KR101950502B1 - Peptide that significantly reduce airway inflammation and Composition comprising the peptide - Google Patents

Abstract

The peptide according to the present invention links a Tat sequence to a GPR (G-protein regulatory) peptide and binds Q (glutamine) to A (alanine (aspartic acid-glutamine-arginine) in the DDQR ), And the GPR peptide is characterized by using the one contained in the AGS (Activators of G-protein Signaling) 3 protein.
According to the present invention, it is possible to provide a novel drug candidate substance which can act on the mechanism of bio-defense and significantly reduce respiratory inflammation.

Description

[0001] Peptides that significantly reduce respiratory inflammation and compositions comprising same [0002]

The present invention relates to a GPR peptide capable of significantly reducing respiratory inflammation and a composition containing the same.

Modern medicine has not been able to provide the fundamental treatment of intractable chronic inflammatory diseases caused by irreversible inflammatory reaction, which is the major respiratory diseases of modern people.

These respiratory diseases are chronic obstructive diseases, inflammatory metabolic diseases, asthma, rhinitis, etc. These are high-rate and low-efficiency diseases which represent the highest level of medical expenses in Korea and the world. Table 1 shows the status of 'DABINDO SOCIETY RATINGS' in '2009 Health and Welfare Department's health insurance finances / salary status'. As shown in Table 1, respiratory inflammatory diseases such as pneumonia, tonsillitis, bronchitis, upper respiratory tract infection, nosephritis, and pharyngitis are highly ranked in the number of medical care and medical care costs.

'08
ranking Admission Outside A bottle name diagnosis
Number Care
salary
cost '08
ranking A bottle name diagnosis
Number Care
salary
cost '08
ranking One  Only one day 216 92,662 One Primary (primary) High blood pressure 19,427 304,865 One 2 Tooth 204 120,381 2 Acute tonsillitis 14,469 231,691 3 3  Old age 176 158,610 4 Acute gastroenteritis 14,329 251,048 2 4 Single delivery by cesarean section 149 140,722 3 Diseases and periapical tissue disorders 11,431 424,110 4 5 Pneumonia of unspecified pathogen 143 101,354 5 Acute upper respiratory tract infections of multiple and unspecified sites 9,319 145,227 5 6 Cerebral palsy 120 168,638 6 Acute coin diphosphate [wound] 8,012 108,247 6 7 Diarrhea and gastroenteritis 106 37,884 8 Chiropractic 7,991 246,678 8 8 Acute suffocation 106 101,786 7 Acute diphtheria 7,825 121,842 7 9 Others 93 92,014 10 Gingivitis (periodontal disease) and periodontal disease 7,377 188,826 9 10 Neural tube column 90 98,144 9 Urinary insulin resistance 7,003 147,392 10

<Unit: thousand won, million won>

In particular, irreversible inflammation in the respiratory tract can lead to severe disability and secondary social problems, which can lead to an exponential increase in economic burdens not only for individuals but also for nations and societies.

So far, strategies to block the activation of inflammatory cells have been used (eg, COX2 inhibitors) as a treatment strategy for inflammatory diseases. However, many therapeutic agents that inhibit the activation pathway of inflammatory cells have not been treated effectively and only transient effects have been observed. Therefore, it is very necessary to develop new alternative treatment strategies.

Intracellular mucus, secretory and overexpression regulatory networks are very important protective mechanisms in vivo that inhibit progression to acute and chronic inflammatory damage. Recently, the deficiency of the bio-defense regulation system in the respiratory or congenital immunological field has been accepted as a new key cause of chronic inflammatory disease.

Candidates for this field of study are essential in light of international research trends to understand endogenous defense mechanisms in an integrated manner.

