Polypeptides as nek7-nlrp3 inhibitors and uses thereof

Abstract

The application discloses a polypeptide as a NEK7-NLRP3 inhibitor and a use thereof. The polypeptide is simple in preparation method and novel in structure; the polypeptide has no obvious toxicity to THP-1 cells; the polypeptide can keep a good alpha helix configuration in an amphiphilic environment; the polypeptide is derived from an alpha helix binding area of NEK7 protein and NLRP3 protein, has high affinity with NLRP3 protein; the polypeptide can interfere with the oligomerization of NLRP3 protein, inhibit the activation of NLRP3 inflammasome, significantly reduce the maturation and secretion of IL-1beta, and then improve the inflammatory microenvironment and reduce pyroptosis, and has good anti-inflammatory activity; the application also relates to a preparation method of the polypeptide, a pharmaceutical combination containing the polypeptide, and a use of the polypeptide alone or in combination with other compounds for preventing or treating inflammation-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to peptides as NEK7-NLRP3 inhibitors and their uses. Background Technology

[0002] The human immune system consists of the innate immune system and the adaptive immune system. The innate immune system primarily uses pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), thus activating the body's first line of defense. Inflammasomes are indispensable multi-protein complexes in this immune process, first reported by Martinon in 2002. Inflammasomes can recognize various danger signals and aggregate. The assembled inflammasome can induce cells to secrete the inflammatory factors interleukin-1β (IL-1β) and IL-18 via caspases, prompting an inflammatory response in the body.

[0003] Among these inflammasomes, the NOD-like receptor containing pyrin domain 3 (NLRP3) inflammasome is the most extensively and thoroughly studied. In the immune response, the NLRP3 inflammasome can promptly clear and defend against invading pathogens or danger signals generated by the body itself, maintaining homeostasis. Notably, when NLRP3 inflammasome activation is dysregulated, it can induce a severe inflammatory response, leading to various related diseases. According to existing research, the NLRP3 inflammasome is involved in the occurrence and development of various diseases, including cardiovascular diseases (such as atherosclerosis, ischemic heart disease, and dilated cardiomyopathy), digestive system diseases (such as non-alcoholic fatty liver disease, type II diabetes, and inflammatory bowel disease), central nervous system diseases (such as depression), and rheumatoid arthritis.

[0004] The NLRP3 receptor protein, located in the cytoplasm, can detect various intracellular stimuli and activate the inflammasome. It consists of a C-terminal leucine-rich repeat (LRR) domain, a nucleotide-binding oligomerization domain (NACHT), and an N-terminal pyrin domain (PYD). The classical activation pathway of the NLRP3 inflammasome can be broadly divided into two phases: initiation and activation, each highly regulated by different signals.

[0005] The initiation phase involves two functions. The stimulatory signal from NLRP3 alone in the cytoplasm is insufficient to activate the NLRP3 inflammasome. Therefore, the first function of the initiation phase is to upregulate the expression of various components of the NLRP3 inflammasome and inflammatory factors. After Toll-like receptors (TLRs) or tumor necrosis factor receptors (TNFRs) bind to their corresponding stimulatory molecules, they promote the entry of nuclear factors into the nucleus, initiating the downstream nuclear factor kappa-B (NF-κB) signaling pathway, resulting in increased expression of NLRP3, pro-IL-1β, and pro-IL-18 within the cell. The second function of the initiation phase is to perform various functional modifications on NLRP3, including ubiquitination, phosphorylation, nitrosation, and acetylation. Post-translational modifications of NLRP3 stabilize it in a self-inhibitory state, preparing it for activation in the second phase.

