CN113143900A

CN113143900A - Application of ML365 in preparation of medicine for preventing and/or treating NLRP3 inflammasome-related diseases

Info

Publication number: CN113143900A
Application number: CN202110418994.9A
Authority: CN
Inventors: 周平正; 庞建新; 吴晓燕; 植源兴; 潘林杰; 曹莹; 吴婷
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2021-07-23
Anticipated expiration: 2041-04-19
Also published as: CN113143900B

Abstract

The invention discloses application of ML365 in preparing a medicament for preventing or treating NLRP3 inflammasome-related diseases for the first time. The inventor researches and finds that ML365 can specifically inhibit ATP-induced activation of NLRP3 inflammasome, and has no influence on Niegericin-induced NLRP3, AIM2 and NLRC4 inflammasome. ML365 as a specific potent inhibitor of TWIK2 can specifically inhibit ATP activation, so that activation of NLRP3 inflammasome is hindered, which can provide theoretical basis for drug development of NLRP3 inflammasome-related diseases in the future and has wide application prospect.

Description

Application of ML365 in preparation of medicine for preventing and/or treating NLRP3 inflammasome-related diseases

Technical Field

The invention relates to the technical field of medicines, in particular to application of ML365 in preparation of a medicine for preventing and treating NLRP3 inflammasome-related diseases.

Background

The inflammasome is the core component of innate immunity (Schroder and Tschopp 2010; Broz and Dixit 2016). Of the numerous inflammasomes, NLRP3 inflammasome is the most clinically relevant and has received extensive attention and research. The NLRP3 inflammasome is unique in that it can be activated by various types of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) signals, such as ATP, crystalloid molecules (urate crystal MSU, silica, asbestos, alum, etc.), toxins secreted by pathogens (Nigericin and maitoxin, etc.), and misfolded proteins associated with neurodegenerative diseases (β -amyloid and α -synuclein), etc. (Davis et al 2011; Heneka et al 2013; Gordon et al 2018). The NLRP3 inflammasome consists of the receptor protein NLRP3, the linker protein ASC, the effector protein pro-caspase-1 (Zoete et al 2014). The Activation comprises two steps of Priming and Activation, wherein the Priming mainly comprises the steps of activating Toll-like receptors (TLRs) acting on cell membranes through stimulation of LPS and the like, activating NF-kB signal channels, and inducing expression of NLRP3, IL-1 beta precursor and IL-18 precursor; the activation is to promote the assembly of an inflammatory corpuscle complex through a specific compound (ATP and the like), a pathogen or a toxin (niger and the like), promote the caspase-1 precursor to carry out self-cleavage to form active caspase-1, further, the caspase-1 carries out enzymolysis to cleave the substrate IL-1 beta precursor and the IL-18 precursor to form a mature form IL-1 beta and IL-18 with biological activity, and the mature form IL-1 beta and IL-18 are released to the outside of cells to cause an inflammatory reaction, promote the fever, the recruitment and activation of immune cells and the generation of secondary cytokines; meanwhile, activated caspase-1 cuts GSDMD protein (Gasderm min D), a pore channel is formed at the N end of the cut GSDMD, water enters cells to swell and break the cells, and cell contents are released to the outside of the cells to cause cell apoptosis (KV et al.2019).

Moderate NLRP3 inflammasome activation is important for clearing microbial infections, however, its abnormal activation is closely related to various autoinflammatories or chronic inflammation. There are a number of metabolic diseases currently reported to be associated with NLRP3, such as sepsis lung injury, rheumatoid arthritis, gout, atherosclerosis, type 2 diabetes, and the like, as well as neurodegenerative diseases including multiple sclerosis, alzheimer's disease, parkinsonism, and the like (hall et al 2008; Strowig et al 2012; Heneka et al 2013; Barclay and Shinohara 2017; Gordon et al 2018; Mart i niez-Garc i a et al 2019; Danielski et al 2020).

The process of NLRP3 inflammasome activation is very complex and the mechanisms that regulate its activation are still under investigation. It is widely accepted that the efflux of intracellular potassium ions is a necessary, sufficient condition for activation of NLRP3 inflammatory bodies (Walev et al 1995;

-Planillo et al.2013；Gong et al.2018；Hafner-

and pelegri i n 2018). Intracellular potassium ion outflow is required in the process that crystal molecules such as ATP, niger and MSU activate NLRP3 inflammasome. On the one hand, NLRP3 inflammasome can be directly activated when extracellular in a potassium-free environment; on the contrary, when extracellularThe activation of NLRP3 inflammasome is blocked in high potassium environments. In addition, the flow of potassium ions specifically modulates NLRP3 inflammasome without affecting AIM2 or NLRC4 inflammasome activation. Therefore, elucidating the material basis of potassium ion efflux (ion channel or pore-forming protein) can find a new idea and a new drug target for the treatment of NLRP3 inflammasome-related diseases.

The K2P family of potassium channels is the latest found family of potassium channels that open near the resting membrane potential, mediating background potassium leakage currents, responsible for stabilizing the cell resting membrane potential (endodi and Czirj a K2010). The K2P channel includes 6 subfamilies, TWIK, TASK, TREK, TALK, THIK and TRESK, respectively, for a total of 15 members. The topology of the K2P channel differs from other potassium channel families by the aggregation of 2 identical subunits (homodimers) or different subunits (heterodimers) to form functional dimers, each subunit comprising 4 transmembrane helical regions (M1-M4), 2 pore regions (P1 and P2), an extracellular cap region (M1P1 loop) and the N-and C-termini of the cytoplasm. Recent studies report that the K2P channel family member, the TWIK2 channel, mediates the efflux of potassium ions during activation of NLRP3 inflammasome in macrophages (Di et al.2018), and is critical for activation of NLRP3 inflammasome. In mouse Bone Marrow Derived Macrophages (BMDM), the mRNA level encoding TWIK2 is the highest of all K2P channels, and activation of NLRP3 inflammasome in BMDM cells can be inhibited by quinine pharmacological inhibition or gene knock-out of the TWIK2 channel, in particular as shown by inhibition of caspase-1 activation, ATP-induced release of IL-1 β, and oligomerization of the apoptosis-associated plaque forming protein ASC. This study suggests that during ATP activation of NLRP3 inflammasome, the TWIK2 channel and P2X7 receptor act synergistically to mediate potassium ion efflux, and activation of NLRP3 inflammasome was significantly hindered in the absence of the TWIK2 channel (Di virgingio et al.2017).

