A cyclic peptide and its uses

Abstract

The present invention discloses a cyclic peptide and its uses. The present invention provides a novel cyclic peptide that has not been reported before. This novel cyclic peptide has a strong affinity for the LRR domain in the NLRP3 protein, and thus can inhibit the activation of the NLRP3 inflammasome. Those skilled in the art know that the NLRP3 inflammasome can be activated by various types of pathogens and danger signals and plays an important role in a variety of diseases, such as inflammatory diseases and tumors. The present invention has demonstrated through experiments that this novel cyclic peptide has excellent anti-tumor activity and anti-inflammatory activity. Thus, it can be seen that the novel cyclic peptide provided by the present invention has the prospect of being developed into a drug for treating diseases that can be treated or alleviated by inhibiting the activation of the NLRP3 inflammasome, such as anti-tumor drugs and anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides, relates to novel polypeptides and their applications, and specifically relates to a cyclic peptide and its uses. Background Art

[0002] As "therapeutic drug molecules", cyclic peptides are the absolute mainstay in the field of cyclic peptide drugs. Compared with linear peptides, cyclic peptides have better structural rigidity and protein resistance, and can obtain a longer half-life and action time. Linear peptides are relatively unstable to exopeptidases, while cyclic peptides formed by cyclization of terminal amino acids can greatly slow down the degradation by exopeptidases. And when a molecule binds to a target, it must adopt a certain conformation. Due to fewer conformational restrictions of linear peptides, the possibility of binding to the target in a suitable conformation per unit time is small, and the entropy penalty is greater. However, the possibility that a cyclic peptide molecule may be in or close to this binding peptide conformation is higher, and it has stronger target affinity.

[0003] The NLR family protein 3 (NLRP3) inflammasome is a large multi-protein complex composed of NLRP3, adaptor protein ASC, and effector protein Caspase-1. The NLRP3 inflammasome can be activated by various types of pathogens and danger signals. Therefore, it plays an important role in various diseases. The normal activation of the NLRP3 inflammasome is beneficial for the host to resist the infection of pathogenic microorganisms and maintain the stability of the internal environment of the body. When the NLRP3 inflammasome is over-activated, various inflammatory diseases will occur (Li Chenguang et al., Research progress of NLRP3 inflammasome inhibitors, Chinese Pharmacological Bulletin, 2024). The NLRP3 inflammasome also plays an important role in the occurrence and development of tumors. Inhibiting the activation of the NLRP3 inflammasome helps to inhibit tumors (Chen Xiuhui et al., Role of NLRP3 inflammasome in tumors and research progress of its inhibitors, Central South Pharmacy, 2023).

[0004] Activation of the NLRP3 inflammasome requires first and second signals. The first signal includes different TLR (Toll-like receptor) ligands such as LPS or other danger signals, etc., which mainly upregulate the protein expression of NLRP3 and pro-IL-1β by activating the NF-κB pathway. The second signal includes some crystalline substances (such as uric acid crystals, silica, asbestos, and aluminum), extracellular ATP, perforating toxins, mitochondrial DNA, and pathogen-related components, etc. Reported regulators of NLRP3 inflammasome activation include double-stranded RNA-dependent protein kinase (PKR), guanylate-binding protein 5 (GBP5), and NIMA-related kinase 7 (Nek7). Different from PKR and GBP5, three independent studies have all suggested that Nek7 plays an important role in the activation process of the NLRP3 inflammasome. Nek7 belongs to the NIMA (never-in-mitosis A) related kinase family and is mainly involved in regulating the mitosis process and DNA damage response. Related studies have shown that the activation of the NLRP3 inflammasome induced by all NLRP3 stimulants (including ATP, nigericin, MSU crystals, and alum) requires Nek7, while the activation of inflammasomes such as NLRC4 and AIM2 does not require Nek7. The catalytic region of Nek7 can interact with the LRRs region of NLRP to form a NLRP3-Nek7 macromolecular complex, and agonists of NLRP3 can enhance this interaction. The activation of NLRP3 mediated by Nek7 is independent of its kinase activity. Nek7 can regulate the oligomerization of NLRP3, the formation of ASC speckles, and the activation of Caspase-1 downstream of K + efflux. Therefore, exploring the response mechanism of Nek7 to NLRP3 agonists will provide new ideas for the study of the molecular mechanism of NLRP3 inflammasome activation.

