A D-protein inhibitor targeting interleukin-6 and its application

Abstract

The present invention relates to a D-protein inhibitor for interleukin-6 and its application. The D-protein inhibitor has: (1) an amino acid sequence of SEQ ID No: 1; or (2) an amino acid sequence having at least 80%, for example, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID No: 1, wherein the amino acids constituting the D-protein inhibitor are all in D configuration except glycine. The D-protein inhibitor of the present invention has a very strong affinity for interleukin-6, and cell experimental data show that its inhibitory effect on interleukin-6 is greatly improved compared with the prior art. The half inhibitory concentration (IC 50 ) is at the nanomolar level, which makes it more advantageous to be developed into a therapeutic drug for interleukin-6-mediated related diseases.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, and in particular relates to a D-protein inhibitor targeting interleukin-6 and application thereof. Background Art

[0002] Compared with small molecule drugs, biological macromolecule drugs such as peptides and antibodies have better biological activity and target protein specificity. Especially when the target protein has a large and flat hydrophobic surface, small molecules are difficult to make into drugs and can only be inhibited by protein drugs. Peptide and protein drugs are composed of L-amino acids and have many limitations of their own, such as being easily hydrolyzed by proteases and easily causing immune reactions. There is an urgent need for a protein drug that is resistant to hydrolysis and does not cause immune reactions to fill the gap in L-protein drugs.

[0003] D-protein drugs are proteins that are naturally chirally symmetrical to L-protein drugs. They are not likely to cause immune responses, cannot be degraded by proteases in the body, and are easy to chemically modify. They have attracted attention in recent years. Currently, several D-protein drugs are undergoing clinical trials abroad, and some have already been launched on the market.

[0004] Since interleukin-6 was first discovered in 1973, nearly 50 years of research have shown that interleukin-6 is involved in a variety of biological events, such as immune regulation, tumor growth, hematopoiesis, and emergency response. Disrupting the formation of hexamer by interleukin-6, interleukin-6 receptor (IL-6R) and gp130 is the key to inhibiting the interleukin-6 signaling pathway. However, the binding interface of this hexamer is too complex and extensive, making it extremely challenging to inhibit its formation with small molecules. There are three positions in interleukin-6 that participate in the formation of hexameric complexes, namely site I, site IIa, and site IIIa. Among them, site I is the site where interleukin-6 binds to the interleukin-6 receptor, which is composed of two α-helices A and C. Site IIa is the junction surface composed of two α-helices A and C of interleukin-6, which is responsible for binding to gp130. Site IIIa is the plane at the top of the α-helical bundle of interleukin-6, which binds to gp130.

[0005] Siltuximab (Johnson & Johnson, USA, currently the only FDA-approved interleukin-6 monoclonal antibody) and two monoclonal antibodies Sirukumab and Clazakizumab that are still in clinical trials all bind to site I of interleukin-6. Such monoclonal antibodies can effectively inhibit the binding of interleukin-6 to its receptor and then block its binding to the membrane protein gp130 to inhibit the downstream signaling pathway. However, there are currently three known signaling pathways for interleukin-6, namely classical signaling, trans-signaling, and trans-presentation. Since trans-presentation is a process in which a cell binds to the interleukin-6 receptor and the interleukin-6 is presented to bind to gp130 on the adjacent cell membrane, this pathway is not interfered by the interleukin-6 site I binding antibody. Targeting interleukin-6 site IIIa (Olokizumab), or targeting the interleukin-6 receptor (Tocilizumab, Sarilumab and Vobarilizumab), or targeting gp130 (Olamkicept) can inhibit these three signaling pathways at the same time. As of 2022, no antibodies binding to interleukin-6 site IIa have been reported.

[0006] Since interleukin-6 is involved in many immune responses, each drug that inhibits the interleukin-6 signaling pathway has one or more corresponding therapeutic diseases. For example, the earliest developed interleukin-6 receptor inhibitor Tocilizumab has passed clinical trials for the following diseases: rheumatoid arthritis, juvenile idiopathic arthritis, multicentric Castleman's disease, giant cell arteritis, cytokine release syndrome and Takayasu arteritis. In addition, adult Still's syndrome, Graves' ophthalmopathy, relapsing polychondritis and ankylosing spondylitis are in the second to third phase of clinical trials.