Although recent studies have shown that the TWIK2 channel mediates the efflux of potassium ions in macrophages and is involved in the activation of NLRP3 inflammasome. However, for a long time, the TWIK channel has been considered non-functional because it exhibits low or no functional expression in exogenous expression systems (Patel et al 2000). There are two main explanations for the non-functionality of the TWIK channel in exogenous expression systems, one being the ubiquitination theory, which suggests that the TWIK channel is expressed on the cell surface but silencing by ubiquitination leads to loss of channel activity in transfected and bulk cells; another is the theory of endosomes, where TWIK channels are believed to withdraw constitutively and rapidly from the cell surface, internalize and pass into the circulating endosomal compartment through an dynamin-dependent mechanism, resulting in the loss or deficiency of TWIK channels on the cell membrane, and no TWIK current was detected, it was found that repeated isoleucine mutations TWIK1-I293/294A at the intracellular C-terminus stabilized the channels on the cell membrane resulting in high current (Rajan et al 2005; Lloyd et al 2011; Hwang et al 2014; Bobak et al 2017). The above difficulties have led to little knowledge of the electrophysiological properties and functional significance of TWIK, and no modulators of the TWIK channel have been reported (Lv et al 2019).

In view of the above, there is a need in the art to find inhibitors of the TWIK2 channel that block NLRP3 inflammasome to treat related inflammatory diseases.

Disclosure of Invention

The first object of the present invention is to provide the use of ML365, a pharmaceutically acceptable salt or a polymorph thereof for the preparation of a medicament for the prevention and/or treatment of NLRP3 inflammasome-related diseases.

It is an object of a second aspect of the present invention to provide the use of ML365, a pharmaceutically acceptable salt or polymorph thereof, for the preparation of an inhibitor of the inflammasome.

The third aspect of the invention aims at providing the application of ML365, pharmaceutically acceptable salts or polymorphs thereof in preparing medicines for treating NLRP3 inflammatory-body abnormal activation diseases.

A fourth aspect of the present invention is directed to the use of ML365, a pharmaceutically acceptable salt or polymorph thereof for the preparation of a potassium ion channel inhibitor.

The fifth aspect of the invention aims to provide a medicament for treating NLRP3 inflammasome-related diseases.

The technical scheme adopted by the invention is as follows:

ML365, chemical name, 2-methoxy-N-[3- [ (3-methylbenzoyl) amino group]Phenyl radical]A benzamide. PubChem was first introduced in 2010 for the synthetic compounds. The compound has the molecular formula of C₂₂H₂₀N₂O₃Molecular weight is 360.4, CAS accession number: 947914-18-3, having the following formula (I):

in a first aspect of the invention, there is provided a use of a compound for the manufacture of a medicament for the prevention and/or treatment of NLRP3 inflammasome-related diseases, said compound being a compound of formula (I), a pharmaceutically acceptable salt or a polymorph thereof;

further, the NLRP3 inflammasome-related diseases include peritonitis, sepsis lung injury, type II diabetes or high fat food-induced obesity, parkinsonism, alzheimer's disease, multiple sclerosis, enteritis, hepatitis, silicosis, asbestosis, silicosis, behcet's disease, and rheumatoid arthritis.

In a second aspect of the present invention, there is provided the use of a compound for the manufacture of an inhibitor of inflammasome, said compound being a compound of formula (I), a pharmaceutically acceptable salt or polymorph thereof;

further, the inflammasome includes NLRP3 inflammasome.

In a third aspect of the invention, the application of a compound in preparing a medicament for treating NLRP3 inflammatory corpuscle abnormal activation diseases is provided, wherein the compound is a compound shown as a formula (I), a pharmaceutically acceptable salt or a polymorphic substance thereof;

further, the NLRP3 inflammatory-body abnormally activated diseases include atherosclerosis, type 2 diabetes, or gout.

In some embodiments of the invention, ML365 is proved to be a specific strong inhibitor of TWIK2, can specifically inhibit ATP-induced activation of NLRP3 inflammasome, and can be used for treating NLRP3 inflammasome abnormal activation diseases.

In a fourth aspect of the present invention, there is provided the use of a compound for the preparation of a potassium ion channel inhibitor, said compound being a compound of formula (I), a pharmaceutically acceptable salt or polymorph thereof;

further, the potassium channel comprises a double-pore potassium channel or a TWIK2 channel.

In some implementations of the invention, it was demonstrated that ML365 is a specific selective inhibitor of the specific TWIK2 channel.

In a fifth aspect of the present invention, there is provided a medicament for treating NLRP3 inflammasome-related diseases, said medicament comprising a compound of formula (I), a pharmaceutically acceptable salt or polymorph thereof;

further, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Still further, the NLRP3 inflammasome-related diseases include peritonitis, sepsis lung injury, type II diabetes or high fat food-induced obesity, parkinsonism, alzheimer's disease, multiple sclerosis, enteritis, hepatitis, silicosis, asbestosis, silicosis, behcet's disease, and rheumatoid arthritis.