Methods of using G protein-coupled receptors to identify compounds useful in the treatment of conditions modulated by PYY, and peptide YY (PYY) secretagogues and PYY WO 2006-003422 (Jan. 12, 2006), USE OF THE RECEPTOR GPR86

Lanier, S.M. AGS proteins, GPR motifs and the siganls processed by heterotrimeric G protein, Biol Cell 96, 369-372 (2004). Cismowski, M.J. et al. Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling. Nat Biotechnol 17, 878-883 (1999). Takesono, A. et al. Receptor-independent activators of heterotrimeric G-protein signaling pathways. J Biol Chem 274, 33202-33205 (1999). Rhee, S., Jiang, H., Ho, C. H., & Grinnell, F. Microtubule function in fibroblast spreading modulates according to the tension state of cell-matrix interactons. Proc Natl Acad Sci USA 104, 5425-5430 (2007). Colvin, R. A. et al. Synaptotagmin-mediated vesicle fusion regulates cell migration. Nat LMunol 11, 495-502 (2010).

It is an object of the present invention to provide a new drug candidates which can replace the existing treatment methods for inhibiting the activation of inflammatory cells in the treatment of respiratory inflammation and can significantly reduce respiratory inflammation by acting on the mechanism of defense against the body .

The peptide according to the present invention links a Tat sequence to a GPR (G-protein regulatory) peptide and binds Q (glutamine) to A (alanine (aspartic acid-glutamine-arginine) in the DDQR ). &Lt; / RTI &gt;

The peptide according to the present invention is characterized by comprising the following amino acid sequence (left N-terminal, right C-terminal).

GRKKRRQRRRPPTMGEEDFFDLLAKSQSKRMDDARVDLAK

The GPR peptide is characterized by using a protein contained in an AGS (Activators of G-protein Signaling) 3 protein.

The peptide according to the present invention is characterized in that it is synthesized by linking a Tat sequence to a GPR (G-protein regulatory) peptide.

The peptide according to the present invention is characterized by comprising the following amino acid sequence (left N-terminal, right C-terminal).

GRKKRRQRRRPPTMGEEDFFDLLAKSQSKRMDDQRVDLAK

The GPR peptide is characterized by using a protein contained in an AGS (Activators of G-protein Signaling) 3 protein.

The composition according to the present invention may comprise any one of the peptides described above.

According to the present invention, it is possible to provide a novel drug candidate substance which can act on the mechanism of bio-defense and significantly reduce respiratory inflammation.

Figure 1 shows the structure of the AGS3 protein.
Figure 2 shows the amino acid sequence of a peptide according to one embodiment of the present invention.
FIG. 3 to FIG. 8 show experimental results using the peptide according to the present invention.

Hereinafter, the present invention will be described in detail.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the understanding why the present invention is not thereby well understood.

Throughout the specification of the present invention, unless the context requires otherwise, the words " comprises " and " comprising " include any step or element, or group of steps or elements, Or groups of elements are not excluded.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[ Peptides  Configuration]

Activators of G-protein signaling (AGS) 3 proteins are present in many cells. Figure 1 shows the structure of the AGS3 protein. As shown in FIG. 1, the AGS3 protein has a modular domain structure composed of seven TPRs (tetracyclic peptide repeats) and four GPR (G-protein regulatory) motifs. Four of these GPR (G-protein regulatory) parts regulate G-protein (G-protein), which plays an important role in intracellular signaling. The most important function of the GPR part is to inhibit signal transduction of the G-protein by binding to the alpha subunit of the G-protein (composed of alpha beta gamma-subunits).

As shown in Fig. 2, the present inventors synthesized a peptide (Tat-GPR) to link the Tat sequence (GRKKRRQRRRPP) to a 28-amino acid GPR peptide so that it can be easily absorbed into cells (left N-terminal, right C -terminal).

In order to demonstrate the functional role of the GPR peptide, as shown in Fig. 2, a modified peptide (Tat) in which glutamine in the DDQR (aspartic acid-aspartic acid-glutamine-arginine) sequence which is the most important part of the GPR peptide was substituted with alanine -GPR Q34A) (left N-terminal, right C-terminal).

The two peptides are peptides that can perform completely different functions.