[0007] Among them, Siltuximab, a monoclonal antibody that binds to interleukin-6, has been approved by the FDA for multicentric Castleman disease. Its development for the treatment of multiple myeloma and amyloid light chain amyloidosis is in Phase II clinical trials. Other symptomatic solid tumors, prostate cancer, metastatic renal cell carcinoma and metastatic renal cancer are in Phase I to II clinical trials. Sirukumab, olokizumab and Clazakizumab are in Phase III, Phase III and Phase II clinical trials for rheumatoid arthritis, respectively. Olamkicept, a monoclonal antibody that binds to gp130, is a symptomatic inflammatory bowel disease and is currently in Phase II clinical trials.

[0008] Patent CN116003530B also discloses a D-protein inhibitor against interleukin-6 (IL-6), however, its biological activity is low and its drugability is insufficient. Therefore, there is still a need to develop an inhibitor against interleukin-6 with improved biological activity. Summary of the invention

[0009] The technical purpose of the present invention is to provide a D-protein inhibitor against interleukin-6 (IL-6).

[0010] Another technical purpose of the present invention is to provide a pharmaceutical composition comprising the D-protein inhibitor of the present application.

[0011] Another technical purpose of the present invention is to provide the use of the D-protein inhibitor in drug preparation.

[0012] In one aspect, the present invention provides a D-protein inhibitor against interleukin-6, wherein the D-protein inhibitor has:

[0013] (1) the amino acid sequence of SEQ ID No: 1; or

[0014] (2) an amino acid sequence having at least 80%, for example, at least 90%, at least 95%, at least 99% sequence identity with SEQ ID No: 1,

[0015] The amino acids constituting the D-protein inhibitor are all in D configuration except glycine.

[0016] In a specific embodiment, the amino acid sequence of the D-protein inhibitor is selected from SEQ ID No:1, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8.

[0017] In a specific embodiment, in the above situation (2), any 1-10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the sequence SEQ ID No: 1 are replaced by similar amino acids.

[0018] In a specific embodiment, the secondary structure of the D-protein inhibitor is as follows: it is composed of 4 α-helices, the first α-helical sequence is SEQ ID No: 2, the second α-helical sequence is SEQ ID No: 3, the third α-helical sequence is SEQ ID No: 4, and the fourth α-helical sequence is SEQ ID No: 5.

[0019] In a specific embodiment, the secondary structure sequence of the D-protein inhibitor is LHHHHHHHHHHHHHHLLLHHHHHHHHHHHHHHHHHHHLLLHHHHHHHHHHHHH HHHHHLLHHHHHHHHHHHHLHHHHHHHHL.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned D-protein inhibitor and a pharmaceutically acceptable carrier.

[0021] In another aspect, the present invention provides use of the above-mentioned D-protein inhibitor and the above-mentioned pharmaceutical composition in preparing a drug for treating a disease.

[0022] In a specific embodiment, the disease is an interleukin-6 mediated related disease.

[0023] In a specific embodiment, the disease is selected from rheumatoid arthritis, juvenile idiopathic arthritis, multicentric Castleman's disease, giant cell arteritis, cytokine release syndrome, Takayasu arteritis, adult Still's syndrome, Graves' ophthalmopathy, relapsing polychondritis, and ankylosing spondylitis.

[0024] In another aspect, the present invention provides use of the above-mentioned D-protein inhibitor and the above-mentioned pharmaceutical composition in the preparation of an interleukin-6 in vivo tracer.

[0025] Beneficial Effects

[0026] 1. The D-protein inhibitor of the present invention solves the problem that L-protein is easily hydrolyzed by proteases in vivo.

[0027] 2. The D-protein inhibitor of the present invention has better thermal stability, which is beneficial to the transportation and storage of drugs.

[0028] 3. The D-protein inhibitor of the present invention exhibits a strong inhibitory activity against interleukin-6, and the binding constant with interleukin-6 is less than 1E10 -8 M, which can specifically bind to human interleukin-6 and block the interaction between interleukin-6 and gp130, has the potential to be developed into a therapeutic drug for interleukin-6 immune-related diseases.

[0029] 4. This application conducts multiple rounds of targeted directed evolution based on the data in patent application CN116003530B and the data of the protein structure model. The D-protein inhibitor of this application maintains the original characteristics in terms of thermal stability and proteolytic stability, but cell experiments confirm that its inhibitory ability is nearly 5000 times higher than the former, so it has made great progress in terms of technical effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Chemical synthesis of D-25367-EVO, A is the RP-HPLC elution curve, B is the mass spectrum of D-25367-EVO, showing that its measured molecular weight is 7390.27 Daltons and the calculated molecular weight is 7391.49 Daltons.

[0031] Figure 2 :D-25367-EVO thermal variable temperature CD spectrum.

[0032] Figure 3 : Co-migration curve of D-25367-EVO and interleukin-6 in molecular sieve Superdex 200Increase10 / 300column (A), and SDS-PAGE gel image (B), in the figure, top: interleukin-6 alone; middle: D-25367-EVO alone; bottom: mixture of D-25367-EVO and interleukin-6 in a molar ratio of 1.1:1.

[0033] Figure 4 :The binding constant of D-25367-EVO and interleukin-6 was determined by biomembrane interference experiment.

[0034] Figure 5 HEK-293T cells were used to verify the effect of D-25367-EVO on the expression of downstream signaling pathways mediated by interleukin-6. The height of each bar graph in the figure is the average of three groups of experiments, and the variance of each three groups of data is shown by a thin line protruding from the bar height.

[0035] Figure 6 : SDS-PAGE gel images of proteolysis experiments of L-25367-EVO and D-25367-EVO.

[0036] Figure 7 : Results of competition experiment between D-25367-EVO and gp130.

[0037] Figure 8 : Experimental results of biomembrane interference experiment of L-25367-combo-4 on D-interleukin-6.

[0038] Fig. 9 : Experimental results of biomembrane interference experiment of L-25367-100-11 on D-interleukin-6.

[0039] Fig.10 : Experimental results of biomembrane interference experiment of L-25367-100-13 on D-interleukin-6.

[0040] Fig.11: Experimental results of L-25367-EVO on biomembrane interference experiment of D-interleukin-6. DETAILED DESCRIPTION

[0041] the term

[0042] In the present application, the term "D-protein inhibitor" means that all amino acids constituting the protein, except glycine, are in D configuration.

[0043] In the present application, in the secondary structure sequence, the letter "L" represents a random region, and the letter "H" represents an α-helix.

[0044] In this application, the term "pharmaceutically acceptable carrier" refers to any kind of solid, semisolid or inert fluid excipient, filler, encapsulating or formulation auxiliary material known to those skilled in the art.

[0045] In the present application, the term "therapeutically effective amount" refers to the amount of the D-protein inhibitor of the present invention contained in the pharmaceutical composition that is sufficient to achieve the intended purpose.

[0046] The present invention is described in detail below through specific embodiments so that those skilled in the art can better understand the present invention. However, these embodiments are not intended to limit the scope of the present invention.

[0047] Preparation Example 1

[0048] The preparation process of the D-protein inhibitor against interleukin-6 (hereinafter named D-25367-EVO) of the present application is described in detail below through specific examples.

[0049] D-25367-EVO was prepared by standard Fmoc solid phase peptide synthesis (Fmoc SPPS) and automatically synthesized by Liberty blue microwave peptide synthesizer (CEM Corporation). Rink Amide AM resin (loading of 0.27 mmol / g or 0.55 mmol / g) was purchased from Tianjin Nankai Hecheng Technology Co., Ltd. The amino acid derivatives used in the experiment were purchased from Jiangsu Shenlang Biotechnology Co., Ltd. (Nantong, China). N,N-Dimethylformamide (DMF), triisopropylsilane (TIPS), trifluoroacetic acid (TFA) and thioanisole were purchased from J&K Scientific Ltd. (Beijing). N,N-Diisopropylcarbodiimide (DIC) and ethyl 2-oximecyanoacetate (Oxyma) were purchased from Shanghai Titan Technology Co., Ltd. 1,2-Ethanedithiol (EDT) was purchased from TCI (Shanghai, China) Development Co., Ltd. Piperidine was purchased from Sinopharm Chemical Reagent Co., Ltd., ether was purchased from Modern Oriental (Beijing) Technology Development Co., Ltd., and acetonitrile was purchased from Mallinckrodt Baker, Inc.