[0005] Targeted inhibition of the NLRP3 inflammasome provides new ideas for the treatment of related diseases. A variety of synthetic small molecules and natural products have emerged as NLRP3 inflammasome inhibitors in the research. They show potential in terms of specificity and efficacy, but most NLRP3 inhibitors entering clinical research are MCC950 analogues, with problems such as a single backbone and serious structural homogenization, which limits the diversity of drugs and the therapeutic effect.

[0006] Based on the discovery of novel cyclic peptides and their activities, the present invention is specifically proposed. Summary of the Invention

[0007] The first object of the present invention is to provide a cyclic peptide, and the second object is to provide the use of the cyclic peptide.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] A cyclic peptide or a pharmaceutically acceptable salt or solvate thereof, wherein the cyclic peptide contains the following sequence:

[0010] LSRMIKHFKKQ.

[0011] Preferably, the cyclic peptide contains the following sequence:

[0012] CLSRMIKHFKKQC.

[0013] More preferably, the sequence of the cyclic peptide is as shown in SEQ ID NO.1 or SEQ ID NO.5:

[0014] SEQ ID NO.1: Ac-CLSRMIKHFKKQC-NH 2 , and the cyclization site is c[1C-13C];

[0015] SEQ ID NO.5: CGDCLSRMIKHFKKQCKRLIC, and the cyclization sites are c[1C-16C] and c[4C-21C].

[0016] Preferably, the cyclic peptide contains the following sequence:

[0017] GDLSRMIKHFKKQKRLI.

[0018] More preferably, the sequence of the cyclic peptide is as shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4:

[0019] SEQ ID NO.2: Ac-GDLSRMIKHFKKQKRLIQ, and the cyclization site is c[8K-18Q];

[0020] SEQ ID NO.3: Ac-CGDLSRMIKHFKKQKRLIC-NH 2 , and the cyclization site is c[1C-19C];

[0021] SEQ ID NO.4: RRRRRRRRRCGDLSRMIKHFKKQKRLIC, and the cyclization site is c[10C-28C].

[0022] Use of the cyclic peptide or a pharmaceutically acceptable salt or solvate thereof for preparing an inhibitor of NLRP3 inflammasome activation, or for preparing a drug for treating or alleviating a disease that can be treated or alleviated by inhibiting NLRP3 inflammasome activation.

[0023] Preferably, the disease that can be treated or alleviated by inhibiting NLRP3 inflammasome activation is a tumor.

[0024] More preferably, the tumor is leukemia.

[0025] More preferably, the disease that can be treated or alleviated by inhibiting NLRP3 inflammasome activation is an inflammatory disease.

[0026] Beneficial effects:

[0027] The present invention provides a novel cyclic peptide that has not been reported before. This novel cyclic peptide has a strong affinity for the LRR domain in the NLRP3 protein, and thus can inhibit the activation of NLRP3 inflammasome. Those skilled in the art know that NLRP3 inflammasome can be activated by various types of pathogens and danger signals and plays an important role in various diseases, such as inflammatory diseases and tumors. The present invention proves through experiments that this novel cyclic peptide has excellent anti-tumor activity and anti-inflammatory activity. Thus, it can be seen that the novel cyclic peptide provided by the present invention has the prospect of being developed into a drug for treating diseases that can be treated or alleviated by inhibiting NLRP3 inflammasome activation, such as anti-tumor drugs and anti-inflammatory drugs. Description of the drawings

[0028] Figure 1 It is the CD spectral structure diagram of cyclic peptides Cy-1, Cy-2, Cy-3, Cy-4, Cy-5 of the present invention dissolved in 1×PBS (containing 50% TFE) in the range of 190 - 260 nm (concentration is 0.1 mg / ml);

[0029] Figure 2 It is the concentration - inhibition rate curve diagram of cyclic peptides Cy-1, Cy-2, Cy-3, Cy-4, Cy-5 of the present invention on the LRR domain of NLRP3 protein in buffer (50 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 8.8). Detailed implementation manners

[0030] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention thereto.