[Function and effect]

Since the most important consideration when introducing peptides into cells is toxicity, the lack of toxicity is demonstrated using apoptosis assays.

The peptides of the present invention were infiltrated into respiratory epithelial cells and then the expression of inflammatory and antiinflammatory factors was confirmed after inflammatory stimulation. As a result, it was confirmed that the modified GPR peptide (Tat-GPR Q34A) reduced the expression of inflammatory factors and increased the expression of anti-inflammatory agents.

Figure 3 shows the expression of inflammatory and anti-inflammatory factors in respiratory epithelial cells. As shown in FIG. 3, the expression of the anti-inflammatory factor (MUC1, TGFβ) was decreased in the Tat-GPR peptide, while the expression of the inflammatory factor (TNFα, IL-6) . In the case of the Tat-GPR Q34A peptide, the expression of inflammatory factors (TNFα, IL-6) decreased while the expression of anti-inflammatory factors (MUC1, TGFβ) Respectively.

The above experiments were also performed in normal human bronchial epithelial cells (NHBE), not cell-line cell lines, and the results are shown in FIG. As shown in FIG. 4, the expression of the anti-inflammatory factors (MUC1, TGFβ) decreased while the expression of the inflammatory factors (TNFα, IL-6) -GPR Q34A peptide, the expression of inflammatory factors (TNFα, IL-6) decreased and the expression of anti-inflammatory factors (MUC1, TGFβ) increased. Thus, the same results were obtained in normal human respiratory epithelial cells, proving to be a common proven effect in the respiratory epithelium rather than in specific cells.

Actin polymerization is one of the cell functions that plays a crucial role in cell migration and immunity. The G-protein receptor called CXCR4 is a mediator that mediates the inflammatory reaction, which causes many inflammatory cells to aggregate and cause inflammation more severely, leading to further disease.

In the present invention, in order to examine whether the modified GPR peptide (Tat-GPR Q34A) inhibits actin polymerization caused by CXCR4, actin polymerization was observed two-dimensionally and three-dimensionally on a slide coated with collagen, Respectively.

As shown in FIG. 5, the actin polymerization activity of the normal GPR peptide (Tat-GPR) was actively increased to increase cell migration and inflammation, but it was found that the modified GPR peptide (Tat-GPR Q34A) have.

These results demonstrate that actin polymerization does not occur any more because inflammation is reduced by modified GPR peptides in the inflammatory microenvironment.

In order to observe the phenomenon occurring in the cells in the cells, rats were subjected to tracheostomy using inflammatory stimulation by tracheostomy again 24 hours after the injection of the peptide (see FIG. 6).

On the third day of inflammatory stimulation, mice were sacrificed and stained with bronchial washing fluid (BAL fluid) and lungs. The results are shown in FIG.

In the AB-PAS method, which only stains mucous cells, it secretes more mucus material by the normal GPR peptide (Tat-GPR) (Figure Ac), but it is significantly reduced by the modified GPR peptide (Tat-GPR Q34A) (Figure Ad).

(T-GPR Q34A) by the H & E staining method, which observes intensive aggregation of inflammatory cells, while more inflammatory cells are concentrated by the normal GPR peptide (Tat-GPR) Suggesting that the recruitment of inflammatory cells is significantly reduced (Figure Bd).

Protein analysis was also performed in the sacrificed rats, and the results are shown in Fig. As shown in FIG. 8, as the concentration of normal GPR peptide (Tat-GPR) increases, the expression of inflammatory factors (TNFα, IL-6) increases, while the anti- inflammatory factors (MUC1, TGFβ) Lt; RTI ID = 0.0 &gt; of &lt; / RTI &gt; In contrast, the modified GPR peptide (Tat-GPR Q34A) showed an anti-inflammatory effect by increasing the expression of anti-inflammatory factors while decreasing the expression of inflammatory factors as the concentration increased.