[0050] First, the Rink Amide AM resin was deprotected from the Fmoc (9-fluorenylmethoxycarbonyl) protecting group at 90°C for 1 minute using a DMF solution containing 10% piperidine and 0.1 M Oxyma. Then, the resin was washed 3 times with DMF. The resin (0.25 mmol), 4 equivalents of Fmoc-protected amino acids (0.2 mM, 5 ml, dissolved in DMF), 4 equivalents of Oxyma (1 mM, 1 ml, dissolved in DMF), and 4 equivalents of DIC (0.5 mM, 2 ml, dissolved in DMF) were mixed and coupled at 90°C for 2 minutes under microwave heating. At the end of the procedure, the peptide was cleaved from the resin with a cleavage solution (TFA / TIPS / thioanisole / water / EDT, volume ratio of 82.5:5:5:5:2.5, vol / vol / vol / vol) for 3 hours. The solution was then concentrated under nitrogen stirring, precipitated with cold ether, and then centrifuged and the supernatant was poured out, and repeated 3 times. The resulting precipitate was a crude peptide, which was subsequently further purified by HPLC (high performance liquid chromatography) to obtain D-25367-EVO (amino acid sequence: GEEEVIEYLTREFKDDPELVRLLREAIEGLLKAGEDPEIVEMLIESLIHITGN PRVAVKLAKEYA (SEQ ID No: 1)). Reverse phase HPLC was performed on a Shimadzu Prominence HPLC. The mobile phase of pump A was acetonitrile (0.1% TFA) and the mobile phase of pump B was deionized water (0.1% TFA). The crude polypeptide was dissolved in water containing 50% acetonitrile and 0.1% TFA, filtered with a 0.22 μm filter, and the purified solution was lyophilized to obtain a pure polypeptide powder, which was named D-25367-EVO. The HPLC and mass spectrometry detection results of D-25367-EVO are shown in Figure 1 shown.

[0051] Test Example 1: Structural Identification of D-25367-EVO

[0052] The secondary structure of D-25367-EVO was measured by circular dichroism spectrometer (Chirascan V100 / Applied Photophysics). The CD spectrum tested the range of 260 to 180 nm, and three CD curves were tested at 25°C, when the temperature was increased to 95°C, and when it was reduced back to 25°C, and the temperature increase was 2°C / min. The test concentration of D-25367-EVO was 0.2 mg / ml dissolved in PBS buffer (pH 7.4).

[0053] The results are as follows Figure 2As shown. The results showed that at 25℃, 95℃ and returning to 25℃, positive peaks were displayed at 208 and 222nm, which was a typical D-type chiral α-helix secondary structure. After further analysis, D-25367-EVO was composed of 4 α-helices. The first α-helix sequence was SEQ ID No: 2 (EEEVIEYLTREF), the second α-helix sequence was SEQ ID No: 3 (PELVRLLREAIEGLLKA), the third α-helix sequence was SEQ ID No: 4 (PEIVEMLIESLIHIT), and the fourth α-helix sequence was SEQ ID No: 5 (PRVAVKLAKEY). Therefore, its secondary structure sequence was LHHHHHHHHHHHHHLLLHHHHHHHHHHHHHHHHLLHHHHHHHHHHHHHH HHHHHHLLHHHHHHHHHHHHHHLHHHHHHHHHH.

[0054] This experimental result also proves that D-25367-EVO has good thermal stability, which is consistent with the D-protein inhibitor disclosed in patent application CN116003530B.

[0055] Test Example 2: Binding analysis of D-25367-EVO and interleukin-6

[0056] The binding analysis of D-25367-EVO and interleukin used the co-migration experiment of analytical molecular sieve chromatography column and the biomembrane interferometry technology respectively.

[0057] Analytical molecular sieve chromatography column method

[0058] D-25367-EVO and interleukin-6, either alone or in a 1.1:1 molar ratio, were passed through a column (Superdex 200 Increase 10 / 300 column) with equal amounts of protein, and the eluted samples were run on SDS-PAGE to verify that D-25367-EVO co-migrated with interleukin-6 as the target protein.