[0006] During the activation phase, molecules such as ATP, cholesterol, nigrain, silica, and high glucose can all participate in the assembly of the NLRP3 inflammasome. The activated NLRP3 PYD domain binds to the PYD domain of an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Subsequently, ASC recruits pro-caspase-1 through CARD-CARD interactions, forming the complex inflammasome. The assembled NLRP3 inflammasome can promote the self-cleavage of pro-caspase-1, forming caspase-1. Caspase-1 cleaves pro-IL-1β and pro-IL-18 to form mature IL-1β and IL-18. Furthermore, it can cleave GSDMD. The N-terminus of GSDMD can form membrane pores on the cell membrane, releasing IL-1β and IL-18 extracellularly. These pyroptosis signals further activate the immune system, leading to a more severe inflammatory response.

[0007] NIMA-related kinase 7 (NEK7) is a multifunctional kinase that influences centrosome replication, mitochondrial regulation, intracellular protein transport, DNA repair, and mitotic spindle assembly. As a downstream of potassium efflux signaling, NEK7 specifically binds to the LRR and NACHT domains of NLRP3, promoting inflammasome assembly. Therefore, NEK7 is essential for NLRP3-stimulated NLRP3 inflammasome activation. NEK7 may become a potential therapeutic target for NLRP3 inflammasome-related diseases.

[0008] In recent years, researchers have conducted in-depth studies on the composition and activation mechanism of the NLRP3 inflammasome and developed small molecule inhibitors such as MCC950, CY-09, OLT1177, and oridonin. However, the safety and mechanism of action of these small molecule inhibitors are not fully understood. The toxicity and clinical safety of small molecule drugs are the main reasons for their development failure. Compared with small molecule drugs, peptides have better binding affinity and specific targeting. At the same time, as endogenous substances, peptide drugs degrade into amino acids, resulting in fewer side effects. Therefore, peptide design based on protein-protein interactions or modification of natural peptides in vivo can provide an effective model for the development of novel anti-inflammatory drugs. Summary of the Invention

[0009] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a polypeptide as an inhibitor of NEK7-NLRP3, which is derived from the α-helical binding region of NEK7 protein and NLRP3 protein and can inhibit the oligomerization of NLRP3 inflammasomes.

[0010] Another object of the present invention is to provide pharmaceutical compositions containing these polypeptides, and their use alone or in combination with other compounds for the prevention or treatment of inflammation-related diseases.

[0011] The objective of this invention can be achieved through the following technical solutions:

[0012] A polypeptide or a salt thereof, wherein the polypeptide is selected from any one of the following (1) to (3):

[0013] (1) A polypeptide with an amino acid sequence as shown in SEQ ID NO.1;

[0014] (2) A polypeptide derived from (1) that exhibits anti-inflammatory activity after substitution, deletion and / or addition of one or more amino acids in the polypeptide amino acid sequence defined in (1);

[0015] (3) The amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of the polypeptide described in item (1) or item (2).

[0016] As a preferred embodiment of the present invention, the polypeptide is N-terminally acetylated and C-terminally amidated.

[0017] A polypeptide or a salt thereof, said polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1, preferably the polypeptide being N-terminally acetylated and C-terminally amidated.

[0018] A polypeptide or a salt thereof, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO.2, preferably the polypeptide is N-terminally acetylated and C-terminally amidated.

[0019] A polypeptide or a salt thereof, said polypeptide comprising an amino acid sequence as shown in SEQ ID NO.3, preferably the polypeptide being N-terminally acetylated and C-terminally amidated.

[0020] A polypeptide or a salt thereof, said polypeptide comprising an amino acid sequence as shown in SEQ ID NO.4, preferably the polypeptide being N-terminally acetylated and C-terminally amidated.

[0021] A pharmaceutical composition comprising the polypeptide or a salt thereof as described in this invention.

[0022] As a preferred embodiment of the present invention, the pharmaceutical composition comprises a medicament for treating inflammation-related diseases and / or a pharmaceutically acceptable carrier.

[0023] The use of the polypeptide or its salt described in this invention, and the pharmaceutical composition thereof, in the preparation of a medicament for treating inflammation-related diseases; the inflammation-related diseases involve the NLRP3 protein.

