KR20130057012A - A composition for inhibiting the activity of immunocyte - Google Patents

Abstract

이와같은 본 발명의 조성물에 따르면, 예컨대 동맥경화, 류마티스 관절염을 포함하는 면역질환로서 세포독성들과 같은 부작용 없이 대식세포의 활성화 과정을 유의하게 억제함으로써 새로운 치료제의 제공이 가능하다는 등의 현저한 효과가 있다.The present invention relates to a composition for inhibiting immune cells. It may contain, as an active ingredient, the coprisin peptide derivative CopA3 (9-mer disulfide dimer) having the following amino acid sequence.

According to such a composition of the present invention, as an immune disease including arteriosclerosis and rheumatoid arthritis, a remarkable effect of providing a new therapeutic agent by significantly inhibiting the activation process of macrophages without side effects such as cytotoxicities can be provided. have.

Description

Composition for Inhibiting the Immune Cell {A Composition For Inhibiting the Activity of Immunocyte}

 The present invention relates generally to a composition for suppressing immune cells, and more particularly, to the cytotoxicity, such as the coprisin peptide derivative CopA3 (9-mer disulfide dimer) extracted from insects such as cedar beetles, as an active ingredient. It relates to a novel immune cell suppression composition that significantly inhibits the activation process of immune cells, such as macrophages without side effects.

In general, atherosclerosis is a disease in which vascular diameter decreases through hyperplasia of smooth muscle cells due to the release of inflammatory cytokines from macrophages and T-cells introduced into the blood vessel wall. Rheumatoid arthritis has also been identified as an inflammatory disease involving macrophages. However, despite the various etiology findings, the therapeutic choice for various inflammatory immune diseases including atherosclerosis and rheumatoid arthritis is very limited, and the previously developed therapeutics also have side effects.

In addition, the development of new drugs for related diseases is urgently needed because the pathogenesis of various diseases is increasingly defined as immunological adverse reactions based on molecular immunology / cell biology studies. At the same time, there is an urgent need to find therapeutic substances with fewer side effects to cure a variety of inflammatory immune diseases, including atherosclerosis, rheumatoid arthritis and broadly allergies.

The present invention has been made to solve the above-mentioned problems of the prior art and to add various other advantages. Particularly, the present inventors have already identified arteriosclerosis and rheumatoid arthritis as macrophage or T cell mediated inflammatory diseases. In addition, the discovery of a controlled drug that can be directly applied to macrophages known to be involved in innate immune response as well as to promote acquired immunity and memory through antigen presentation is a new therapeutic agent that can be applied to immune diseases as a whole. From this point of view, the efficacy of the insect-derived peptide CopA3 found by the present inventors to strongly inhibit LPS-induced macrophage activation can be evaluated very positively for drug development. Furthermore, the non-cellular toxicity of CopA3 against macrophages has been confirmed. Therefore, it can be judged that there is no major problem in clinical application, and finally, at the cellular level, the CopA3 inhibition mechanism is due to the inhibitory effect on intracellular transcripts (STAT1 and STAT5) activated by LPS stimulation. Since it was proved, the inventors focused on not only CopA3 discovered through the present invention but also on the development of new drugs based on the structure.

The present invention is a novel novel coprisine peptide derivative CopA3 (9-mer disulfide dimer) extracted from insects such as cedar beetles, for example, as an active ingredient, which can suppress the activation process of immune cells such as macrophages without cytotoxicity. It is an object to provide a composition for inhibiting immune cells.

The object is achieved by a composition for inhibiting immune cells provided according to the present invention.

The composition for suppressing immune cells provided according to one aspect of the present invention contains a coprisin peptide derivative CopA3 (9-mer disulfide dimer) having the following amino acid sequence as an active ingredient.

In one embodiment, it is a regulator that inhibits the process of activation of immune cells.

In another embodiment, the immune cell is a macrophage.

In another embodiment, the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is D-type.

In another embodiment, the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is derived from an insect, preferably isolated from Copris tripartitus.

In another embodiment, the coprisin peptide derivative CopA3 (9-mer disulfide dimer) may be used for the treatment of immune diseases including atherosclerosis and rheumatoid arthritis.

In another embodiment, the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is located at the alpha-helix 9 of the antibiotic peptide Coprisin (43 amino acids) isolated from cedar beetle. Selected amino acids are further synthesized, and further substituted histidine with leucine among the 9 amino acids, and finally synthesized by dimers using a thiol residue of cysteine located in the middle of the 9-mer coprisin analogue sequence Immune cell suppression composition.

According to the present invention having the above-described constitution, immune cells such as macrophages, without side effects such as cytotoxicity, using copricin peptide derivative CopA3 (9-mer disulfide dimer) extracted from insects such as cedar beetles as an active ingredient A novel immune cell suppression composition can be provided that can significantly inhibit the activation process of the drug, thereby providing the possibility of developing a new therapeutic agent without side effects for the treatment of immune diseases such as arteriosclerosis, rheumatoid arthritis, or allergies, etc. Can provide a significant effect.

1 is a schematic diagram illustrating a peptide sequence of the coprisin peptide derivative CopA3 (9-mer disulfide dimer) which is the main component of the composition for suppressing immune cells according to an embodiment of the present invention.
Figure 2 is a graph showing the efficacy of inhibiting the LPS-stimulated induced macrophage activation process of the coprisin peptide derivative CopA3 (9-mer disulfide dimer), the main component of the composition for suppressing immune cells according to an embodiment of the present invention.
Figure 3 is a graph showing the efficacy of inhibiting the mouse celiac macrophage activation process of the coprisin peptide derivative CopA3 (9-mer disulfide dimer), the main component of the composition for suppressing immune cells according to an embodiment of the present invention.
Figure 4 is a graph showing the cell nontoxic effect on macrophages of the coprisin peptide derivative CopA3 (9-mer disulfide dimer), the main component of the composition for suppressing immune cells according to an embodiment of the present invention.
FIG. 5 shows STAT1 and STAT5 transcription factors of the coprisin peptide derivative CopA3 (9-mer disulfide dimer), the main component of the composition for suppressing immune cells according to one embodiment of the present invention, activated by LPS stimulation in macrophages. Graph showing the inhibitory effect on

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. To fully disclose the scope of the invention to those skilled in the art, and is defined by the claims of the present invention.

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 subject matter of the present invention rather unclear. The following terms are defined in consideration of the functions in the embodiments of the present invention, which may vary depending on the intention of the user, the intention or the custom of the operator. Therefore, the definition should be based on the contents throughout this specification.

Hereinafter, a composition for suppressing immune cells according to a specific embodiment of the present invention will be described with reference to the accompanying drawings.

In the present invention, the immune activation response against Raw264.7 macrophages to Lipopolysaccharide (hereinafter LPS) constituting the outer membrane of bacteria, in particular Gram-negative bacteria, as a complex of fat and polysaccharide was evaluated.

LPS acts as an endotoxin in the human body and is known as a cytokine-inducing substance by capturing the CD14 / TLR4 / MD2 receptor complex to interfere with the immune system and stimulate macrophages. It has already been shown that the cytokine secretion and immune activity are increased when this material is treated with raw macrophage, a mouse macrophage.

In all experiments performed in the present invention, 1 μg / ml concentration of CopA3 was pretreated for 1 hour and LPS was treated at a concentration of 1 μg / ml. After treatment with LPS and CopA3 in raw 264.7 macrophages, the changes in the secretion of proinflammatory cytokine (IL-6, TNF-) were measured using an ELISA kit to evaluate macrophage activation. In addition, we evaluated the inhibitory activity of CopA3 on the activation process of macrophages isolated from mouse abdominal cavity. The non-toxicity of CopA3 against Raw264.7 macrophages was also evaluated by MTT assay and TUNEL assay. Finally, Western blotting was used to confirm the activation phosphorylation of STAT1 and STAT5, which are dependent on LPS, to investigate the inhibition of CopA3.

Hereinafter, the present invention will be described more specifically by way of examples.

These examples are only to specifically describe the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. In addition, although not shown in the specific examples, it is possible to prepare an autoimmune disease therapeutic composition containing an antimicrobial peptide according to the present invention as an active ingredient having an immunosuppressive action against immune cells such as macrophages of humans or mammals. Will be taken for granted.

Example 1 Peptide Sequence Comparison of Coprisin and Coprisin Analogue CopA3

The inventors confirmed that the alpha-helix site of the antibiotic peptide Coprisin (43 amino acids) isolated from cedar beetle was a site showing antibiotic effect. Based on this, nine amino acids located in the alpha-helix region were selected for further synthesis and still showed strong antibiotic effects. Researchers and others further substituted histidine with leucine among 9 amino acids and confirmed that the antibiotic effect was increased. Finally, we tried to evaluate the efficacy on immune cells by synthesizing dimer using thiol residue of cysteine located in the middle of 9-mer coprisin analogue sequence. This peptide finally synthesized as a dimer was named CopA3, and all peptides used in the present invention were CopA3.

Example 2 Efficacy of CopA3 Inhibiting LPS-stimulated Induction Macrophage Activation Process

In the case of IL-6 and TNF-alpha cytokines, extracellular secretion is evaluated as an indicator of macrophage activation, so seeding Raw264.7 macrophages on a 12-well plate and pretreating 1 μg / ml CopA3 for 1 hour And 1 μg / ml LPS was treated for 24 hours and then the secretion was measured to evaluate the degree of activation of macrophages. To this end, after collecting the supernatant, IL-6 and TNF-alpha were measured using an ELISA kit. 100 μl of the supernatant was added to a 96-well plate and the reaction was performed at room temperature for 2 hours, and then the first antibody reaction was performed for 1 hour. Then, the secondary antibody was added and further reacted for 30 minutes, and then a coloring solution (stop solution) was added and measured at a wavelength of 580 nm. Both IL-6 and TNF-alpha showed a two-fold higher increase in the LPS treatment group. However, in the group treated with CopA3, the amount of secretion of the inflammatory cytokines was confirmed to be significantly reduced. The extent of the decline was reduced to near baseline.

Example 3: Evaluation of the Inhibitory Activity of CopA3 on Activation of Macrophage Cells in Rat Peritoneum

Next, the inventors tried to determine whether CopA3 could also inhibit the activation of macrophages isolated from mouse abdominal cavity. To this end, 10 ml of sterile saline was injected into the abdominal cavity of rats, shaken, and physiological saline was recovered again using a syringe. The cells were centrifuged at 1000 rpm for 5 minutes and the seeded cells were seeded in h-DMEM (w / 10% FBS) medium. In the existing literature, it is known that intraperitoneal immune cells are composed of macrophages, B cells and T cells. Therefore, 1 hour after seeding, washing with cold PBS to remove unadhered immune cells (B cells, T cells) can recover only macrophages attached to the bottom of the culture vessel. The macrophages prepared through the above procedure were treated with LPS or LPS and CopA3 for 15 hours, and IL-6 and TNF- were measured again. As shown in FIG. 3, all cytokines were increased in the group treated with LPS, but the secretion was significantly decreased in the group treated with CopA3. The activation of whole peritoneal cells in response to LPS stimulation was also significantly reduced with CopA3 treatment, but the decrease was relatively small compared to the response in single macrophages.

The above results suggest that the inhibitory effect of CopA3 is a specific response to macrophages rather than B cells or T cells.

Example 4: Toxic effect of CopA3 on Raw264.7 cells

To determine whether CopA3 induces apoptosis or cytotoxicity in Raw264.7 macrophages, we performed MTT assay and TUNEL assay, which is a cell health assessment. After seeding Raw264.7 macrophages into 1 x 10 ⁴ in a 96-well plate, the culture was maintained for one day and used for the experiment. The cells were treated with CopA3 (1 μg / ml) alone or CopA3 and LPS (1 μg / ml) simultaneously for 52 hours, followed by MTT assay. As shown in (A) of FIG. 4, CopA3 alone treatment group was found to increase the cell health about 60% compared to the control group unexpectedly. In the LPS and CopA3 treatment groups, no significant decrease in health was observed compared to the control group. Next, the researchers performed TUNEL assay to determine the effect of apoptosis after CopA3 treatment in Raw264.7 macrophages. Macrophages were maintained in culture and then treated with CopA3 (1 μg / ml) or staurosporine (1 μM) for 52 and 24 hours, respectively. After removing the medium and adding 4% paraformaldehyde, the cells were fixed in the incubator for 10 minutes. The cells were then perforated with 0.1% triton X-100 and reacted at 37 for 2 hours in a TUNEL buffer mixed with equilibrium buffer, nucleotide mixture and rTdT. Irrespective of DNA cleavage, propidium iodide was added and stained simultaneously to stain nuclear DNA of whole cells. After the reaction, the cells were washed three more times with PBS, finished with a mounting solution and a cover slide, and cell images were measured using a fluorescence microscope. As shown in FIG. 4 (B), CopA3 did not induce DNA cleavage in the nucleus, but it was confirmed that staurosporine used as a positive control caused very high. These experiments demonstrated that CopA3 did not reduce cell health or cause cell death for macrophages.

Example 5 Inhibitory Effect of CopA3 on STAT1 and STAT5 Transcription Factors Activated by LPS Stimulation in Macrophages.

Transcription proteins known to be dependent on LPS stimulation include signal transducers and activators of transcription-1 and -5 (STAT1 and STAT5). In immune cells, these proteins are attached to phosphates by LPS stimulation, resulting in phospho-STAT1 and phospho-STAT5, which are then activated and involved in inflammatory cell responses. By evaluating the active phosphorylation of these proteins, we tried to elucidate the molecular mechanism of the inhibitory effect of CopA3. To this end, CopA3 was pretreated in Raw264.7 macrophages for 1 hour, LPS was treated at 1 μg / ml for 2 hours, and the active phosphorylation of STAT1 and STAT5 was confirmed by Western blotting.

Treatment cells were lysed and loaded at the same concentration on a 10% SDS-PAGE gel, followed by electrophoresis of the protein. After that, the membranes were transferred and reacted overnight using phospho-STAT1 and phospho-STAT5 antibodies. Phosphorylation was evaluated by color development after reaction at room temperature for 2 hours using a secondary antibody. As in Figure 5 (A), LPS stimulation significantly increased the active tyrosine phosphorylation of STAT1 and STAT5. However, when CopA3 was pretreated, the degree was significantly reduced.

Next, the reporter assay was performed to determine whether CopA3 treatment also inhibited the transcriptional regulation of STAT1 and STAT5. Intracellular transcription that is altered by response to LPS stimulation and CopA3 treatment using an artificial luciferase vector (a reporter with 8 repeated STAT action sites as an 8X-GAS luciferase assay vector) with a STAT family protein-specific action site at the promoter site The amount change was measured.

Transfection of 8X-GAS luciferase assay vector regulated specifically to STAT family protein using lipofectamine was performed on the macrophages, followed by simultaneous treatment of LPS or LPS and CopA3 for 24 hours. As in FIG. 5B, LPS alone treatment significantly increased reporter activity (SATA1 and STAT5 activated by LPS stimulation triggered the reporter). However, in the group treated with CopA3, the increase was significantly decreased. This result shows that STAT phosphorylation reduced by CopA3 treatment leads to decreased activity of glycolytic protein that regulates transcription.

As already mentioned, the macrophage activation process has been considered as a major etiology in immune diseases such as atherosclerosis and rheumatoid arthritis. Therefore, it is reasonable to develop a therapeutic agent for immune diseases such as atherosclerosis and rheumatoid arthritis by using the inhibitory effect of macrophages activated by CopA3 peptide isolated from insects. In the present invention, CopA3 was confirmed to be non-cytotoxic to macrophages and the development of therapeutic agents is expected to be positive because it clearly identified intracellular molecular changes on the inhibitory mechanism. In addition, if various attempts are made to synthesize new substances based on the peptide structure, it will increase the immunosuppressive effect and speed up the discovery of powerful new substances without cytotoxicity.

As described above, the present invention revealed that the coprisin peptide derivative CopA3 (9-mer disulfide dimer) isolated from Copris tripartitus significantly inhibited the activation of Raw264.7 macrophages, which are immune mediating cells. As a novel immunosuppressive agent. It was demonstrated that interleukin-6 (IL-6) and TNF-alpha, which are proinflammatory cytokines secreted through macrophages in the process of stimulation by lipopolysaccharide (LPS) stimulation, were significantly reduced by pretreatment with CopA3. The present invention also demonstrated that the activation of the transcription factors STAT1 and STAT5, which are known to be activated LPS stimuli dependently, is inhibited by CopA3, thereby inhibiting the macrophage activation process.

Claims (8)

A composition for inhibiting immune cells, comprising as an active ingredient a coprisin peptide derivative CopA3 (9-mer disulfide dimer) having the following amino acid sequence.

The composition of claim 1, wherein the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is a regulator that inhibits the process of activating immune cells. The composition for inhibiting immune cells according to claim 2, wherein the immune cells are macrophages. The composition of claim 1, wherein the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is D-type. The composition of claim 1, wherein the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is derived from an insect. The composition of claim 5, wherein the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is isolated from copris tripartitus. The composition of claim 1, wherein the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is used for the treatment of immune diseases including atherosclerosis and rheumatoid arthritis. The 9-mer disulfide dimer (CopA3) according to claim 1, wherein the coprisin peptide derivative CopA3 (9-mer disulfide dimer) is a 9 amino acid located at the alpha-helix site of the antibiotic peptide Coprisin (43 amino acids) isolated from cedar beetle Selected and further synthesized, and further substituted histidine with leucine among 9 amino acids, and finally synthesized by dimer using a thiol residue of cysteine located in the middle of the 9-mer coprisin analogue sequence Inhibiting composition.

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