PEPTIDE FOR TREATING RHEUMATOID ARTHRITIS AND USE THEREOF

Abstract

A peptide for treating rheumatoid arthritis and use thereof are provided, and a peptide consisting of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or a polynucleotide encoding the same, and a pharmaceutical composition for treating rheumatoid arthritis or a health functional food composition for improving rheumatoid arthritis, each including the peptide, are provided.

Claims

1. A peptide consisting of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

2. A pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a peptide consisting of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or a polynucleotide encoding the same as an active ingredient.

3. The pharmaceutical composition of claim 2, wherein the composition suppresses secretion of inflammatory cytokines.

4. The pharmaceutical composition of claim 2, wherein the cytokines are one selected from interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and a combination thereof.

5. The pharmaceutical composition of claim 2, wherein the N-terminal of the peptide binds to a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG), or the C-terminal of the peptide binds to a protecting group selected from the group consisting of an amino group (—NH.sub.2) and azide (—NHNH.sub.2).

6. The pharmaceutical composition of claim 2, wherein the composition is concurrently, separately, or sequentially administered in combination with an anti-inflammatory agent other than the peptide.

7. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier.

8. A health functional food composition comprising a peptide consisting of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 as an active ingredient.

9. A method for screening for a therapeutic agent of rheumatoid arthritis, comprising: (a) contacting apolipoprotein B100 (apoB100) with a subject sample comprising ENO1 (α-enolase) together with a candidate substance; and (b) comparing an apolipoprotein B100-ENO1 binding level, measured from the sample in contact with the candidate substance, with a control group to which no candidate substance is administered.

10. The method of claim 9, wherein the sample is a blood sample.

11. The method of claim 9, wherein the apolipoprotein B100-ENO1 binding level is measured by at least one method selected from the group consisting of two-hybrid assay, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), UV or chemical crosslinking assay, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), fluorescence polarization assay (FPA), and in vitro pull-down assay.

12. The method of claim 9, further comprising, when the binding level of apolipoprotein B100 to ENO1, measured from the sample in contact with the candidate substance, is reduced compared to that of the control group, determining the candidate substance as a therapeutic agent of rheumatoid arthritis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0047] FIG. 1 shows the results of observation of ENO1 expressed on surfaces of peripheral blood mononuclear cells isolated from rheumatoid arthritis patients, as measured using a confocal microscope.

[0048] FIG. 2 shows the results of comparison of changes in the secretion of (A) IL-1β, (B) IL-6, and (C) TNF-α, after treating peripheral blood mononuclear cells isolated from rheumatoid arthritis patients with apoB100 (HC: healthy control group, RA: rheumatoid arthritis patient group, *p<0.05).

[0049] FIG. 3 shows the results of (A) immunoblotting, (B) confocal microscopy, (C) BIACORE system, and (D) ELISA assay, to identify apoB100-ENO1 binding.

[0050] FIG. 4 shows the results of comparison of changes in the secretion of (A) IL-1β, (B) IL-6, and (C) TNF-α, after peripheral blood mononuclear cells isolated from rheumatoid arthritis patients were treated with ENO1siRNA to inhibit the expression of ENO1 and then treated with apoB100 (ConsiRNA: control group, ENO1siRNA: ENO1 expression inhibited group).

[0051] FIG. 5 shows the results of comparison of changes in (A) weight, (B) ankle thickness, and (C) arthritis score, after treating an LDLR knockout mouse and a wild-type mouse with apoB100 (LDLR K/O: LDLR knockout mouse, WT: wild-type mouse).

[0052] FIG. 6 shows the results of comparison of changes in the secretion of (A) IL-1β, (B) IL-6, and (C) TNF-α, after treating LDLR knockout mouse and a wild-type mouse with apoB100 (LDLR K/O: LDLR knockout mouse, WT: wild-type mouse).

[0053] FIG. 7 shows apoB100 motifs binding to ENO1 in peripheral blood, as identified by peptide micro array.