The action and stability of the peptides constructed according to the present invention have been demonstrated through experiments and the present invention can be used to develop new peptide drugs that inhibit intracellular signal transduction receptors that are not COX-2 inhibitors that inhibit existing inflammation .

The results showed that the GPR peptide (Tat-GPR Q34A) showed very good efficiency, performance, and physiological functions, as the results in cells, the results in normal respiratory epithelial cells, and the results in laboratory animals are all the same.

The present invention can provide candidates for new drug development by providing basic data for the development of inflammatory therapeutic agents in the respiratory tract.

Studies on endogenous inflammation treatment networks are expected to offer novel therapeutic alternatives for intractable and chronic inflammatory diseases and are expected to have very high expectations and prospects for peptides.

In the present invention, systematic and integrated / integrated studies on the specific signaling network blocking / suppression and specific anti-inflammatory functions and regulatory mechanisms of inhibitory protein candidates in experimental animal models are creative and functional.

The present invention is expected to provide an internationally competitive core base in the development of a fundamental diagnostic or therapeutic method for acute / chronic inflammatory diseases.

Peptides according to the present invention are expected to play a very leading / creative role when compared with the international research flow to understand mutually integrated understanding of the pathology of metabolic and inflammatory diseases in recent years.

The global market for diagnostic kits using genetic information has reached 4 trillion won as of 2010. Thanks to the remarkable development of biotechnology and the steady interest in the health of the general public, / Therapeutics-related technology is expected to steadily increase, it is necessary to provide a new drug using the peptide of the present invention having excellent anti-inflammatory effect.

As described above, an optimal embodiment has been disclosed in the drawings and specification. Although specific terms have been employed herein, they are used for purposes of illustration only and are not intended to limit the scope of the invention as defined in the claims or the claims. Therefore, those skilled in the art will appreciate that various modifications and equivalent embodiments are possible without departing from the scope of the present invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> KOSIN UNIVERSITY INDUSTRY-ACADEMY COOPERATION <120> Peptide that significantly reduces airway inflammation and          Composition comprising the peptide <130> PN16-0659-KR <140> 10-2017-0012928 <141> 2017-01-26 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Tat-GPR <400> 1 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Thr Met Gly Glu   1 5 10 15 Glu Asp Phe Phe Asp Leu Leu Ala Lys Ser Gln Ser Lys Arg Met Asp              20 25 30 Asp Gln Arg Val Asp Leu Ala Lys          35 40 <210> 2 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Tat-GPR Q34A <400> 2 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Thr Met Gly Glu   1 5 10 15 Glu Asp Phe Phe Asp Leu Leu Ala Lys Ser Gln Ser Lys Arg Met Asp              20 25 30 Asp Ala Arg Val Asp Leu Ala Lys          35 40

Claims (7)

A Tat sequence is linked to a GPR (G-protein Regulatory) peptide consisting of the amino acid sequence of SEQ ID NO: 1 and Q (glutamine) is converted from DDQR (aspartic acid-glutamine-arginine) in the amino acid sequence of the GPR peptide to A Alanine) as an active ingredient. The composition for preventing and treating respiratory inflammatory diseases according to claim 1,
The method according to claim 1,
Wherein the GPR peptide comprises the following amino acid sequence (left N-terminal, right C-terminal).
GRKKRRQRRRPPTMGEEDFFDLLAKSQSKRMDDARVDLAK
The method according to claim 1,
Wherein the GPR peptide is one contained in an AGS (Activators of G-protein Signaling) 3 protein.
The method according to claim 1,
Wherein said composition reduces the expression of inflammatory factors and increases the expression of antiinflammatory agents.
5. The method of claim 4,
Wherein the inflammatory factor is TNF? Or IL-6.
5. The method of claim 4,
Wherein the anti-inflammatory agent is MUC1 or TGF [beta].
The method according to any one of claims 1 to 6,
Wherein the respiratory inflammatory disease is at least one disease selected from the group consisting of chronic obstructive diseases, inflammatory metabolic diseases, asthma, rhinitis, and the like.

Images