[0031] Example 1: Preparation and characterization of cyclic peptide

[0032] The target cyclic peptide was synthesized by a conventional cyclic peptide synthesis method in the art. The cyclic peptide sequence is as follows, where "Ac" represents N-terminal acetylation of the cyclic peptide and "NH 2 " represents C-terminal amidation of the cyclic peptide:

[0033] SEQ ID NO. Name Sequence Cyclization site SEQ ID NO.1 Cy-1 <![CDATA[Ac-CLSRMIKHFKKQC-NH 2 > c[1C-13C] SEQ ID NO.2 Cy-2 Ac-GDLSRMIKHFKKQKRLIQ c[8K-18Q] SEQ ID NO.3 Cy-3 <![CDATA[Ac-CGDLSRMIKHFKKQKRLIC-NH 2 > c[1C-19C] SEQ ID NO.4 Cy-4 RRRRRRRRRCGDLSRMIKHFKKQKRLIC c[10C-28C] SEQ ID NO.5 Cy-5 CGDCLSRMIKHFKKQCKRLIC c[1C-16C]&c[4C-21C]

[0034] The specific synthesis method is as follows:

[0035] The cyclic peptide was synthesized manually by the solid-phase peptide synthesis (SPPS) method; the synthesis scale was 0.1 mmol, and 40-RAM amphiphilic Rink amide resin was loaded at 0.4 mmol / g, with a 5-fold excess used for the protected amino acids; all protected amino acids and the coupling agent HATU were pre-dissolved in DMF to prepare a stock solution with a concentration of 0.5 M; under vortex stirring, the resin was first swollen in 6 ml of DMF for 30 minutes. After removing the DMF by filtration, the Fmoc group was deprotected by adding a 20% piperidine / DMF solution (6 ml) and vortexing for 1 minute; after performing the above steps twice, the resin was washed 3 times with DMF, and then the required amino acid (1 ml, 0.5 mmol), N,N-diisopropylethylamine (DIEA) (0.164 ml, 1 mmol), and the HATU coupling agent (1 ml, 0.5 mmol) were added in sequence, and the mixture was stirred for 5 minutes under vortex stirring. Between two couplings, this operation was performed twice by washing with DMF; the deprotection of Fmoc and the coupling of amino acids were repeated until the expected linear sequence was obtained; after the synthesis of the linear peptide, cyclization was carried out by the iodine oxidation method. I 2 (25 mg, 0.1 mmol) was added, and then 6 mL of DMF was added. The reaction was carried out for 2 hours under vortex stirring. After completion, the resin was washed 3 times with DMF. After cyclization, the final deprotection of the Fmoc group was carried out, and an acetylation reaction was carried out for 10 minutes with a DMF solution of acetic anhydride containing DIEA (1 / 1 / 8). The final cleavage of the peptide was carried out in the presence of a solution (10 ml) of trifluoroacetic acid, triisopropylsilane, and water (95 / 2.5 / 2.5); after filtration, the solution was concentrated and dissolved in ether; the precipitate was centrifuged and the mother liquor was decanted; the above steps were carried out twice, and the precipitate was dissolved in water and freeze-dried to obtain the fully deprotected peptide; after freeze-drying, the cyclic peptide was purified by preparative reverse-phase HPLC with acetonitrile / water elution in the presence of 0.1% TFA; the obtained cyclic peptide preferably had a purity greater than 95%.

[0036] The cross-linked compound was purified on a reverse-phase C18 column by high-performance liquid chromatography to obtain a pure compound; the chemical composition of the pure product was confirmed by LC / MS mass spectrometry and amino acid analysis. The mass spectrometry data and purity of each target cyclic peptide are as follows:

[0037] SEQ ID NO. Name Calculated m / z (M+2H) Observed m / z (M+2H) Purity (%) SEQ ID NO.1 Cy-1 831.5 831.3 95.16 SEQ ID NO.2 Cy-2 1126.4 1126.2 99.35 SEQ ID NO.3 Cy-3 1172.9 1172.8 98.72 SEQ ID NO.4 Cy-4 1855.3 1855.1 99.61 SEQ ID NO.5 Cy-5 1254.6 1254.2 98.18

[0038] Example 2: Evaluate whether the cyclic peptide maintains an α-helical structure in an amphiphilic cell membrane-mimicking environment

[0039] To further understand the effect of the α-helical structure on the biological activity of the cyclic peptide, and taking advantage of the characteristic of trifluoroethanol in promoting the formation of the α-helical secondary structure, circular dichroism (CD) spectroscopy was used to test and analyze the cyclic peptide; in the far-ultraviolet region (185 - 245 nm) of the CD spectrum, typical secondary structures of the cyclic peptide have their respective characteristic absorption peaks; among them, the α-helix shows double negative peaks at 208 nm and 222 nm, and a positive peak at 195 nm; the β-sheet shows a weak negative peak at 215 nm; the random coil has a negative peak at 200 nm and a small and broad positive peak at 220 nm.

[0040] The secondary structure of the cyclic peptide was determined by circular dichroism spectroscopy. The CD spectrum was measured using a J-810 spectrometer at 25 °C in a quartz cell with an optical path length of 1.0 mm, and was recorded by scanning in the range of 190 to 260 nm; the speed was 50 nm / min, the bandwidth was 1 nm, and the response was 1 s; the cyclic peptide was dissolved in a 50% trifluoroethanol solution (cell membrane-mimicking environment) prepared with PBS to prepare a cyclic peptide solution with a concentration of 0.1 mg / mL; each spectrum is the result of averaging and accumulating 3 times; then the obtained CD spectrum was converted to the mean residue ellipticity using the following formula:

[0041] [θ] 222 =([θ] obs ×MW) / (L×ρ);

[0042] where: [θ] 222 is the mean residue ellipticity at 222 nm (deg*cm2*dmol-1), [θ]obs is the observed ellipticity corrected for the buffer at 222 nm wavelength (mdeg), MW is the average residue molecular mass of the cyclic peptide, ρ is the mass concentration of the cyclic peptide (mg / ml), and L is the cell path length of 1 mm.

[0043] The α-helix content was calculated according to the following formula:

[0044] α-helix content (%) = -([θ] 222 × 100) / 40000.

[0045] The obtained CD spectrum is shown as the mean residue ellipticity [θ] (deg*cm 2 *dmol -1 ) versus the wavelength λ (nm).

[0046] As Figure 1As shown, in the amphiphilic cell membrane-mimicking environment, both cyclic peptides Cy-1 and Cy-4 have good α-helical structures, showing a positive peak at 195 nm and double negative peaks at 208 nm and 222 nm; the α-helix contents of Cy-1 - Cy-5 are 16.69%, 14.99%, 39.42%, 8.75%, and 18.58% respectively.

[0047] Example 3: Evaluation of the binding inhibitory activity of cyclic peptides against the LRR domain in NLRP3 protein

[0048] I. Experimental materials

[0049] The recombinant protein of the LRR domain was purchased from Zhongding Biotechnology Co., Ltd., Tris-HCl, NaCl, and EDTA were all purchased from Sangon Biotech, and the fluorescent probe FITC-Acp-LSRMIKHFKKQ-NH 2 was synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.; a multifunctional microplate reader (Thermo), 384-well low-edge black flat-bottomed polystyrene NBS microplates (Corning), etc.

[0050] II. Experimental methods

[0051] 1. Experimental principle: To detect the binding inhibitory activity of Cy-1, Cy-2, Cy-3, Cy-4, and Cy-5 against the LRR domain in NLRP3 protein, the inhibition rate was determined based on the fluorescence polarization technique. With the fluorescence-labeled detection technology, the fluorescent substance was labeled on a specific substance to convert the originally weak reaction signal into a stronger fluorescent signal, playing a role in signal enhancement. At the same time, a polarizer and an analyzer were added to the detection system. When a beam of light emitted from the light source becomes vertically polarized light after passing through the vertical polarizer, the sample is excited by the vertically polarized light to generate polarized fluorescence. This fluorescence can measure the intensity of the horizontally or vertically polarized fluorescence related to the sample concentration after passing through the analyzer. A working curve was made through the fluorescence intensity and the sample concentration for quantitative analysis. When the fluorescent probe binds to the protein, the molecular weight becomes larger and the rotation speed becomes slower, and the mP value will increase. Therefore, when the compound is an active substance, the fluorescent probe competes with the compound for binding to the protein, and the binding of the probe to the protein decreases, and the mP value decreases; when the compound is an inactive substance, the fluorescent probe is more likely to bind to the protein, and the mP value increases. According to the change of the mP value in the system, the inhibition rate of the cyclic peptide was calculated to obtain the binding inhibitory activity of the cyclic peptide against the LRR domain.

[0052] 2. Detection method: 10 mmol·L -1Cy-1, Cy-2, Cy-3, Cy-4 and Cy-5 were serially diluted 2-fold (starting concentration 100 μmol·L -1 , with a total of 12 concentration gradients) in a fluorescence polarization reaction solution (50 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 8.8), and added to a 384-well plate, 20 μL per well. Three replicate wells were set for each group. Then, 375 nmol·L - 1 LRR was added, 20 μL per well, and incubated at room temperature for 30 min. Finally, 10 nmol·L -1 fluorescent probe was added to the 384-well plate (both were final concentrations), 20 μL per well, and incubated at room temperature for another 30 min. The mP value was detected using a multifunctional microplate reader. The inhibition rate of the cyclic peptide on the LRR domain was calculated as follows (using the well containing 2% DMSO as the negative control well and the well containing only 10 nM fluorescent probe as the positive control well):

[0053] Inhibition rate (%) = (1 - (mP value of drug well - mP value of positive well) / (mP value of negative well - mP value of positive well)) * 100%.

[0054] III. Experimental Results

[0055] The binding inhibition activities of Cy-1, Cy-2, Cy-3, Cy-4 and Cy-5 on the LRR domain in NLRP3 protein are as Figure 2 shown, and their IC 50 values are 0.8117 μM, 4.274 μM, 0.4926 μM, 0.3418 μM and 0.5426 μM respectively, indicating that the cyclic peptides of the present invention all have extremely strong binding inhibition activities on the LRR domain in NLRP3 protein.

[0056] Example 4: Evaluation of the anti-tumor activity of cyclic peptides by CCK8 method

[0057] I. Experimental Materials

[0058] 1. Instruments and Reagents

[0059] Instruments: laminar flow hood, centrifuge (Thermo), cell incubator, E1x800 microplate reader (BioTek, Vermont, USA), etc.; 1640 medium (Nanjing KeyGen Biotech Co., Ltd.), fetal bovine serum from Brazil, CCK8 reagent was purchased from TargetMol.

[0060] 2. Experimental Cells

[0061] THP-1 cells (human monocytic leukemia cells) were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0062] II. Experimental Methods

[0063] 1. Culture of THP-1 cells

[0064] (1) Complete medium: 90% RPM-1640 medium and 10% fetal bovine serum.

[0065] (2) Incubator environment: 37°C, 95% air and 5% carbon dioxide.

[0066] (3) Cell seeding: Take THP-1 cells in good condition, transfer them to a centrifuge tube and centrifuge (800 rpm, 5 min). Discard the supernatant, add medium, resuspend by pipetting, count the cells, supplement the medium to make the cell concentration about 100,000 cells / ml, and pipette gently to mix evenly. Take a 96-well plate, add 100 μl of cell suspension to each well, so that the number of cells in each well is about 10,000 cells / well, ensuring that the cell concentration in each well is uniform. After adding, gently shake the 96-well plate to disperse the cells evenly, and place it in the incubator for culture.

[0067] 2. CCK8 assay

[0068] The experiment was divided into a blank control (DMSO) and a drug administration group (cyclic peptide Cy-4). The anti-tumor activity of cyclic peptide Cy-4, which has the strongest inhibitory activity on the binding of the LRR domain in NLRP3 protein, was determined by using the CCK8 method. After treating THP-1 cells with a density gradient of the test cyclic peptide or DMSO for 24 hours, 10 μL of CCK8 solution was added to each well of a 96-well plate and incubated for 3 hours. The absorbance value (OD value) was recorded at 450 nm using an E1x800 microplate reader. The calculation of the inhibition rate of the cyclic peptide on cell growth is as follows (using the cell wells without drugs as the blank control): Inhibition rate (%) = (1 - OD value of drug wells / OD value of blank wells) × 100%. The IC 50 value was obtained according to the inhibition rates at different concentrations.

[0069] III. Experimental results

[0070] The results showed that compared with the control group, cyclic peptide Cy-4 had strong inhibitory activity on human monocytic leukemia cells THP-1, and the IC 50 = 8.023 μM.

[0071] Example 5: Evaluation of the anti-inflammatory activity of cyclic peptides by ELISA method

[0072] I. Experimental materials

[0073] 1. Instruments and reagents

[0074] Instruments: laminar flow hood, centrifuge (Thermo), cell incubator, full wavelength microplate reader (Thermo), etc.: RPMI-1640 medium (Nanjing KeyGen Biotech Co., Ltd.), fetal bovine serum from Brazil, Human IL-1beta Uncoated ELISA Kit purchased from Thermo Fisher Scientific, Phorbol 12-myristate 13-acetate (PMA, Abcam), LPS (Sigma-Aldrich), ATP (Sigma-Aldrich).

[0075] 2. Experimental cells

[0076] THP-1 cells (human monocytic leukemia cells) are from the Cell Bank of the Chinese Academy of Sciences.

[0077] II. Experimental methods

[0078] 1. Culture of THP-1 cells

[0079] (1) Complete medium: 90% RPMI-1640 medium, 10% fetal bovine serum.

[0080] (2) Incubator environment: 37°C, 95% air, 5% carbon dioxide.

[0081] (3) Cell plating and induced differentiation: Take THP-1 cells in good condition, transfer them to a centrifuge tube and centrifuge (800 rpm, 5 min), discard the supernatant, add medium, resuspend by pipetting, count, supplement the medium to make the cell concentration about 5×10⁵ cells / ml, add PMA to a final concentration of 100 ng / ml, and pipette evenly. Take a 96-well plate, add 100 μl of cell suspension to each well, so that the number of cells per well is about 50,000 cells / well, ensuring that the cell concentration in each well is uniform. After adding, gently shake the 96-well plate to disperse the cells evenly, and place it in the incubator for 12 h to induce their differentiation into macrophages, which is a commonly used anti-inflammatory cell model in this field.

[0082] (4) Modeling and drug administration: Gently aspirate the supernatant with a pipette, add the pre-prepared Cy-4 solution along the wall. The blank group and the model group add an equal amount of RPMI-1640 medium, and culture in the incubator for 24 h. Prepare a 10 μg / ml LPS working solution with RPMI-1640 medium, add an appropriate amount to each well to make the final concentration of LPS 500 ng / ml, continue to culture for 3 h. Prepare a 100 mg / ml ATP working solution with PBS, add an appropriate amount to each well to make the final concentration of ATP 5 mM, and continue to culture for 1 h.

[0083] 2. Enzyme-linked immunosorbent assay (ELISA)

[0084] (1) Grouping: DMSO blank group, LPS+ATP model group as negative control, and LPS+ATP+Cy-4 administration group as experimental group.

[0085] (2) Method: Use Human IL-1beta Uncoated ELISA Kit to determine the anti-inflammatory activity of the cyclic peptide Cy-4, which has the strongest inhibitory activity on the binding of the LRR domain in NLRP3 protein. After the freshly collected THP-1 cell samples were allowed to stand at room temperature for 20 min, they were centrifuged at 4 °C and 3000 g for 10 min, and the supernatant could be used for detection and analysis. The THP-1 cell samples and IL-1β standard were added to the 96-well plates coated with antibodies respectively. After adding biotinylated antibodies, they were incubated on a shaker at 37 °C for 1 h, then the plates were washed 5 times, enzyme-conjugated working solution was added, and they were incubated in the dark at 37 °C for 30 min, then the plates were washed 5 times. Chromogenic substrate was added and incubated in the dark at 37 °C for 15 min. Stop solution was added to terminate the reaction. The absorbance value was measured at a wavelength of 450 nm with a full-wavelength microplate reader within 10 min. The inhibition rate of Cy-4 on interleukin IL-1β was calculated as follows: Inhibition rate (%) = 1 - (OD value of drug well - OD value of blank well) / (OD value of negative control well - OD value of blank well). Three replicate wells were set, and the inhibition rate results of the cyclic peptide on IL-1β were expressed as the mean ± SEM of each group of data.

[0086] III. Experimental Results

[0087] The results showed that the cyclic peptide Cy-4 had strong inhibitory activity on interleukin IL-1β. The inhibition rate of 10 μM Cy-4 on interleukin IL-1β was (37.02 ± 6.78)%, and the inhibition rate of 50 μM Cy-4 on interleukin IL-1β was as high as (104.34 ± 0.12)%.

[0088] In summary, the present invention provides a novel cyclic peptide that has not been reported before. This novel cyclic peptide has extremely strong affinity for the LRR domain in NLRP3 protein, and thus can inhibit the activation of NLRP3 inflammasome. Those skilled in the art know that NLRP3 inflammasome can be activated by various types of pathogens and danger signals and plays an important role in many diseases, such as inflammatory diseases and tumors. The present invention proves through experiments that this novel cyclic peptide has excellent anti-tumor and anti-inflammatory activities. Thus, the novel cyclic peptide provided by the present invention has the prospect of being developed into drugs for treating diseases that can be treated or alleviated by inhibiting the activation of NLRP3 inflammasome, such as anti-tumor drugs and anti-inflammatory drugs.

[0089] The role of the above embodiments is to specifically introduce the substantial content of the present invention. However, those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A cyclic peptide or a pharmaceutically acceptable salt or solvate thereof, characterized in that: The sequence of the cyclic peptide is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5: SEQ ID NO.1: Ac-CLSRMIKHFKKQC-NH2, cyclization site is c[1C-13C]; SEQ ID NO.2: Ac-GDLSRMIKHFKKQKRLIQ, cyclization site is c[8K-18Q]; SEQ ID NO.3: Ac-CGDLSRMIKHFKKQKRLIC-NH2, cyclization site is c[1C-19C]; SEQ ID NO.4: RRRRRRRRRCGDLSRMIKHFKKQKRLIC, cyclization site is c[10C-28C]; SEQ ID NO.5: CGDCLSRMIKHFKKQCKRLIC, the cyclization sites are c[1C-16C] and c[4C-21C].

2. Use of the cyclic peptide with a sequence as shown in SEQ ID NO. 4 in claim 1 or a pharmaceutically acceptable salt or solvate thereof for preparing a drug for a disease that can be treated or alleviated by inhibiting NLRP3 inflammasome activation, wherein the disease that can be treated or alleviated by inhibiting NLRP3 inflammasome activation is leukemia or inflammatory disease.

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