[0054] FIG. 8 shows the results of comparison of changes in the secretion of (A) IL-1β, (B) IL-6, and (C) TNF-α, after treating peripheral blood mononuclear cells isolated from rheumatoid arthritis patients with apoB100-derived peptides #1 to #5 (HC: healthy control group, RA: rheumatoid arthritis patient group, NBP: non-binding peptide treated group, *p<0.05, ***p<0.001).

[0055] FIG. 9 shows the results of comparison of changes in the secretion of (A) IL-1β, (B) IL-6, and (C) TNF-α, after treating CD14(−) peripheral blood mononuclear cells and CD14(+) peripheral blood mononuclear cells with apoB100-derived peptides #1 to #5 (NBP: non-binding peptide treated group, *p<0.05, **p<0.01, ***p<0.001).

[0056] FIG. 10 shows the results of comparison of changes in the secretion of (A) IL-1β, (B) IL-6, and (C) TNF-α, after treating peripheral blood mononuclear cells isolated from rheumatoid arthritis patients with peptides for inhibiting the secretion of inflammatory cytokines (apoB100 #1, apoB100 #3, and apoB100 #5) (NBP: non-binding peptide treated group, *p<0.05).

[0057] FIG. 11 shows the results of comparison of changes in (A) weight, (B) ankle thickness, and (C) arthritis score, after treating K/B×N rheumatoid arthritis animal models with peptides for inhibiting the secretion of inflammatory cytokines (apoB100 #1, apoB100 #3, and apoB100 #5).

[0058] FIG. 12 shows the results of comparison of (A) synovial inflammation, (B) bone erosion, (C) cartilage damage, and (D) leukocyte infiltration, after treating K/B×N rheumatoid arthritis animal models with peptides for inhibiting the secretion of inflammatory cytokines (apoB100 #1, apoB100 #3, and apoB100 #5).

MODE OF DISCLOSURE

[0059] Hereinafter, the present disclosure will now be described in greater detail with reference to the accompanying Examples below. However, these Examples are for illustrative purposes only, and should not be construed as being limited to the scope of the inventive concept in any way.

EXAMPLES

Example 1. Effect of ENO1-apoB100 Binding on Secretion of Inflammatory Cytokine

[0060] In this example, in order to identify the relation between rheumatoid arthritis and ENO1 expression on peripheral blood mononuclear cells, the ENO1 expression thereof was observed. In addition, it was attempted to identify the effect of binding ENO1 (α-enolase) to apolipoprotein B100 (apoB100) isolated from a rheumatoid arthritis patient and selected as an ENO1 (α-enolase) binding ligand on the secretion of inflammatory cytokine in the peripheral blood of the rheumatoid arthritis patient through proteomic analysis using immunoprecipitation (IP)/mass spectroscopy (MS). In detail, after ENO1 immunoblotting was performed on mononuclear cells obtained from the rheumatoid arthritis patient and peripheral blood mononuclear cells were treated with apoB100, changes in the secretion of IL-1β, IL-6 and TNF-α were compared. Meanwhile, a group in which cells isolated from healthy individuals are used (HC: healthy control) was determined as the control group.

[0061] As shown in FIG. 1, the observation results showed that the ENO1 level expressed on the surface of the mononuclear cell isolated from the rheumatoid arthritis patient was significantly high, compared to the control group. In addition, the mononuclear cells isolated from the rheumatoid arthritis patients were treated with apoB100, and the results showed that the secretion levels of all of IL-1β, IL-6, and TNF-α as inflammatory cytokines were all markedly increased in the peripheral blood mononuclear cells, compared to the healthy control group, as shown in FIG. 2.

[0062] In addition, ENO1-apoB100 binding was identified by immunoblotting, confocal microscopy, BIACORE system, and ELISA assay, and the results confirmed that, as shown in FIG. 3, ENO1 and apoB100 established relatively strong binding.