[0059] The results are as follows Figure 3 As shown. Figure 3 It can be seen that when interleukin-6 and D-25367-EVO are mixed, the peak of the complex is earlier than that of each monomer, which means that the volume of the complex is larger. And from the gel image, it can be seen that the peak corresponding to the complex is a complex of the two.

[0060] Biomembrane interferometry (Octet RED96e / ForteBio)

[0061] Biofilm interferometry uses the Octet instrument and a SA (streptavidin) probe that specifically binds biotin to bind biotinylated L- or D-interleukin-6. For D-interleukin-6, a biotin molecule is attached to the amino terminus of the protein during synthesis. The biotinylation of L-interleukin-6 is based on an Avi tag at the amino terminus, which is linked to L-interleukin-6 by a biotin ligase.

[0062] The experimental process is:

[0063] (1) Baseline 1: The SA probe was immersed in phosphate-buffered saline buffer containing 20,000 ppm Tween-20, pH 7.4 (hereinafter referred to as PBST) for 60 seconds.

[0064] (2) Immobilization: dilute biotinylated interleukin-6 in PBST to a final concentration of 10 μg / ml, and immerse the SA probe in this solution for 120 seconds;

[0065] (3) Blocking: (Blocking solution is 10 μg / ml biotin in PBST solution), immerse the SA probe in the blocking solution for 60 seconds.

[0066] (4) Baseline 2: Immerse in PBST for 60 seconds,

[0067] (5) Binding: Immerse in L or D-25367-EVO solution diluted with PBST for 600 seconds at different concentrations of 10, 5, 2.5, 1.25, and 0.63 nM, respectively.

[0068] (6) Dissociation: Soak in PBST for 600 seconds.

[0069] (7) The binding constant between interleukin-6 and D-25367-EVO was determined to be 3.03 ± 0.04 nM (see Figure 4 ).

[0070] from Figure 4 It can be seen that the D-protein inhibitor of the present application exhibits a binding constant of 3.03±0.04 nM with L-interleukin-6, which is nearly ten times higher than the binding constant (28.3 nM) disclosed in patent CN116003530B.

[0071] Test Example 3: D-25367-EVO Cell Activity Experiment

[0072] Interleukin-6-mediated JAK-STAT signaling has been shown to be quantitatively regulated using human placental secretory alkaline phosphatase expression.

[0073] In this experiment, 1×10 4 Secretory alkaline phosphatase was expressed in HEK-293T cells to sense extracellular interleukin-6. Transfected cells were incubated with different concentrations of D-25367-EVO and human interleukin-6 in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% carbon dioxide for 48 hours. The cell culture supernatant was then heat-inactivated (65°C, 30 minutes), and 80 μl of the supernatant was mixed with 120 μl of substrate solution [100 μl of twice-concentrated secretory alkaline phosphatase detection buffer containing 20 mmol homoarginine, 1 mmol magnesium chloride, 21% (vol / vol) diethanolamine pH 9.8 and 20 μl of substrate solution containing 120 mmol p-nitrophenyl phosphate]. Interleukin-6 signaling was assessed by measuring the production of secretory alkaline phosphatase in the cell culture medium. The absorbance was recorded at a wavelength of 405 nm (37° C.) using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments, Inc.) and the unit was enzyme activity unit U / L.

[0074] The results are as follows Figure 5 As described above, it can be seen from the figure that D-25367-EVO can significantly inhibit the interleukin-6 mediated signaling pathway.

[0075] from Figure 5 It can be seen that the half-maximal inhibitory concentration of the D-protein inhibitor of the present application is about 1 nM, which is nearly 5000 times higher than that in CN116003530B. In this experiment, it is close to the efficacy of siltuximab, the only FDA-approved monoclonal antibody drug for interleukin-6 on the market. Therefore, it has more practical value and development value than the D-protein inhibitors disclosed in the prior art.

[0076] Test Example 4: Hydrolysis experiment of D-25367-EVO protease

[0077] In the proteolysis experiment, the protein concentration was 0.2 mg / ml, and it was incubated at 37°C in trypsin and pepsin solutions, respectively, while the chiral mirror peptide L-25367-EVO (same amino acid sequence but composed of L-amino acids) of D-25367-EVO was used as a control. In the experiment, the final concentrations of trypsin and pepsin were both 2.2 mg / ml, and samples were taken at 6 and 20 hours of incubation, and verified by SDS-PAGE running.

[0078] Please see the results Figure 6 .from Figure 6 It can be seen that the D-25367-EVO of the present application is not easily hydrolyzed by proteases and shows good in vivo stability. Therefore, the D-protein inhibitor of the present application is consistent with that in patent CN116003530B in terms of protease stability.

[0079] Test Example 5: Competition experiment between D-25367-EVO and gp130

[0080] The principle of this experiment is based on the biomembrane interference experiment. Human gp130 with AVI label is solidified on the SA probe, and then gp130 is detected whether it is combined with a solution containing different interleukin-6 components (refer to the specific process described in Test Example 2 for the experimental steps). During the experiment, the extracellular region of interleukin-6 and interleukin-6 receptor (IL-6R) is first formed into a complex to simulate the complex formed by soluble interleukin-6 receptor (sIL-6R) and interleukin-6 in the blood circulation. Then, different concentrations of D-25367-EVO inhibitor are added to inhibit the possibility of the extracellular region complex of interleukin-6 and interleukin-6 receptor (IL-6R) 10nM and gp130 forming a six-original complex. The control group in the experiment is a negative control without adding an inhibitor, and a positive control with 10nM of siltuximab. The experimental steps are consistent with the description of Test Example 2.

[0081] Results Figure 7 .from Figure 7 It can be seen that D-25367-EVO of the present application can simulate the complex formed by capturing soluble interleukin-6 receptor (sIL-6R) and interleukin-6 in the blood, and its efficacy is close to that of siltuximab.

[0082] Test Example 6: Site protection analysis

[0083] In protein engineering, Schumacher et al., 1996, Science 271: 1854-1857, Chang et al., 2015, Angew Chem Int Ed Engl 54: 11760-11764, Zhou et al., 2020, Angew Chem Int Ed Engl 59: 15114-15118, Marinec et al., 2021, ACS Chem Biol 16: 548-556, etc. A large number of literatures have confirmed that the screening or optimization of D-type polypeptides or proteins requires first screening the D-type target protein through the L-type drug library, and then obtaining functional D-type drugs through chiral conversion. Therefore, the following biomembrane interference experiment results of L-protein inhibitors are used to illustrate the possibility and feasibility of the mutation of D-protein inhibitors involved in this application.

[0084] During multiple rounds of targeted optimization of the existing patent CN116003530B, the inventors also found several outstanding optimized versions (L-25367-combo-4: SEQ ID NO: 6, L-25367-100-11: SEQ ID NO: 7, L-25367-100-13: SEQ ID NO: 8), all of which have similar physical and chemical properties to L-25367-EVO, and were subjected to biomembrane interference experiments (the experimental steps refer to the specific process described in Test Example 2).

[0085] Figure 8-Figure 10 The experimental results of the biomembrane interference experiment of natural chiral (L-type) inhibitors of different modified amino acid sequences of L-25367-EVO on D-interleukin-6 are shown respectively. Fig.11 The graph shows the experimental results of the biomembrane interference experiment of L-25367-EVO (as a control group) on D-interleukin-6. Figure 8-11 The experimental results show that although there are some sequence differences, it does not affect its binding to the target protein.

Claims

1. A D-protein inhibitor against interleukin-6, the amino acid sequence of the D-protein inhibitor is: SEQ ID No: 1, in, The amino acids constituting the D-protein inhibitor are all in D configuration except glycine.

2. The D-protein inhibitor according to claim 1, wherein The secondary structure of the D-protein inhibitor is as follows: it is composed of 4 α-helices, the first α-helical sequence is SEQ ID No: 2, the second α-helical sequence is SEQ ID No: 3, the third α-helical sequence is SEQ ID No: 4, and the fourth α-helical sequence is SEQ ID No:

5.

3. A pharmaceutical composition comprising a therapeutically effective amount of the D-protein inhibitor according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. Use of the D-protein inhibitor according to claim 1 or 2 or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating a disease, wherein the disease is an interleukin-6-mediated related disease, and the disease is selected from rheumatoid arthritis, juvenile idiopathic arthritis, multicentric Castleman's disease, giant cell arteritis, cytokine release syndrome, Takayasu arteritis, adult Still's syndrome, Graves' ophthalmopathy, relapsing polychondritis and ankylosing spondylitis.

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