[0024] The inflammatory-related diseases mentioned are NLRP3 protein-related inflammations, such as diabetes, atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease, gout, chronic kidney disease, cold pyridine-associated periodic syndrome, non-alcoholic fatty liver disease, inflammatory bowel disease, and neurodegenerative diseases.

[0025] The use of the polypeptide or its salt, or the pharmaceutical composition described in this invention, in the preparation of NEK7-NLRP3 inhibitors.

[0026] A method for treating an inflammatory-related disease, the method comprising administering one of the aforementioned polypeptides to a patient in need.

[0027] Furthermore, the aforementioned inflammatory-related diseases are NLRP3 protein-related inflammations; such as diabetes, atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease, gout, chronic kidney disease, cold pyridine-associated periodic syndrome, non-alcoholic fatty liver disease, inflammatory bowel disease, and neurodegenerative diseases.

[0028] The polypeptide of this invention originates from the α-helix binding region of NEK7 and NLRP3 proteins; the polypeptide has a high affinity for NLRP3 protein and inhibits the oligomerization of NLRP3 inflammasomes; the polypeptide can maintain a good α-helix conformation in an amphiphilic environment; the polypeptide has no significant toxicity to THP-1 cells.

[0029] The polypeptide of the present invention is designed based on the interaction between NEK7 and NLRP3 proteins. NEK7 is a multifunctional kinase that influences centrosome replication, mitochondrial regulation, intracellular protein transport, DNA repair, and mitotic spindle assembly. As a downstream of potassium ion efflux, NEK7 can bind to the LRR and NACHT domains of NLRP3, promoting the assembly of the inflammasome. Specifically, the N-terminal PYD domain of NLRP3 binds to the N-terminal PYD domain of ASC, subsequently recruiting pro-caspase-1 from the CARD domain of ASC to form the inflammasome complex. The assembled NLRP3 inflammasome can promote the self-cleavage of pro-caspase-1, forming caspase-1. Activated caspase-1 can act as a next-stage signal, participating in the cleavage of pro-IL-1β and pro-IL-18, forming mature IL-1β and IL-18, which are then secreted and released extracellularly, inducing pyroptosis. NEK7 is essential for stimulating the activation of the NLRP3 inflammasome, and includes ATP, melanin, monosodium urate crystals, and alum. Therefore, NEK7 may become a potential treatment for NLRP3 inflammasome-related inflammatory diseases. At the target site, the strategy employed is to use computer-aided drug design to extract the α-helical peptide from the folded subdomains of the polypeptide secondary structure that form the backbone of protein-protein interactions. The peptide is then synthesized and modified using solid-phase polypeptide synthesis, potentially yielding an active polypeptide drug lead that selectively acts on the NLRP3 protein. Specifically, a polypeptide is developed that mimics the α-helix of NEK7 to interfere with the NEK7-NLRP3 interaction. The selected polypeptides contain slightly different key amino acids and are named Pep-1, Pep-2, Pep-3, and Pep-4, respectively.

[0030] The sequences are listed in Table 1 below:

[0031] Table 1

[0032] SEQ ID NO. name sequence SEQ ID NO.1 Pep-1 <![CDATA[Ac-LSRMIKHFKKQ-NH2]]> SEQ ID NO.2 Pep-2 <![CDATA[Ac-LSRMIKHFKKQKR-NH2]]> SEQ ID NO.3 Pep-3 <![CDATA[Ac-LSRMIKHFKKQKRLI-NH2]]> SEQ ID NO.4 Pep-4 <![CDATA[Ac-GDLSRMIKHFKKQKRLI-NH2]]>

[0033] “Ac” represents N-terminal acetylation of a polypeptide, and “NH2” represents C-terminal amidation of a polypeptide.

[0034] The preparation method of polypeptides is as follows:

[0035] The methods for synthesizing the polypeptides described herein are known in the art; however, the following exemplary methods are used; it should be understood that various steps may be performed in an alternative order or sequence to obtain the desired polypeptide; synthetic chemical transformation and protecting group methods (protection and deprotection) that can be used to synthesize the polypeptides described herein are known in the art.