[0063] Accordingly, in this example, it was additionally verified whether the binding of ENO1 and apoB100 crucially affected inflammation responses due to rheumatoid arthritis. First, in a case where the ENO1 expression was inhibited by treating the peripheral blood mononuclear cell isolated from the rheumatoid arthritis patient with ENO1 siRNA, it was identified whether apoB100 treatment increased the secretion of inflammatory cytokines, like in the conventional case. In addition, since LDL existing in vivo in a state in which it binds to apoB100 protein may potentially involve the inflammation responses, LDLR knockout mouse and C57BL/6 wild-type mouse control group were treated with the serum of K/B×N mouse (referred to as K/B×N serum, hereinafter) and ApoB100, and resulting changes in vivo were compared. Meanwhile, addition of K/B×N mouse serum functions to induce rheumatoid arthritis through deposition of autoantibodies existing in the K/B×N serum, and is one of conventional techniques for manufacturing rheumatoid arthritis animal models by autoimmune responses.

[0064] As a result, as shown in FIG. 4, when the ENO1 expression of peripheral blood mononuclear cells was inhibited, an increased secretion level of inflammatory cytokines was not observed by the apoB100 treatment. In addition, as shown in FIGS. 5 and 6, the results indicate that changes in the ankle thickness, arthritis score, and inflammatory cytokine secretion of the LDLR knockout mouse were similar to or higher than those of the control group, suggesting that the inflammation response is not relevant to LDL or LDL receptors.

[0065] That is, to sum up, the experimental results indicate that the onset of rheumatoid arthritis is closely related with the ENO1 expression of peripheral blood mononuclear cells, and it is particularly confirmed that the binding of ENO1 and apoB100 in peripheral blood plays a crucial role in the inflammation response of the relevant disease.

Example 2. Deduction of Peptides for Inhibiting Secretion of Inflammatory Cytokine

[0066] In this example, it was attempted to deduce peptides capable of inhibiting the secretion of inflammatory cytokine by inhibiting the binding of ENO1 in peripheral blood to apoB100, on the basis of the experimental results of Example 1. In detail, as shown in FIG. 7, apoB100 motifs binding to ENO1 were identified by peptide micro array, 5 peptides in total were obtained. Detailed information regarding the peptides is given in Table 1 below.

TABLE-US-00001 TABLE 1  Number Sequence information Remarks #1 AICKEQHLFLPF  Represented with  (SEQ ID NO: 1) “22” in FIG. 7 #2 CKEQHLFLPFSY  Represented with  (SEQ ID NO: 2) “23” in FIG. 7 #3 SYKNKYGMVAQV  Represented with  (SEQ ID NO: 3) “28” in FIG. 7 #4 KNKYGMVAQVTQ  Represented with  (SEQ ID NO: 4) “29” in FIG. 7 #5 TKKMGLAFESTK  Represented with  (SEQ ID NO: 5) “44” in FIG. 7

[0067] Thereafter, peripheral blood mononuclear cells obtained from the healthy control and rheumatoid arthritis patients were treated with the peptides #1 to #5 (which are named apoB100 #1, apo100 #2, apoB100 #3, apoB100 #4, and apoB100 #5, respectively), and changes in the secretion of IL-1β, IL-6 and TNF-α were compared. Meanwhile, a non-binding peptide treated group (NBP) was determined as the control group, and the results of this example are indicated as the mean values of the secretion levels for each six of rheumatoid arthritis patients and healthy individuals.

[0068] As a result, as shown in FIG. 8, the secretion levels of IL-1β, IL-6 and TNF-α were significantly increased in the rheumatoid arthritis patients, compared to the healthy control (HC) group, which is the same as described above. In addition, when the mononuclear cells isolated from the rheumatoid arthritis patients were treated with apoB100 #2 or apoB100 #4, the results showed that the secretion levels of all of IL-1β, IL-6, and TNF-α were markedly increased in the peripheral blood mononuclear cells, compared to the healthy control group, while the secretion levels of IL-1β, IL-6, and TNF-α in the peripheral blood mononuclear cells treated with apoB100 #1, apoB100 #3 or apoB100 #5, the secretion levels of IL-1β, IL-6, and TNF-α were decreased.

