 12 to 15, characterized in that the inhibitory peptide is introduced in combination with a compound used to treat or prevent inflammation-related conditions. A kit comprising at least one inhibitory peptide according to any one of claims 1 to 6 and a control peptide.