[0036] The peptides of this invention can be prepared by chemical synthesis methods, as is well known to those skilled in the art; one method for preparing the peptides described herein is using solid-phase peptide synthesis (SPPS); the C-terminal amino acid is linked to a cross-linked polystyrene resin via an acid-labile bond to a linker molecule; this resin is insoluble in the solvent used in the synthesis, thus allowing for relatively simple and rapid washing away of excess reagents and byproducts; the N-terminus is protected by an Fmoc group, which is stable in acid but can be removed by a base; any side chain functional groups are protected by base-stable but acid-labile groups. The N-terminus of the synthetic peptide is acetylated, while the C-terminus is amidated.

[0037] In some embodiments, the contact step is carried out in a solvent selected from proton solvents, aqueous solvents, organic solvents, and mixtures thereof; for example, the solvent may be selected from H2O, THF, THF / H2O, tBuOH / H2O, DMF, DIEA, CH3CN or CH2Cl2, ClCH2CH2Cl, or mixtures thereof; in a specific embodiment, DMF is used.

[0038] The peptides were purified and characterized using standard methods.

[0039] In a specific embodiment, the method for preparing the polypeptide according to the present invention includes at least the following steps:

[0040] A plurality of peptides containing protecting groups are provided, each peptide immobilized on a solid support; a deprotecting agent is exposed to the immobilized peptide to remove the protecting groups from at least partially immobilized peptides; at least partially deprotecting agent is removed; protected amino acid residues are dissolved in a solvent, preferably DMF; a coupling agent, preferably HATU, is used; a base agent, preferably DIEA, is used; the protected amino acid residues and the coupling agent are exposed to the immobilized peptide such that at least partially activated amino acid residues bind to the immobilized peptide to form newly bound amino acid residues; and at least partially activated amino acid residues not bound to the immobilized peptide are removed; the final peptide is exposed to a lysis agent for final deprotection; the final peptide is precipitated, purified, and lyophilized, preferably to obtain a purity greater than 95%.

[0041] The beneficial effects of this invention are:

[0042] The polypeptide described in this invention exhibits a high α-helix content and a stable α-helix conformation in a cell membrane-simulated amphiphilic environment. Figure 1 ).

[0043] The polypeptide described in this invention has no significant cytotoxicity to THP-1 cells (Table 3).

[0044] The IC50 of the peptides Pep-1, Pep-2, Pep-3, and Pep-4 described in this invention on the inhibition of IL-1β secretion 50The concentrations were 23.75 μM, 27.15 μM, 20.60 μM, and 20.02 μM, respectively. Figure 2 ).

[0045] The peptides Pep-1, Pep-2, Pep-3, and Pep-4 described in this invention exhibit high affinity for the LRR domain of the NLRP3 protein, with affinities of 2.27 μM, 3.08 μM, 2.57 μM, and 6.80 μM, respectively. Figure 4 ). Attached Figure Description

[0046] Figure 1 The CD spectrum of the 0.1 mg / ml polypeptide dissolved in 1×PBS (containing 50% TFE) in the present invention in the range of 190-260 nm;

[0047] Figure 2 The IC50 of Pep-1, Pep-2, Pep-3, and Pep-4 in this invention inhibits IL-1β secretion. 50 Analysis chart;

[0048] Figure 3 This invention presents a semi-quantitative analysis of the ratio of the gray value of the target protein in THP-1 cells to the gray value of the internal reference β-actin.

[0049] Figure 4 Affinity analysis diagram of the peptide of this invention with the LRR domain in the NLRP3 protein.

[0050] A: Affinity analysis diagram of Pep-1 and the LRR domain in NLRP3 protein in this invention;

[0051] B: Affinity analysis diagram of Pep-2 and the LRR domain in NLRP3 protein in this invention;

[0052] C: Affinity analysis diagram of Pep-3 and the LRR domain in NLRP3 protein in this invention;

[0053] D: Affinity analysis diagram of Pep-4 and the LRR domain in NLRP3 protein in this invention. Detailed Implementation

[0054] Example 1: Preparation and characterization of peptides:

[0055] 1.1 Synthetic polypeptides

[0056] The peptides were synthesized artificially using the SPPS method. The synthesis scale was 0.1 mmol, with 40-RAM amphiphilic Rink amide resin loaded at 0.4 mmol / g, and an excess of 5 equivalents for the protected amino acids. All protected amino acids and the coupling agent HATU were pre-dissolved in DMF to prepare a 0.5 M stock solution. The resin was first swollen in 6 ml of DMF for 15 minutes under vortex stirring. After removing the DMF by filtration, the Fmoc groups were deprotected by adding 20% ​​piperidine / DMF solution (6 ml) and vortexing for 1 minute. This step was repeated twice. The resin was then washed three times with DMF, followed by the sequential addition of the desired amino acid (1 ml, 0.5 mmol), N,N-diisopropylethylamine (DIEA) (0.164 ml, 1 mmol), and HATU coupling agent (1 ml, 0.5 mmol). The mixture was stirred under vortex stirring for 5 minutes, and this operation was performed twice between two couplings by washing with DMF. The deprotection of Fmoc and coupling of amino acids were repeated until the desired linear sequence was obtained. Once the linear peptide was synthesized, the final deprotection of the Fmoc group was performed, followed by acetylation in a DMF solution (1 / 1 / 8) containing acetic anhydride (DIEA) for 10 minutes. The final cleavage of the peptide was carried out in a solution (10 ml) of trifluoroacetic acid, triisopropylsilane, and water (95 / 2.5 / 2.5). After filtration, the solution was concentrated and dissolved in diethyl ether. The precipitate was centrifuged and the mother liquor was decanted. The above steps were performed twice, and the precipitate was dissolved in water and lyophilized to obtain a completely deprotected peptide. After lyophilization, the peptide was purified by elution with acetonitrile / water in the presence of 0.1% TFA on a preparative reversed-phase HPLC. The peptide thus obtained preferably had a purity greater than 95%.

[0057] 1.2 Characterization

[0058] The cross-linked compound was purified by high performance liquid chromatography on a reversed-phase C18 column to obtain a pure compound; the chemical composition of the pure product was confirmed by LC / MS mass spectrometry and amino acid analysis.

[0059] The results are shown in Table 2 below:

[0060]

[0061] Example 2: Evaluation of whether the peptide retains its α-helix structure in an amphiphilic cell membrane simulated environment:

[0062] To further understand the influence of α-helix structure on peptide bioactivity, and taking advantage of the fact that trifluoroethanol promotes the formation of α-helix secondary structures, circular dichroism (CD) analysis was performed on the peptides. In the far-ultraviolet region (185–245 nm) of the CD spectrum, typical peptide secondary structures exhibited their own characteristic absorption peaks. Specifically, the α-helix showed double negative peaks at 208 nm and 222 nm, and a positive peak at 195 nm; the β-sheet showed a weak negative peak at 215 nm; the random coil showed a negative peak at 200 nm and a small, broad positive peak at 220 nm.

[0063] The secondary structure of the peptide was determined by circular dichroism spectroscopy. CD spectra were measured using a J-810 spectrometer at 25°C with a quartz cell and a path length of 1.0 mm. The spectra were recorded by scanning in the range of 190 to 260 nm at a speed of 50 nm / min, a bandwidth of 1 nm, and a response time of 1 s. A 0.1 mg / mL peptide solution was prepared by dissolving the peptide in 50% trifluoroethanol solution (cell membrane simulation environment) prepared from PBS. Each spectrum was the result of three average summations. The obtained CD spectra were then converted to the average residual ellipticity using the following formula:

[0064] [θ] 222 =([θ] obs *MW) / (L*ρ)

[0065] Where [θ] 222 It is the average residual ellipticity at 222 nm (deg*cm2*dmol-1), [θ] obs The observed ellipticity (mdeg) is corrected for buffer at a wavelength of 222 nm. MW is the average molecular weight of the peptide residues, ρ is the mass concentration of the peptide (mg / ml), and L is the optical path length of the cuvette (1 mm).

[0066] The α-helical content is calculated using the following formula:

[0067] α-helical content (%) = -([θ] 222 *100) / 4000

[0068] The resulting CD spectrum is shown as the average residual ellipticity [θ] (deg*cm2*dmol-1) versus wavelength λ (nm).

[0069] like Figure 1As shown, in an amphiphilic cell membrane simulation environment, all peptides exhibited a good α-helix structure, showing a positive peak at 195 nm and two negative peaks at 208 nm and 222 nm; the α-helix contents of Pep-1, Pep-2, Pep-3, Pep-4 and Pep-5 were 37.06%, 30.82%, 61.37%, 42.02%, and 29.40%, respectively.

[0070] Example 3: Evaluation of peptide cytotoxicity using the MTT assay:

[0071] Cytotoxicity was determined using an MTT assay. MTT was purchased from Sigma (St. Louis, MO). It was dissolved in PBS to a stock solution concentration of 5 mg / mL and stored at -20°C. After treating THP-1 cells with a density gradient of the test peptide or DMSO for 24 hours, 20.0 μL of MTT solution (5 mg / mL) was added to each well of a 96-well plate and incubated for 4 hours. Then, the solution was removed and 150.0 μL of DMSO was added to each well to dissolve the water-soluble MTT-formazan crystals. The absorbance (OD value) was recorded at 570 nm using an E1x800 microplate reader (BioTek, Vermont, USA). The cell growth inhibition rate of the compound was calculated as follows:

[0072] Inhibition rate (%) = 1 - OD value of drug well / OD value of blank well, with cell wells without drug and inducing factor serving as blank controls. The cytotoxicity evaluation of a polypeptide of this invention is shown in Table 3.

[0073] Table 3

[0074] SEQ ID NO. name <![CDATA[MTT IC 50 (μM)]]> SEQ ID NO.1 Pep-1 ＞100 SEQ ID NO.2 Pep-2 ＞100 SEQ ID NO.3 Pep-3 ＞100 SEQ ID NO.4 Pep-4 ＞100 SEQ ID NO.5 Pep-5 ＞100

[0075] As shown in Table 3, the polypeptide of the present invention has no significant toxicity to THP-1 cells.

[0076] Example 4: Preliminary evaluation of the anti-inflammatory activity of the peptide and its inhibitory effect on IL-1β secretion in THP-1 cells using ELISA. 50 :

[0077] The secretion of IL-1β in cell culture supernatant was detected using a double-antibody sandwich ELISA. Freshly collected THP-1 cell samples were incubated at room temperature for 20 min, then centrifuged at 3000g for 10 min at 4℃. The supernatant was then used for analysis. THP-1 cell samples and IL-1β standards were added separately to antibody-coated 96-well plates. After adding biotinylated antibody, the plates were incubated at 37℃ for 1 h on a shaker, washed 5 times, and then enzyme-conjugated working solution was added. The plates were incubated at 37℃ in the dark for 30 min, followed by 5 washes. A chromogenic substrate was added, and the plates were incubated at 37℃ in the dark for 15 min. The reaction was terminated by adding stop solution. The absorbance was measured at 450 nm using a Thermo microplate reader within 10 min. The yield of the compound to interleukin IL-1β was calculated as follows:

[0078] Inhibition rate (%) = 1 - (OD value of drug wells - OD value of blank wells) / (OD value of negative control wells - OD value of blank wells). Cell wells without drugs and inducing factors were used as blank controls, and cell wells without drugs but with LPS and ATP were used as negative controls.

[0079] In this invention, MCC950 was used as a positive control, DMSO as a negative control, and cell lysis buffer as a blank background. Three replicates were used. The inhibition rate of the peptide against IL-1β is expressed as the average value of each group ± SEM. The inhibition rate of one peptide against interleukin IL-1β in this invention is shown in Table 4 below:

[0080] Table 4

[0081]

[0082] "N / A" indicates that no activity was shown.

[0083] As shown in Table 4, some of the peptides in this invention exhibit inhibitory activity against interleukin IL-1β. This provides a foundation for developing highly effective, safe, novel, and specific NLRP3 peptide inhibitors, and for treating NLRP3-related inflammatory diseases.

[0084] In this invention, Pep-1, Pep-2, Pep-3, and Pep-4 inhibit the secretion of interleukin IL-1β by an IC50. 50 like Figure 2 As shown, Pep-1, Pep-2, Pep-3, and Pep-4 inhibited the secretion of interleukin IL-1β in a concentration-dependent manner, maintaining the stability of the cellular microenvironment and effectively alleviating pyroptosis. The IC50 values ​​of Pep-1, Pep-2, Pep-3, and Pep-4 for inhibiting IL-1β secretion were... 50 The effective concentrations were 23.75 μM, 27.15 μM, 20.60 μM, and 20.02 μM, respectively, all exhibiting good anti-inflammatory activity. This provides new insights for the development of anti-inflammatory drugs.

[0085] Example 5: Monitoring the effect of peptides on target proteins using Western blotting:

[0086] Intracellular protein preparation: Cell supernatant was aspirated from six-well plates, washed twice with PBS buffer on ice, and cells were scraped from the plates using a cell scraper and collected. Centrifuged at 2000 rpm for 5 min, and RIPA protein lysis buffer was added at a ratio of 1:5 (somatic cell volume: lysis buffer volume). Lysis was performed on ice for 60 min, followed by centrifugation at 12000 rpm for 10 min. The supernatant was collected as whole-cell protein. 4 μL was used for BCA assay to determine protein concentration. The remaining protein supernatant was added by volume to 5× loading buffer and denatured in a boiling water bath for 10 min. After aliquoting, the supernatant was stored at -20°C.

[0087] Protein preparation from cell supernatant: Aspirate cell supernatant into a pre-chilled EP tube, slowly add 100% trichloroacetic acid to a final concentration of 10%, incubate on ice for 1 hour to precipitate the protein, centrifuge at 12000 rpm and 4°C for 10 min, remove the supernatant, wash the precipitate with acetone, centrifuge at 12000 rpm and 4°C for 10 min, repeat three times, then remove the acetone, add 1× loading buffer by volume, denature in a boiling water bath for 10 min, aliquot and store at -20°C.

[0088] Based on the BCA quantification results, 40-80 μg of sample per lane was loaded. SDS-PAGE with different gel concentrations was used for separation according to the molecular weight of the target protein. The protein was concentrated at constant voltage (84V) for 25 min, followed by constant voltage (120V) gel electrophoresis for 55 min. Wet transfer at 200 mA for 90 min transferred the protein to a PVDF membrane (Millipore, USA). The PVDF membrane was blocked at room temperature for 1 h with shaking in 3% BSA-TBST (pH 7.4, 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20). Then, 3% primary antibodies prepared in BSA-TBST were added: rabbit anti-cleaved-IL-1β (1:1000, Cell Signaling Technology, USA), rabbit anti-IL-1β (1:1000, Cell Signaling Technology, USA), and mouse anti-β-actin (1:1000, Beyotime, China). The membrane was incubated overnight at 4°C. After rinsing with TBST for 10 min × 3 times, HRP-labeled goat anti-mouse secondary antibody (1:4000) and HRP-labeled goat anti-rabbit secondary antibody (1:4000) were added, and the mixture was incubated at room temperature with shaking for 100 min. After rinsing with TBST for 10 min × 3 times, ECL (Beyotime, China) luminescent substrate was added for color development. The images were developed using an Image Quant LAS4000 mini (GE). The ratio of the gray value of the target protein to the gray value of the internal control β-actin was used for semi-quantitative analysis. Figure 3 ).

[0089] Western blot experiments showed that Pep-1 in this invention can significantly inhibit the expression of intracellular protein Pro-IL-1β, and can also reduce the maturation and secretion of IL-1β in a concentration-dependent manner.

[0090] Example 6: Evaluation of the affinity of the peptide for the LRR domain in the NLRP3 protein:

[0091] To determine the affinity of Pep-1, Pep-2, Pep-3, and Pep-4 for the LRR domain in the NLRP3 protein, label-free isothermal titration calorimetry (ITC) was used for real-time determination. The MicroCalPEAQ-ITC isothermal titration microcalorimeter was used to measure the heat absorbed or released during molecular interactions. The reference cell was typically filled with pure water, the sample cell with LRR protein (20 μM), and the titrator with one of Pep-1, Pep-2, Pep-3, or Pep-4 (700 μM). During the experiment, the ligands were precisely and controllably added to the sample cell under well-mixed conditions, typically in increments of 0.4 μL to 3 μL, until the molar amount of ligand in the sample cell was 2–3 times the amount of protein in the sample cell. Each titration generates a heat pulse. By integrating the heat from each drop and normalizing the concentration, the molar heat release (kcal / mol) is generated and plotted against the molar ratio (ligand / sample). Then, by fitting an appropriate binding model, thermodynamic information such as binding affinity (KD), stoichiometry (n), and interaction enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) is obtained.

[0092] The affinity between the peptide and the LRR domain of NLRP3 protein was determined by ITC. The measurement was performed using a MicroCal PEAQ-ITC isothermal titration microcalorimeter at 25°C. The reference DP value was 8 μcal / s, the rotation speed was 750 rpm, the feedback mode was hight, the number of drops was 19, the titration volume was 3 μL, the titration interval was 150 s, and the peptide volume was 700 μM and 70 μL, while the LRR protein volume was 20 μM and 300 μL. The molar heat release (kcal / mol) was generated by integrating the heat from each drop and normalizing the concentration, and then plotted against the molar ratio (peptide / LRR protein). The affinity value (KD) was obtained by fitting an appropriate binding model.

[0093] Set up a control experiment:

[0094] Titration A: Peptide is added to LRR protein sample;

[0095] Titration B: Buffer solution for titrating peptides into LRR protein samples;

[0096] Titration C: Add LRR protein sample to peptide buffer;

[0097] Titration D: The buffer solution for titrating the LRR protein sample with peptide buffer;

[0098] Final experimental result = Titration A - Titration B - Titration C + Titration D

[0099] The affinity of Pep-1, Pep-2, Pep-3, and Pep-4 for the LRR domain in the NLRP3 protein is as follows: Figure 4 As shown, the affinity constants KD values ​​are 2.27 μM, 3.08 μM, 2.57 μM, and 6.80 μM, respectively, indicating that the peptide of the present invention can directly bind to NLRP3 and has high affinity.

[0100] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A polypeptide or a salt thereof, characterized by, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

1.

2. The polypeptide or salt thereof according to claim 1, characterized by, The polypeptide is acetylated at the N-terminus and amidated at the C-terminus.

3. A polypeptide or a salt thereof, characterized by, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

2.

4. The polypeptide or salt thereof according to claim 3, characterized by The polypeptide is acetylated at the N-terminus and amidated at the C-terminus.

5. A polypeptide or a salt thereof, characterized by, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

3.

6. The polypeptide or salt thereof according to claim 5, characterized by The polypeptide is acetylated at the N-terminus and amidated at the C-terminus.

7. A polypeptide or a salt thereof, characterized by, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

4.

8. The polypeptide or salt thereof according to claim 7, characterized by The polypeptide is acetylated at the N-terminus and amidated at the C-terminus.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polypeptide or salt thereof according to any one of claims 1-8.

10. The pharmaceutical composition of claim 9, wherein, The pharmaceutical composition comprises a pharmaceutically acceptable carrier.

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