The invention has the beneficial effects that:

the inventor finds that ML365 is a strong selective inhibitor of TWIK2 channel through electrophysiological experiments. I/I of ML365 (10. mu.M) on inhibition of TWIK2 channel₀0.35 plus or minus 0.02; further studies found that ML365 inhibits IC of TWIK2 channel₅₀It was 4.07. mu.M. And the compound does not affect the function of the TWIK1 channel or the THIK1 channel.

In addition, in vitro research experiments show that ML365 can specifically inhibit the activation of NLRP3 inflammasome induced by ATP and has no influence on the activation of NLRP3 inflammasome induced by Nigericin, AIM2 and NLRC4 nonclassical inflammasome. These results are consistent with the important role of the TWIK2 channel in NLRP3 inflammasome.

Further, through the study on the molecular mechanism of ML365 for inhibiting the action of NLRP3 inflammasome, the activity of ML365 for inhibiting NLRP3 inflammasome is remarkably reduced after knocking down TWIK2, which indicates that ML365 inhibits NLRP3 inflammasome by blocking TWIK2 channel.

Based on the beneficial results, the invention provides a new development cut for preparing the medicament for preventing or treating NLRP3 inflammasome-related diseases, and is expected to become a lead compound of TWIK2 channel inhibitors in the future. This can provide theoretical basis for the future drug development of NLRP3 inflammasome-related diseases.

Drawings

FIG. 1: the THIK1 channel was subcloned into the plasmid map of pcDNA3.1 (+).

FIG. 2: typical electrophysiology amperograms (a in fig. 2) and current density histograms (B in fig. 2) for the wild-type TWIK2(wtTWIK2) and mutant TWIK2 (muttwik 2) channels.

FIG. 3: to illustrate the modulating effects of compounds such as ML365 on the TWIK2 channel. A is the chemical structure diagram of the six K2P channel modulators used in this study; b is a typical current diagram before and after 10. mu.M ML365 acts on mutWIK 2; c is a summary of the effect of K2P channel modulators on muttwik 2 channels.

FIG. 4: to illustrate the physiological properties of ML365 on the inhibition of the TWIK2 channel. A is a typical current plot of the effect of 10. mu. MML365 on the mutWIK2 channel; b is a summary plot of the inhibitory effect of ML365(10 μ M) on mutWIK2 at different voltages (-60 to +80 mV); c is the dose-response curve for ML365 inhibiting the mutpik 2 channel.

FIG. 5: typical electrophysiology amperograms (a in fig. 5) and current density histograms (B in fig. 5) for the wild-type TWIK1(wtTWIK1) and mutant TWIK1 (muttwik 1) channels.

FIG. 6: to illustrate the effect of compounds such as ML365 on the TWIK1 channel. A is a typical graph before and after the action of 10 mu MML365 on muTWIK 1; b is a summary of the effect of the K2P channel modulators tested on muttik 1.

FIG. 7: to illustrate the effect of 20 μ M ML365 on mutWIK1 and THIK1 channels. A is a time current plot of the effect of 20 μ M ML365 on mutWIK 1; b is a time current diagram of the effect of 20 mu M ML365 on the THIK 1; c is a summary plot of the effect of 20. mu.M ML365 on muTWIK1 and THIK1 channels.

FIG. 8: to illustrate the effect of compounds such as ML365 on the THIK1 channel. A is the result of real-time fluorescent quantitative PCR (RT-qPCR) analysis of TWIK and THIK subfamily channels in BMDM cells, RAW264.7 cells and BV-2 cells; b is a typical graph before and after ML365(10 mu M) acts on the THIK 1; c summarizes the effect of the K2P channel modulators tested on the THIK 1.

FIG. 9: to demonstrate that ML365 specifically inhibits ATP-induced activation of NLRP3 inflammasome. A is a result graph of the effect of different concentrations of ML365 on activation of NLRP3 inflammasome under LPS and ATP induction in cell lysates or supernatants of BMDM cells detected by immunoblotting (WB); B. c is a result graph of the effect of different concentrations of ML365 on the generation of IL-1 beta and TNF-alpha under the induction of LPS and ATP in cell supernatant of BMDM cells detected by enzyme-linked immunosorbent assay (ELISA); d is a result graph of the effect of different concentrations of ML365 on activation of NLRP3 inflammasome under the induction of LPS and Nigericin in cell lysate or supernatant of BMDM cells detected by immunoblotting (WB); E. f is a result graph of the effect of ML365 with different concentrations on the generation of IL-1 beta and TNF-alpha under the induction of LPS and Nigericin in cell supernatant for detecting BMDM cells by enzyme-linked immunosorbent assay (ELISA); g, H, I diagram shows the effect of ML365 (5. mu.M) on IL-1 beta secretion during AIM2, NLRC4 and activation of non-classical inflammasome in ELISA. Wherein LPS + poly (dA: dT) is a stimulator of the inflammatory bodies of AIM 2; pam3CSK4 with transfected lps (clps) induced activation of non-classical inflammasome, Pam3CSK4 with Flagellin induced activation of NLRC4 inflammasome.

FIG. 10: to demonstrate that ML365 inhibits ATP-induced activation of NLRP3 inflammasome (a in fig. 10) but has no effect on Nigericin-induced activation of NLRP3 inflammasome (B in fig. 10).

FIG. 11: it was demonstrated that knock-down of TWIK2 abolished the inhibitory effect of ML365 on ATP-induced activation of NLRP3 inflammasome. A is the result of detecting the TWIK2 channel reduction by real-time fluorescent quantitative PCR; b is a result graph of ML365(5 mu M) on activation of NLRP3 inflammatory bodies under LPS and ATP induction in BMDM cells before and after TWIK2 knockdown by an immunoblotting method (WB); c is the result chart of enzyme-linked immunosorbent assay (ELISA) generated by IL-1 beta corresponding to B.

Detailed Description

ML365, chemical name, 2-methoxy-N- [3- [ (3-methylbenzoyl) amino group]Phenyl radical]A benzamide. PubChem was first introduced in 2010 for the synthetic compounds. The compound has the molecular formula of C₂₂H₂₀N₂O₃Molecular weight is 360.4, CAS accession number: 947914-18-3, having the formula (I):

ML335, CAS:1069498-96-9, 1- [ [4- (acetylamino) phenyl ] methyl ] -4- (2-phenylethyl) -4-piperidinecarboxylic acid ethyl ester, the chemical structural formula is shown in formula (II):

the invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto. The experimental procedures in the following examples are conventional ones unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 construction of the THIK1 plasmid

Human THIK1(Gene ID:56659), which was cloned into pcDNA3.1(+) vector after HindIII and ApaI restriction enzyme sites were introduced at N-and C-termini, respectively, by searching for the Open Read Frame (ORF) sequence using EditSeq software based on the human THIK1 Gene sequence published by Genebank. The map of the THIK1 plasmid vector is shown in FIG. 1.

Example 2 experiment of electrophysiological finding that ML365 potently inhibits the TWIK2 channel

Firstly, experimental materials:

1. plasmid: the plasmids used were human TWIK1-WT, TWIK1-I293/294A/K274E and human TWIK2-WT, TWIK2-Y308A/I289A/L290A, wild-type channels of human TWIK1(Gene ID:3775) and TWIK2(Gene ID:9424), respectively, and functional channels with corresponding mutation sites (capable of exhibiting electrophysiological activity in heterologous expression systems), as given by Prof.

2. Control drugs: K2P channel modulators include dcpiib (Apexbio B6780, 98%) supplied by aexbio, quinine supplied by alatine bio, fluoxetine (dow T0450, 99.3%), supplied by shanghai ceramic, ML335 supplied by MCE, and TKDC supplied by SPECS;

ML 365: supplied by MCE corporation, usa;

COS-7 cells: purchased from atcc (american type culture collection);

0.25% pancreatin: supplied by Thermo fisher corporation, usa.

3. Solution:

(1) K2P channel whole cell patch clamp experimental external liquid: KCl 2.5mM, MgCl₂ 3mM，CaCl₂Dissolving 1mM, HEPES 10mM and NaCl 145mM in double distilled water, adjusting pH to 7.4 with NaOH solution, and storing at 4 ℃ for later use;

(2) K2P channel whole cell patch clamp experiment internal liquid: EGTA5mM, MgCl₂1mM HEPES 10mM KCl 140mM in double distilled water, adjusting pH to 7.4 with KOH solutionAnd storing at 4 ℃ for later use;

(3) preparing antibiotics: 50mg/mL kanamycin (kanamycin) 0.5g, 100mg/mL ampicillin (ampicillin sodium salt) 1g, the corresponding powder was dissolved in 10mL autoclaved double distilled water, filter sterilized, aliquoted, stored at-20 ℃ and, before use, as 1: 1000 into the culture medium;

(4) preparing an LB culture medium: NaCl1g, yeast extract 0.5g, peptone 1g, dissolved in 100mL double distilled water (if prepared with solid culture medium, 1.5% agar is added), high pressure steam sterilized, cooled to about 65 deg.C, added with corresponding antibiotic, and stored at 4 deg.C for use;

(5) preparation of 50 XTAE electrophoresis solution: na (Na)₂EDTA-2H 2O3.72g, Tris-base 24.2g and glacial acetic acid 5.71g, and double distilled water is added into the mixture to be 100mL for standby and diluted by 50 times for use;

(6) WB blocking buffer: 2.5g of skimmed milk powder and 50 mL of TBST, and uniformly mixing by vortex for later use;

(7) WB TBST buffer: TBS powder 1 bag, Tween 201mL, add double distilled water to 2L, spare;

(8) WB transfer buffer: 5.8g of Tris-base, 2.9g of glycine, 0.37g of sodium dodecyl sulfate and 200 mL of methanol, and adding double distilled water to 1L for later use;

(9) electrophoresis buffer solution for WB: 3.3g of Tris-base, 18.8g of glycine and 1g of sodium dodecyl sulfate, and adding double distilled water to 1L for later use;

(10) compound solution: DCIB, fluoxetine, quinine, ML335, TKDC and ML365 are respectively dissolved in dimethyl sulfoxide (DMSO) to prepare 100mM or 20mM mother liquor, and the mother liquor is stored at-20 ℃ for later use. Before use, a proper volume of compound mother liquor is taken to prepare the target concentration.

II, an experimental method:

1. plasmid extraction: putting the required consumables into a super clean bench in advance, and turning on ultraviolet irradiation for 30 min; 5mL of LB liquid medium containing the corresponding antibiotic (50. mu.g/mL kanamycin or 100. mu.g/mL ampicillin) was added to the autoclaved glass tube; picking the monoclone colony after the corresponding plasmid is transformed by using a small gun head which is sterilized by high pressure, directly driving the gun head into a test tube, and wrapping the mouth of the test tube by using aluminum foil paper; placing the test tube into a shaking table at 37 ℃, and culturing for 16-20h at the rotating speed of 250 rpm; transferring the bacteria liquid into a centrifugal tube, centrifuging for 1min at 10000Xg, pouring out the supernatant, and collecting the bacteria liquid precipitate; adding 250 mu L of Solution I/RNase A into the bacterial liquid sediment of the centrifugal tube, and blowing and beating the sediment until the sediment is fully resuspended; adding 250 mu L of Solution II into the centrifugal tube, slightly turning over for a plurality of times, and standing for 2min until the bacterial liquid is clear; adding 350 mu L of Solution III into the centrifugal tube again, and immediately and gently turning for a plurality of times (avoiding forming local precipitates); 13000xg, centrifugating for 10 min; the column was placed in a 2mL collection tube, the supernatant from the column was transferred to the column, 13000Xg, and centrifuged for 1min to remove the waste from the tube. Adding 500 mu L HBC buffer, 13000Xg HBC buffer into the adsorption column, centrifuging for 1min, and completely discharging waste liquid in the collection pipe; adding 700 mu L of Wash buffer into the adsorption column, 13000Xg, and centrifuging for 1 min; the waste liquid in the collecting pipe is cleaned and repeated once; putting the adsorption column back into the collection tube, and centrifuging at 13000Xg for 2min to dry the adsorption column. Putting an adsorption column into a new centrifuge tube, adding 50 μ L deionized water into the center of the adsorption column, standing at room temperature for 2min, 13000Xg, and centrifuging for 1 min; adding the eluate in the centrifugal tube into the adsorption column again, standing at room temperature for 2min, 13000Xg, and centrifuging for 1 min; the eluent in the centrifuge tube is the plasmid obtained by extraction, and the concentration and purity of the plasmid are measured by a spectrophotometer. Storing at-20 deg.C;

2. cell transfection: when the cell fusion degree in the 12-well plate reaches 60-70%, transfection can be carried out, and the K2P channel plasmid with the green fluorescent protein marker is transfected into COS-7 cells by using Lipofectamine 2000; two 1.5mL centrifuge tubes were added with 125uL of Opti-MEM medium. Add 10. mu.L of lipofectamine2000 reagent to one of the tubes and 3000ng of plasmid to be transfected to the other tube, mix by gentle vortex, and let stand at room temperature for 5 min. Adding the diluted plasmid into diluted lipofectamine2000, slightly swirling and mixing uniformly, and standing for 5min at room temperature; sucking the old culture medium of the cells to be transfected, adding new complete culture medium, gently dripping the plasmid-lipid complex into the cell culture medium, returning to 37 deg.C, and containing 5% CO₂Culturing in a cell culture box; after 24h of transfection, the cells were trypsinized and added again to the columnSoaking the polylysine on a cover glass before, and performing a patch clamp experiment after the cells adhere to the wall;

3. whole-cell patch-clamp records K2P channel current: and (3) 24-48 h after transfection, taking the cover glass inoculated with the cells, and selecting the cells emitting green fluorescence under a fluorescence inverted microscope to perform an electrophysiological experiment. After sequential dosing, channel currents were recorded by a hardware Digidata 1550B data acquisition system and multicamp 700B patch clamp amplifier and software pClamp 10. The data acquisition frequency was 20kHz and the digital filtering was 2 kHz. The series resistance compensation is set to 60-80%. The electrode is formed by drawing a borosilicate glass capillary tube through an electrode drawing instrument, and the resistance is 2-5M omega after the electrode is filled with internal liquid. Data is collected.

4. Data analysis and processing:

electrophysiological data were processed using the campfit 10.6 software for this experiment and further analyzed using GraphPad Prism 6 software. Dose-effect curves are given by the hill equation f ═ 1/[1+ (C/EC)₅₀)n]Fitting, wherein f ═ I_compound/I_control，EC₅₀Refers to the concentration of drug required to produce half of the maximal effect, [ C]Is the drug concentration and n is the hill coefficient. At least three independent transfections were performed for each experiment. Differences between groups were compared by paired t-test or Student t-test, and a P value less than 0.05 considered that the two groups of data were statistically different.

Thirdly, analyzing an experimental result:

the results show that ML365 is a potent inhibitor of the TWIK2 channel. The TWIK2 mutant (muttwik 2) which introduced three mutations (I289A/L290A/Y308A) at the C-terminus of wild-type TWIK2(wtTWIK2) increased its expression on the cell membrane, thereby producing a detectable current in transfected cells. The inventors introduced these three mutations into wtTWIK2 in transfected COS-7 cells resulting in an approximately 14-fold increase in current (as shown in the results in figure 2), which made further screening for TWIK2 modulators possible. The inventors subsequently examined the effect of ML365 et al K2P channel modulators on muttik 2 channels. Of the compounds tested, only ML365 showed a strong inhibitory effect on mutWIK2 (as shown by the results in FIG. 3), I/I at a concentration of 10. mu.M₀0.35. + -. 0.02 (as shown in the results of FIG. 4). Followed byThe inventors then examined the effect of compounds such as ML365 on another member of the K2P channel family, either TWIK1 or THIK 1. Similar to TWIK2, the current of mutant TWIK1 (mutpik 1) with three mutation sites (I293A/I294A/K274E) introduced in wild-type TWIK1(wtTWIK1) was significantly increased (as shown in the results of fig. 5). ML365 (10. mu.M) showed no inhibition of the mutWIK1 channel (as shown by the results in FIG. 6), even at higher concentrations (20. mu.M) (as shown by the A results in FIG. 7). Likewise, the inventors also tested the effect of ML365, et al K2P channel modulators on THIK 1. As shown by the results in fig. 8, ML365(10 μ M) had little inhibitory effect on the THIK1 channel, even at higher concentrations (20 μ M) (as shown by B in fig. 7). These results indicate that ML365 is a selective inhibitor of the TWIK2 channel.

The results show the specificity, the strong effect and the selectivity of ML 365. Therefore, the compound can provide a strong and effective tool compound for the future research of K2P channels, particularly for the TWIK subfamily, and is expected to provide a structural framework for a lead compound of a TWIK2 channel inhibitor in the future.

Example 3 in vitro study of ML365 inhibition of NLRP3 inflammatory body inhibition

Firstly, experimental materials:

ML 365: supplied by MCE corporation, usa;

recombinant mouse M-CSF: supplied by MCE corporation, usa;

DMEM medium: supplied by Thermo fisher corporation, usa;

fetal bovine serum FBS: supplied by Thermo fisher corporation, usa;

phosphate buffered saline PBS: supplied by Thermo fisher corporation, usa;

nigericin: supplied by InvivoGen of France

ATP: supplied by Sigma company, usa;

dimethyl sulfoxide (DMSO): supplied by Sigma company, usa;

LPS: supplied by Sigma company, usa;

OPTI-MEM: supplied by Thermo fisher corporation, usa;

chloroform (chloroform): from Shanghai Aladdin Biochemical;

methanol: supplied by Shanghai Merling, Inc.;

MCC 950: supplied by MCE corporation, usa;

RIPA lysate: supplied by Shanghai Biyuntian corporation;

protease inhibitor cocktail: by Jiangsu Kaiky corporation;

phosphatase inhibitor cocktail: by Jiangsu Kaiky corporation;

western primary anti-dilution: from Shanghai Biyuntian corporation;

BCA protein quantification kit: supplied by Thermo fisher corporation, usa;

PAGE gel Rapid preparation kit: supplied by Shanghai Yazyme Inc.;

PVDF film: supplied by Roche, switzerland;

sodium dodecyl sulfate SDS: supplied by Shanghai Merling, Inc.;

glycine: supplied by Shanghai Merline corporation;

TBS buffer powder: supplied by warrior, inc;

tween 20: supplied by Sigma company, usa;

and (3) skim milk powder: supplied by Shanghai assist in san Francisco;

ECL luminescent liquid: supplied by jedbis, guangzhou;

protein Marker: supplied by Thermo fisher corporation, usa;

anti-IL-1 β antibody: supplied by R & D Systems, usa;

anti-caspase-1 antibodies: supplied by AdipoGen life science, USA;

anti-beta-actin antibodies: supplied by shanghai ericsson;

anti-TWIK 2 antibody: supplied by Santa Cruz, USA;

mouse IL-1. beta. uncoated ELISA kit: supplied by Thermo fisher corporation, usa;

II, solution: the same as in example 2.

Thirdly, an experimental method:

extraction of BMDM cells: ultraviolet sterilizing a superclean bench for 30min in advance, preparing primary instruments and 2 middle dishes, and respectively filling alcohol and PBS; taking a C57BL/6 mouse aged 6-8 weeks, bleeding carotid artery, killing the mouse, soaking the mouse in 75% ethanol for disinfection, and separating thighbone and shinbone of hind limb under aseptic condition; after muscles and connected tissues are removed by a scalpel, soaking thighbones and shinbones in alcohol, transferring the thighbones and the shinbones into PBS (phosphate buffered saline) for rinsing, and transferring the thighbones and the shinbones into DMEM (DMEM); cutting off epiphysis at two ends by using a scalpel, exposing a marrow cavity, sucking DMEM (DMEM) by using a 1mL syringe to wash the marrow until the inner surface of the marrow cavity is whitish, and blowing and sucking culture solution for multiple times by using a pipette to generate single cell suspension; filtering the cell suspension by using a 70 mu M filter screen to a new culture dish, transferring the filtered cell suspension to a centrifuge tube, centrifuging at 1500rpm for 10min, and removing the supernatant; resuspending the cell sediment with complete culture medium (DMEM + 10% FBS + 1% double antibody +20ng/ml M-CSF) of BMDM cells, fully blowing uniformly, inoculating the cells into a 6-hole plate according to proper density, and placing the 6-hole plate into a cell culture box for culture; changing the solution every 2-3 days, and obtaining mature BMDM cells after 7 days;

activation and compound modulation of NLRP3 inflammasome: after BMDM cells are mature, discarding the old culture medium, adding OPTI-MEM culture medium, adding LPS (5 mu g/mL) for pre-excitation, and treating overnight; treatment was performed for 2h with DMSO or ML365 at the corresponding concentration. An agonist of NLRP3 was added to activate the inflammasome (ATP,5mM,1 h; niger, 10. mu.M, 30 min); collecting the supernatant and the cells, and respectively extracting proteins for immunoblotting analysis;

3. western blot experiments:

(1) preparation of supernatant protein samples by precipitation: centrifuging the collected cell culture supernatant at 13000rpm for 10min, and collecting the supernatant in a centrifugal tube in a new centrifugal tube; mixing 500 μ L supernatant with 500 μ L methanol and 150 μ L chloroform, vortexing, centrifuging at 13000rpm for 10min, and removing the upper organic solvent layer without contacting the middle protein layer; adding 800 mu L of methanol, whirling and uniformly mixing, centrifuging at 13000rpm for 10min, pouring out supernatant liquid, and reversely buckling the orifice of a centrifugal tube on absorbent paper to suck the residual liquid; standing at room temperature for about 10min, drying protein precipitate, adding 1 × loading buffer (diluted with RIPA), resuspending, decocting at 100 deg.C for 5min, and storing at-20 deg.C;

(2) preparation of cell protein sample: removing the culture solution by suction, washing the cells for 2 times by using ice PBS, completely sucking away the PBS, adding 60 mu L of RIPA lysate containing a phosphatase inhibitor and a protease inhibitor into each hole, fully scraping the cells by using a cell scraper after cracking the cells on ice for 30min, collecting the cells into a 1.5ml centrifuge tube, oscillating the cells for 1h at 4 ℃, centrifuging the cells for 15min at 4 ℃ and 13000rpm, and transferring the supernatant into another new centrifuge tube;

(3) protein quantification: preparing working solution according to the number of samples (solution A: solution B is 50:1), sucking 6 mul of protein sample, diluting 5 times by PBS, adding 200 mul of working solution and 25 mul of diluted protein sample into each hole of a 96-hole plate, mixing uniformly, blowing away bubbles by a blowing cylinder, incubating for 30min at 37 ℃, measuring absorbance at 570nm by an enzyme labeling instrument, calculating the concentration of the protein sample, calculating the sample amount required by normalizing to the same protein amount, RIPA lysate and 5 × loading buffer, adding into a centrifugal tube, mixing uniformly, boiling for 5min at 100 ℃, and preserving for later use at-20 ℃;

(4) preparing glue: preparing polyacrylamide gel by using a PAGE gel rapid preparation kit of Shanghai Yazyme company;

(5) loading: firstly, fixing the prepared polyacrylamide gel in an electrophoresis module, then placing the electrophoresis module into an electrophoresis tank according to the indication of an electrode, filling electrophoresis buffer solution into the electrophoresis tank, and vertically pulling out a comb upwards. Adding a protein Marker into lanes on two sides, and then sequentially adding 10 mu L of corresponding protein samples into the sample adding holes;

(6) electrophoresis: performing electrophoresis at a constant voltage of 80V for 20min, and adjusting the voltage to 120V for about 60 min;

(7) electric film transfer: cutting PVDF membrane with appropriate size, and soaking in methanol for 1min for activation. Taking out the gel after the electrophoresis is finished, carefully peeling one glass plate, removing the concentrated glue on the upper layer, and mounting the separation glue on the lower layer and the other glass plate in the following sequence: the filter paper comprises a negative pole clamping plate, a spongy cushion, three layers of filter paper, separation glue, a PVDF membrane, three layers of filter paper, a spongy cushion and a positive pole clamping plate. After the device is installed, the electric film-transferring device is placed into an electrophoresis tank according to the indication of the electrode, the electrophoresis tank is filled with film-transferring buffer solution, an ice bag with proper size is placed into the electrophoresis tank, and a cover is covered. The electrophoresis tank is filled with ice blocks to form a low-temperature environment to prevent the temperature from being too high. Setting the voltage to be 100V, and carrying out film rotation for 90 min;

(8) and (3) sealing: after the membrane is transferred, taking out the PVDF membrane, soaking the PVDF membrane in a closed buffer solution, and slowly oscillating the PVDF membrane on a shaking table at room temperature for 60 min;

(9) dressing, breeding and cleaning primary antibody: preparing an anti-dilution solution in advance according to the required antibody concentration. Taking out the PVDF membrane, soaking in TBST, rinsing for 2-3 times to wash the confining liquid, tightly spreading the PVDF membrane on the inner wall of a centrifugal tube containing primary anti-dilution liquid, and placing at 4 ℃ for rolling and laying overnight; soaking the PVDF membrane in TBST, and cleaning for 5min each time on a shaking table in a shaking way for 6 times;

(10) dressing and washing secondary antibody: preparing a second antibody diluent according to the required antibody concentration in advance, tightly paving a PVDF membrane on the inner wall of a centrifugal tube containing the second antibody diluent, and rolling and breeding for 1h at room temperature; soaking the PVDF membrane in TBST, and cleaning for 5min each time on a shaking table in a shaking way for 6 times;

(11) chemical development: preparing ECL chemiluminescence liquid, absorbing excess water from a PVDF membrane by using absorbent paper, spreading the PVDF membrane on a black plastic plate of a multifunctional imaging analyzer in a flatwise manner, uniformly dropwise adding the ECL chemiluminescence liquid on the PVDF membrane surface, removing bubbles, and then putting the PVDF membrane into the multifunctional imaging analyzer for scanning to obtain a picture.

4. Enzyme-linked immunosorbent assay (ELISA):

(1) determining the number of the holes of the enzyme label plate required by the detection, and adding 1 hole as a TMB blank developing hole, wherein the total number is the number of the samples plus 9; when duplicate detection is performed, 2. Diluting PBS (10X) by 10 times with distilled water to prepare Coating Buffer (1X), then diluting Capture Antibody (1:250) in the 1X Coating Buffer for Coating ELISA plate holes, adding 100 mu L of each hole, sealing the plate, and incubating at 4 ℃ overnight;

(2) pouring off liquid in the holes, adding 250 mu L of Wash Buffer into each hole for washing for 3 times, soaking for 1min each time, and beating the plate reversely and opposite to the absorbent paper for a plurality of times to remove residual liquid;

(3) diluting the Dilute Concentrate (5X) with distilled water to ELISA/ELISPOT Concentrate (1X), adding 200. mu.L of ELISA/ELISPOT Concentrate (1X) per well, and incubating at room temperature for 1 h;

(4) preparing a standard (1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25 pg/mL, 15.625pg/mL, 7.8125pg/mL, diluted with distilled water);

(5) the liquid in the wells was decanted, washed 1 time with Wash Buffer, and 100. mu.L of the corresponding cell culture supernatant sample or standard was added to each well. Adding a sealing plate membrane on the ELISA plate, and incubating for 2h at room temperature;

(6) detection Antibody (250X), Streptavidin-HRP (250X) were diluted to working solution with ELISA/ELISPOT Diluent (1X), respectively;

(7) the plate was washed 5 times according to the procedure in (2), and the prepared Detection antibodies were added in the order of 100uL per well (except for TMB blank chromogenic wells). Adding a sealing plate membrane on the ELISA plate, and incubating for 1h at room temperature;

(8) the plate was washed 5 times according to the procedure in (2), and prepared Streptavidin-HRP was added in 100uL per well in sequence. Adding a sealing plate film on the ELISA plate, and reacting for 30min at 37 ℃;

(9) washing the plate for 5 times according to the step (2), adding 100uL of 1X TMB color development solution into each hole, and incubating for 15min at room temperature;

(10) 100uL Stop Solution per well, blue turning yellow immediately;

(11) measuring OD value at 450nm wavelength by using an enzyme-labeling instrument;

(12) and drawing a standard curve and calculating the concentration of the sample.

Fourthly, analyzing an experimental result:

the results show that ML365 blocks ATP-induced activation of NLRP3 inflammasome. As shown in the results of FIG. 9, ML365 dose-dependently inhibited ATP-induced activation of the NLRP3 inflammasome in BMDMs, and immunoblot (WB) results indicated a decrease in mature IL-1 β and caspase-1 expression levels in the cell supernatant (shown as A in FIG. 9); enzyme-linked immunosorbent (ELISA) results showed that the release of IL-1. beta. was significantly reduced (as shown in B in FIG. 9). In the same supernatant, ML365 had little effect on TNF-. alpha.expression (as shown in FIG. 9, panel C). In contrast, ML365 (even 10 and 20 μ M) had no effect on Nigericin induced activation of NLRP3 inflammasome (as shown in D, E, F in figure 9). The effect of ML365 on ATP-or Nigericin-induced activation of the NLRP3 inflammasome was also demonstrated in human THP-1 macrophages (as shown by the results in FIG. 10). The inventors next investigated the specificity of ML365 for ATP-induced activation of NLRP3 inflammasome. The inventors examined the effect of ML365 on non-classical inflammatory bodies. The data show that ML365 does not inhibit activation of non-classical inflammasome (as shown by G in figure 9). Furthermore, ML365 did not inhibit AIM2 or NLRC4 inflammasome-induced IL-1 β production (as shown in H, I in fig. 9). These results indicate that ML365 specifically inhibits ATP-induced NLRP3 inflammasome, consistent with the important role of the TWIK2 channel in NLRP3 inflammasome.

Example 4 molecular mechanism study of ML365 inhibiting NLRP3 inflammasome

Firstly, experimental materials:

lipofectamine RNAiMAX supplied by Thermo Fisher corporation of America

And others: same as example 3

II, an experimental method:

extraction of BMDM cells: same as example 3

2. Transfection of siRNA BMDM cells were plated in 6-well plates (2.5X 10 per well)⁵Individual cells) were then transfected with 50nMsiRNA with 7.5 llipifectamine RNAiMAX, 48 hours later the expression level of the TWIK2 channel was measured by real-time fluorescent quantitative PCR (RT-qPCR) or inflammasome stimulation was performed and the effect of ML365 on NLRP3 inflammasome activation after TWIK2 knockdown was examined.

Activation and compound modulation of NLRP3 inflammasome: same as example 3

Thirdly, analyzing an experimental result:

the results indicate that the absence of TWIK2 blocks the inhibition of ATP-induced activation of NLRP3 inflammasome by ML 365. To test whether ML365 inhibited the activation of NLRP3 inflammasome by blocking the TWIK2 channel, the inventors knocked down the TWIK2 channel using small interfering rna (sirna) (a in fig. 11). Knock-down of TWIK2 significantly reduced IL-1 β production in ATP-activated BMDMs (B, C in FIG. 11). Furthermore, the inventors found that by knocking down TWIK2, the inhibitory effect of ML365 on ATP-induced activation of NLRP3 inflammasome was greatly reduced (B, C in fig. 11). This result further confirms that ML365 blocks ATP-induced activation of NLRP3 inflammasome by inhibiting the TWIK2 channel.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The use of a compound for the manufacture of a medicament for the prevention and/or treatment of NLRP3 inflammasome-related diseases, said compound being a compound of formula (I), a pharmaceutically acceptable salt or polymorph thereof;

2. the use of claim 1, wherein the NLRP3 inflammasome-related diseases include peritonitis, sepsis lung injury, type II diabetes or high fat food-induced obesity, parkinson's syndrome, alzheimer's disease, multiple sclerosis, enteritis, hepatitis, silicosis, asbestosis, silicosis, behcet's disease, and rheumatoid arthritis.

3. Use of a compound for the manufacture of an inhibitor of inflammasome, said compound being of formula (I), a pharmaceutically acceptable salt or polymorph thereof;

4. the use of claim 3, wherein the inflammasome comprises an NLRP3 inflammasome.

5. The use of a compound in the preparation of a medicament for treating NLRP3 inflammatory-corpuscle abnormal activation diseases, wherein the compound is a compound shown as a formula (I), a pharmaceutically acceptable salt or a polymorphic substance thereof;

6. use of a compound for the preparation of a potassium channel inhibitor, said compound being a compound of formula (I), a pharmaceutically acceptable salt or polymorph thereof;

7. the use according to claim 6, wherein said potassium channel comprises the double-pore potassium channel TWIK2 channel.

8. A medicament for treating NLRP3 inflammasome-related diseases, which comprises a compound of formula (I), a pharmaceutically acceptable salt or a polymorph thereof;

9. the medicament of claim 8, further comprising a pharmaceutically acceptable excipient.

10. The medicament of claim 8 or 9, wherein the NLRP3 inflammasome-related diseases include peritonitis, sepsis lung injury, type II diabetes or high fat food-induced obesity, parkinson's syndrome, alzheimer's disease, multiple sclerosis, enteritis, hepatitis, silicosis, asbestosis, silicosis, behcet's disease and rheumatoid arthritis.