[0069] Meanwhile, previous research has identified that the increased expression level of ENO1 in the blood of a rheumatoid arthritis patient was mostly derived from CD14(+) peripheral blood mononuclear cells. Therefore, in this example, it was attempted to indirectly identify whether the binding of ENO1 to apoB100 involved the responses by comparing changes in the secretion levels of IL-1β, IL-6, and TNF-α, after treating the CD14(−) or CD14(+) peripheral blood mononuclear cells with the peptides #1 to #5. Meanwhile, a non-binding peptide treated group (NBP) was determined as the control group.

[0070] As a result, as shown in FIG. 9, the CD14(+) peripheral blood mononuclear cells showed a similar propensity to the result shown in FIG. 8, while no changes were observed in the CD14(−) peripheral blood mononuclear cells, further corroborating that the responses were closely related with the binding of ENO1 to apoB100.

[0071] On the basis of such experimental results, apoB100 #1, apoB100 #3, and apoB100 #5 were derived as peptides for inhibiting the secretion of inflammatory cytokine.

Example 3. Verification of Efficacy in Treating Rheumatoid Arthritis

[0072] In this example, it was attempted to verify the efficacy of the peptides (apoB100 #1, apoB100 #3, and apoB100 #5) for inhibiting the secretion of inflammatory cytokine, as in Example 2, in treating rheumatoid arthritis.

[0073] 3-1. Decreased Secretion of Inflammatory Cytokine in Peripheral Blood Derived from Rheumatoid Arthritis Patient

[0074] After peripheral blood cells derived from rheumatoid arthritis patients were treated with apoB100 #1, apoB100 #3, and apoB100 #5, changes in the secretion of IL-1β, IL-6 and TNF-α were identified. A non-binding peptide treated group (NBP) was determined as the control group.

[0075] As a result, as shown in FIG. 10, the secretion levels of IL-1β, IL-6 and TNF-α were significantly decreased with treatment with apoB100 #1, apoB100 #3 or apoB100 #5, and specifically, the treatment efficacy was highest when the treatment was made with apoB100 #3.

[0076] 3-2. Identification of Efficacy in Treating Rheumatoid Arthritis Animal Models

[0077] K/B×N rheumatoid arthritis animal model (K/B×N serum transfer arthritis mouse) was established by inducing moderate rheumatoid arthritis by administering K/B×N serum and apoB to the mouse, and peptides for inhibiting the secretion of inflammatory cytokine were subcutaneously injected to the affected region of the animal model. Afterwards, changes in the ankle thickness and arthritis score resulting after peptide injection were identified. In addition, under the same conditions as described above, changes in the synovial inflammation, bone erosion, cartilage damage and leukocyte infiltration were compared. Meanwhile, groups treated with K/B×N serum and apoB only were determined as control groups, and groups treated with K/B×N serum and apoB, and a non-binding peptide treated group (NBP) were determined as comparison groups.

[0078] As a result, as shown in FIG. 11, the apoB100 #1, apoB100 #3 or apoB100 #5 treatment significantly decreased the ankle thickness and arthritis score increased due to rheumatoid arthritis. In addition, as shown in FIG. 12, similarly to the above results shown in FIG. 11, the synovial inflammation, bone erosion, cartilage damage and leukocyte infiltration were suppressed with treatment with the peptides. That is, to sum up, the experimental results indicate that the apoB100 #1, apoB100 #3, or apoB100 #5 can be used as the effective ingredient of a therapeutic agent for rheumatoid arthritis.

[0079] The foregoing description of the present disclosure has been provided for illustration, and it will be understood by those of ordinary skill in the art that various changes in form and details may be readily made therein without departing from the technical idea and essential features of the present disclosure. Therefore, it should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation.