KR102074215B1 - Binding sites of β-carotene to NLRP3 PYD domain and screening method for therapeutic agent of gout using same - Google Patents

Abstract

The present invention relates to a NLRP3 PYD domain binding site of β-carotene and a method for screening a gout therapeutic agent using the same, wherein the NLRP3 PYD domain to which NLRP3 inflammasome activity inhibitor β-carotene directly binds Specific sites are directed to methods of screening gout agents or gout inflammatory diseases. The screened compounds can be usefully used as a gout or gout inflammatory therapeutic.

Description

Binding sites of β-carotene to NLRP3 PYD domain and screening method for therapeutic agent of gout using same}

The present invention relates to a NLRP3 PYD domain binding site of β-carotene and a screening method of a gout therapeutic agent using the same, wherein the NLRP3 PYD domain of β-carotene, an NLRP3 inflammasome activity inhibitor, directly binds to Specific sites are directed to methods of screening gout agents or gout inflammatory diseases.

Gouty arthritis is caused by the deposition of uric acid crystals in the joints and surrounding tissues. This disease occurrence is directly related to serum uric acid levels, and agents that reduce the serum level of uric acid are used to treat patients with gouty arthritis. These agents work by inhibiting the synthesis of uric acid, such as xanthine oxidase inhibitor allopurinol, or by increasing the release of uric acid, such as uric acid urine.

Recently, NLRP3 [NOD-like receptor family, pyrin domain containing 3] has been identified as a receptor that causes inflammatory signals in response to uric acid crystals. NLRP3 is a member of the Nod-like receptor family (NLR) and detects endogenous hazard signals such as microbial invasion and uric acid crystals. In the presence of this signal, NLRP3 forms inflammasomes with the adapter proteins ASC and pro-casease-1. Pro-Casephase-1 is cleaved and separated into Kephase-1 to become an active form.Casephase-1 cleaves the pro-IL-1β precursor to activate IL-1β and is secreted into the extracellular environment.

Recent structural determination studies have identified potential mechanisms associated with NLRP3 activation, suggesting that NLRP3 PYD may be an appropriate molecular target for the development of selective small molecule inhibitors of NLRP3 inflamasomes (FIG. 1A). . However, to date, no small molecules that directly bind to NLRP3 or specifically regulate NLRP3 inflammasome activity have been reported.

Although components of NLRP3 inflamasomes (e.g. NLRP3, NEK7, ASC, and caspase-1) and related signaling mechanisms [e.g. Priming, mitochondrial damage, potassium efflux and chloride efflux] can be targeted to inhibit NLRP3 inflammasome activation, but only direct targeting of NLRP3 can inhibit NLRP3 inflammasomes. Can be selectively suppressed. Several NLRP3 inflammasome inhibitors have been developed, such as sulforaphane, caffeic acid phenetyl ester, β-hydroxybutyrate, CY-09 and MCC950, but no compounds that directly and specifically target NLRP3 have been found. For the prevention or treatment of the disease, it is still unclear whether NLRP3 can be selectively targeted.

Korea Patent Registration No. 10-1731303 (Registered April 24, 2017)

It is an object of the present invention to react NLRP3 pyrine domain (PYD) with a candidate compound; And selecting candidate compounds that directly bind to at least one residue selected from the group consisting of Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 of the NLRP3 pyrine domain (PYD) in the reacted reactant. To provide a method for screening a gout treatment or a gout inflammatory disease comprising the step.

In order to achieve the above object, the present invention comprises the steps of reacting the NLRP3 pyrine domain (PYD) and the candidate compound; And selecting candidate compounds that directly bind to at least one residue selected from the group consisting of Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 of the NLRP3 pyrine domain (PYD) in the reacted reactant. It provides a method for screening a gout treatment comprising the step.

In addition, the present invention comprises the steps of reacting the NLRP3 pyrine domain (PYD) and the candidate compound; And selecting candidate compounds that directly bind to at least one residue selected from the group consisting of Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 of the NLRP3 pyrine domain (PYD) in the reacted reactant. It provides a method of screening for a gout inflammatory disease treatment comprising the step.

The present invention relates to a NLRP3 PYD domain binding site of β-carotene and a method for screening a gout therapeutic agent using the same, wherein the NLRP3 PYD domain to which NLRP3 inflammasome activity inhibitor β-carotene directly binds Specific sites are directed to methods of screening gout agents or gout inflammatory diseases. The screened compounds can be usefully used as a gout or gout inflammatory therapeutic.

1 shows a virtual screening method for the PYD domain of NLRP3.
FIG. 2 shows that β-carotene inhibits the activity of NLRP3 inflammasomes in primary macrophages, but does not inhibit the activity of AIM2 inflamasomes. A and CE, cell culture supernatants and cell lysates were immunoblotted against pro-caspase-1, caspase-1 (p10), pro-IL-1β and IL-1β. B, IL-1β secreted in the cell culture supernatant is analyzed by ELISA. The figures are expressed as means ± SEM (n = 3). #, Significant difference compared to control alone (p <0.05). *, MSU, ATP, nigericin and poly dA: dT alone showed significant differences (p <0.05).
3 shows that inhibition of inflammasome activation by β-carotene is dependent on NLRP3. The figures are expressed as means ± SEM (n = 3). (A) NLRP3 + ASC + caspase-1, (b) ASC + caspase-1 and (c) Caspase-1 alone.
4 shows that β-carotene inhibits the interaction of NLRP3 and ASC. (A) LPS-primed mouse primary macrophages were treated with β-carotene for 1 hour and then stimulated with monosodium uric acid (MSU) crystals (500 μg / ml) for 6 hours. Cell lysates were immunoprecipitated with anti-NLRP3 antibody and immunoblotting was performed as indicated. (B) 293T cells were transfected with NLRP3 or ASC expressing plasmids and treated with β-carotene for 16 hours. Cell lysates were immunoprecipitated with anti-NLRP3 antibody and immunoblotting was performed as indicated. (C) 293T cells were transfected with NLRP3 or ASC expressing plasmids and treated with β-carotene. Cells were fixed and infiltrated and stained for NLRP3 (red) and ASC (green). Nuclei were stained with 4 ', 6-diamidino-2-phenylindole (DAPI; blue). Representative results of at least two independent experiments are shown.
5 shows the results that β-carotene binds to PYD of NLRP3. (A) Sensograms of β-carotene that bind to NLRP3 (PYD) recombinant protein in the presence of the command activator (0.005% Tween-20) were obtained by surface plasmon resonance (SPR) analysis. Different concentrations of β-carotene were aligned at the start of the infusion and represented by overlay plots. (B) Sensograms of β-carotene binding to recombinant ASC protein in the presence of the commander (0.005% Tween-20) were obtained by surface plasmon resonance (SPR) analysis. Different concentrations of β-carotene were aligned at the start of the infusion and represented by overlay plots. The table shows the kinetic parameters for binding between β-carotene and recombinant protein, calculated using a simple 1: 1 interaction model. The maximum expected binding level (Rmax) was calculated using the Biocore T200 evaluation software.
Figure 6 shows the result that β-carotene binds to the pyrine domain (PYD) of NLRP3. Β-carotene senso that binds to the recombinant protein of (A) PYD of wild type NLRP3, (B) PYD of A69K mutated NLRP3 and (C) PYD of V72K mutated NLRP3 in the presence of a commander (0.005% Tween-20) The grams (Sensograms) were obtained by surface plasmon resonance (SPR) analysis. Different concentrations of β-carotene were aligned at the start of the infusion and represented by overlay plots. The table shows the kinetic parameters for binding between β-carotene and recombinant protein, calculated using a simple 1: 1 interaction model. The maximum expected binding level (Rmax) was calculated using the Biocore T200 evaluation software.
7 shows that Ala 69, Val 72, Trp 73, Tyr 84 and Glu 91 are essential for the binding of β-carotene and PYD of NLRP3. (A) Luciferase activity of the cell lysates was analyzed. (B) IL-1β secreted in the cell culture supernatant was measured by ELISA. The figures are expressed as means ± SEM (n = 3). (C) 293T cells were transiently transfected with NLRP3, mutant NLRP3, ASC or capsase-1 expressing plasmids. After 8 hours, cells were treated with β-carotene (20 μM) for 16 hours. Cell culture supernatants and cell lysates were then immunoblotted with pro-caspase-1, caspase-1 (p10), pro-IL-1β and IL-1β.
8 shows that β-carotene inhibits ASC particle formation depending on Ala69, Val72, Trp73, Tyr84 and Glu91 in 293T cells. Cells were fixed and infiltrated and stained for NLRP3 (red) and ASC (green). Nuclei were stained with 4 ', 6-diamidino-2-phenylindole (DAPI; blue). Representative results of at least two independent experiments are shown.
FIG. 9 shows that in mouse airbag inflammation model, oral administration of β-carotene attenuates uric acid crystal-induced activation of NLRP3 inflammasomes. The supernatant derived from air pocket exudate was subjected to immunoblotting for (A) caspase-1 (p10) and IL-1β, (B) assay for caspase-1 enzyme activity and (C to E) IL-1β, IL-18 and TNF- This is the result of ELISAs analysis for α. (F) Airbag tissue was fixed for histological testing and H & E staining was used. Purple areas indicate invasive neutrophils. (G) Myeloperoxidase (MPO) activity reflecting neutrophil recruitment levels in air bag exudates. Values in the bar graph are represented by means ± SEM (n = 6 mice). #, Significant difference compared to control alone (p <0.05). *, Significant difference compared to MSU alone (p <0.05). Veh, control. β-car, β-carotene.
Figure 10 shows that in mouse paws, oral administration of β-carotene alleviates uric acid crystal-induced gout. (A) Shows foot thickness over time. (B) Representative photographs of hind paws and H & E staining results are shown. (C) Neutrophils invaded in hind paw tissue appeared as purple areas in H & E staining (400X). (D) Myeloperoxidase (MPO) activity of the supernatant derived from the foot tissue lysate was analyzed. (E) Foot tissue lysates were analyzed by immunoblotting of pro-caspase-1, caspase-1 (p10), pro-IL-1β and IL-1β. (F) Caspase-1 enzyme activity analysis result. (G to I) ELISAs analysis results for IL-1β, IL-18, TNF-α. Values in the line and bar graphs are represented by means ± SEM (n = 6 mice). #, Significant difference compared to control alone (p <0.05). *, Significant difference compared to MSU alone (p <0.05). Veh, control. β-car, β-carotene.

Hereinafter, the present invention will be described in more detail.

We identified β-carotene as an NLRP3 inflammasome inhibitor. β-carotene binds directly to the NLRP3 PYD domain and inhibited NLRP3 inflammasome activation. In vivo, β-carotene has been shown to directly target NLRP3 to inhibit NLRP3 inflammasome activation, confirming the remarkable therapeutic effect in NLRP-induced acute gouty arthritis mouse model, and completing the present invention. It was.

The present invention comprises the steps of reacting the NLRP3 pyrine domain (PYD) and the candidate compound; And selecting candidate compounds that directly bind to at least one residue selected from the group consisting of Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 of the NLRP3 pyrine domain (PYD) in the reacted reactant. It provides a method for screening a gout treatment comprising the step.

Specifically, the selected candidate compounds may inhibit NLRP3 inflammasome activation, and more specifically, the inhibition of NLRP3 inflammasome activation may be to block binding of NLRP3 PYD and ASC PYD, but is not limited thereto. It doesn't happen.

In detail, the selected candidate compound may be β-carotene, but is not limited thereto.

In addition, the present invention comprises the steps of reacting the NLRP3 pyrine domain (PYD) and the candidate compound; And selecting candidate compounds that directly bind to at least one residue selected from the group consisting of Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 of the NLRP3 pyrine domain (PYD) in the reacted reactant. It provides a method of screening for a gout inflammatory disease treatment comprising the step. Preferably, the gout inflammatory disease may be gouty arthritis, but is not limited thereto.

Specifically, the selected candidate compounds may inhibit NLRP3 inflammasome activation, and more specifically, the inhibition of NLRP3 inflammasome activation may be to block binding of NLRP3 PYD and ASC PYD, but is not limited thereto. It doesn't happen.

In detail, the selected candidate compound may be β-carotene, but is not limited thereto.

The amino acid sequence of the NLRP3 pyrine domain (PYD) described in the present invention may be represented by SEQ ID NO: 1, but is not limited thereto.

On the other hand, the ASC PYD domain plays an important role in inflamasomes, which are known to directly bind to the PYD domain of NLRP3 when inflamasomes are formed. ASC (apoptosis-associated speck-like protein) plays an important role in inflamasome adapters. As a protein, it is composed of two protein domains consisting of a PYRIN-PAAD-DAPIN (PYD) domain at the N-terminus and a caspase-recruitment domain (CARD) at the C-terminus.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

1. Select Database

We used a commercially available ZINC natural compounds database (http://zinc.docking.org/) for virtual screening, which includes more than 6 million commercially available compounds. Protocols include deduplication, salt stripping, mixture splitting, functional group normalization and charge neutralization. The ionizable compounds were then converted to the most appropriate charge form at pH 7.4, and other tautomers were generated using the LigPrep module in Schrodinger. For chiral compounds with unknown chiral centers, all structures in which the chirality of each chiral center was combined were produced using the LigPrep module in Schrodinger.

2. Target Manufacturing and Grid Generation

The crystal structure of the human NLRP3 protein pyrin domain (PYD) was retrieved from RCSB PROTEIN DATA BANK (RCSB PDB, http://www.rcsb.org, PDB code: 3QF2). The protein structure was applied to the docking calculation and performed with the Glide module in the Schrodinger molecular simulation package. The Protein Preparation Wizard using the OPLS force field was used, and the structure was confirmed by suppressing minimization to mitigate steric collisions. Minimization was terminated when the root-mean square deviation (RMSD) reached the highest value or 0.3 dB. The cocrystal ligand was removed and the final structure was used as the receptor mode of the present invention. During grid generation and ligand docking, default Glide settings were used. Docking calculations of all molecules and related ligands (beta-carotene) were performed via the Glide program.

3. Virtual Screening and Visual Inspection

Using Glide SP mode, all structures were docked and scored. Of the top-ranked molecules, undesirable compound groups and substructures, such as toxic or overly degraded molecules, were filtered through visual inspection.

4. Protein Expression and Purification

The expression and purification methods used in the present invention were performed as previously reported (Bae and Park JBC 2011). Specifically, human NLRP3 PYD (amino acid 3-110) in a self-made pOKD vector was expressed by induction at 20 ° C. overnight in E. coli BL21 (DE3). Target proteins were purified by nickel affinity and size-exclusion chromatography. Superdex 200 gel filtration column 10/30 (GE healthcare) pre-equilibrated with 20 mM Sodium Citrate and 150 mM NaCl solution at pH 5.0 was used for size-exclusion chromatography. About 18 ml of eluted NLRP3 PYD was concentrated to 4-5 mg / ml and used for crystallization.

5. Crystallization, chemical loading and data collection

Previously introduced crystallization conditions were used equally to obtain NLRP3 PYD crystals. 1 μl protein solution (4-5 mg ml- ¹ protein in 20 mM sodium citrate, pH 5.0 and 150 mM NaCl) and 1 μl stock solution (0.2 M ammonium sulfate, 0.4 M stock solution per 0.4 ml stock solution, pH 4.6, 30 The mixture containing% Polyethylene glycol monomethyl ether 2.000) was equilibrated to allow initial crystals to grow on the plate. After the optimization step, the best crystals were produced under the conditions of 0.24 M ammonium sulfate, 0.1 M sodium acetate trihydrate pH 4.7, 25% polyethylene glycol monomethyl ether 2.000. Crystals grew to a maximum size of 0.1 × 0.1 × 0.5 mm within 4 days. Prior to freezing with liquid nitrogen, the crystals were immersed in 5 mM β-carotene-containing buffer for 24 hours. Diffraction data was collected at the SB-II (5C) beamline at Pohang Accelerator Laboratory (PAL) (South Korea). Data processing and scaling was performed using HKL2000.

6. Determination and Analysis of Structure

The complex structure was determined by molecular replacement phasing method using Phaser. The previously revealed NLRP3 PYD structure (PDB code: 3QF2) was used as the search model. The structure was completed by iterative manual building and refinement in Coot and PHENIX Refine. The quality of the model was confirmed using PROCHECK. Molecular structure images were generated using the PyMOL Molecular Graphics System.

7. Animal and Cell Culture

Mice (C57BL / 6) were purchased from Orient Bio (Seoul, South Korea). Mice were bred under sterile conditions in a room where temperature (23 ± 3 ° C.) and relative humidity (40-60%) were controlled. Mice were acclimated in animal facilities for at least a week before the experiment. Animal breeding and testing protocols were performed according to the guidelines of the Catholic University Institutional Animal Care and Use Committee (IACUC) (permission # 2014-015). Bone marrow-derived primary macrophages (BMDMs) were prepared by separating bone marrow from C57BL / 6. Macrophages and 293T cells (human embryonic kidney cells) were Dulbecco's modified with 10% (v / v) fetal bovine serum (Invitrogen, Carlsbad, Calif.), 10,000 units / ml penicillin and 10,000 μg / ml streptomycin (Invitrogen) Incubated in eagle medium (DMEM).

8. Reagents

Esherichia LPS isolated from coli was List Biological Laboratory Inc. Purchased from Campbell, CA and dissolved in endotoxin-free water. β-carotene was purchased from Sigma-Aldrich and stock solution was prepared by DMSO. Antibodies against mouse caspase-1 and ASC were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Antibodies against NLRP3 were purchased from Adipogen (San Diego, Calif.). Antibodies against interleukin-1β (IL-1β) were purchased from R & D Systems (Minneapolis, MN). Caspase-1 activity kits for animal models were purchased from Ab-cam (Cambridge, Mass.).

9. Plasmid

The pcDNA 3.1 nV5-hNLPR3 expression plasmid was provided by You-Me Kim (Pohang University of Science and Technology, Pohang, South Korea). Expression plasmids for ASC and caspase-1 were provided by Giulio Superti-Furga (Austrian Academy of Sciences, Wien, Austria). iGLuc plasmid was provided by Veit Hornung (University of Bonn, Bonn, Germany). Transient transfection and luciferase assays were performed as previously reported.

10. Foot Gout Mouse Model

C57BL / 6 mice (7-8 weeks old) were orally administered 0.5 ml sterile water with β-carotene (30 mg / kg) or control (0.02% DMSO). After 1 hour, MSU crystals (2 mg in 0.1 ml sterile endotoxin-free PBS) or PBS were injected subcutaneously into the plantar surface of the right foot. Foot thickness was observed over time. Twenty four hours after injection of MSU crystals, the foot tissue was treated with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na ₃ VO). ₄ , 10 μg / ml aprotinin, and 10 μg / ml leupeptin), and supernatants were collected for MPO analysis, ELISAs and immunoblot analysis. For histological analysis, the sagittal sections of the plantar were fixed with 10% paraformaldehyde and stained with H & E.

11. Myeloperoxidase ( Myeloperoxidase ; MPO Activity analysis

MPO activity was determined by quantitative determination of neutrophil segregation in air-pouch washes and plantar tissue homogenates using MPO colorimetric activity assay kit (Bio-vision, Milpitas, CA).

12. Inflamasome  Activation Analysis

BMDMs were dispensed on 6-well plates at a density of 2 × 10 ⁶ cells / ml and treated with LPS for 4 hours. To exclude the effect on β-carotene on LPS, β-carotene was added after washing LPS with PBS. Cells were treated with β-carotene for 1 hour and stimulated with NLRP3 inflammasome activators such as MSU crystals, ATP and nigericin dissolved in serum free medium. The cells were lysed in RIPA buffer and immunoblot analysis was performed. The degradation of pro-caspase-1 to caspase-1 (p10) and cleavage of pro-IL-1β to IL-1β in the supernatant were identified as indices of inflammasome activation and measured by immunoblot analysis. The supernatant was precipitated by addition of 1 volume of methanol and 0.25 volume of chloroform, followed by centrifugation at 20,000 g for 10 minutes. The upper layer was removed and 1 volume of methanol was added. The mixture was centrifuged at 20,000 g for 10 minutes to obtain protein precipitate, dried at room temperature and then Laemmli buffer (0.25 M Tris-HCl, pH 6.8, 0.4% glycerol, 10% SDS, 0.2% 2-mercaptoethanol and 0.64% bromophenol blue). The sample was digested on SDS-PAGE and then electrophoresed to polyvinylidene difluoride membrane. The membrane was probed with the primary antibody and related HRP-conjugated secondary antibody. Each band was visualized using an ECL system.

13. Enzyme-linked Immunosorbent Assay (ELISA)

IL-1β and IL-18 levels were measured using the enzyme-linked immunosorbent assay (ELISA) kit (R & D systems, Minneapolis, MN), according to the manufacturer's instructions. Standard curve concentrations were 15.6-1000 pg / ml for IL-1β and 25.6-1000 pg / ml for IL-18. IL-1α, IL-6, KC and MCP-1 levels were measured using the MILLIPLEX MAP Mouse Cytokine / Chemokine Kit (Millipore, Billerica, Mass.). Fluorescence intensity was analyzed by MAGPIX® (Luminex Corporation, Austin, TX).

14. Air pouch  Inflammation model

Local inflammation was induced using the airbag model. 5 ml sterile air was injected subcutaneously under the back of C57BL / 6 mice (7-8 weeks of age) to create an air bag. After 3 days, 5 ml sterile air was further injected into the bag. On day 4, 0.2 ml sterile water containing control (0.02% DMSO) or β-carotene (30 mg / kg) was orally administered and after 1 hour MSU crystals (3 mg in 1 ml sterile endotoxin-free PBS) Injected. Six hours after MSU crystal injection, mice were sacrificed and air pocket fluids were collected in 2 ml PBS containing 5 mM EDTA. The wash was centrifuged at 1200 rpm for 2 minutes and the supernatant was used for cytokine ELISAs. For immunoblot analysis, air bag washes were precipitated to obtain protein precipitates. For histological analysis of the air sacs, sagittal sections of air sacs were fixed with 10% paraformaldehyde and stained with hematoxylin and eosin (H & E).

15. In vitro caspase -1 activity assay

In vitro caspase-1 activity was measured according to the manufacturer's instructions using a fluorometric caspase-1 assay kit with recombinant human caspase-1 (Bio-vision). Fluorescence was recorded using SpectraMaxM5 (Molecular Devices, Sunnyvale, CA) at 400 nm after excitation at 505 nm.

16. Surface Plasmon  Resonance (Surface plasmon  resonance; SPR ) analysis

Reconstitution of NLRP3 The PYD domain protein was prepared as previously reported (J Biol Chem 286, 39528-39536, 2011). CM5 sensor chip (cat. # BR-1005-30, GE Healthcare, Buckinghamshire, UK) was activated with 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide, The PYD protein of NLRP3 covalently immobilized to form a carboxymethylated dextran matrix surface. Binding and dissociation states were observed with Biacore T200 (GE Healthcare) for affinity measurements. β-carotene was dissolved in basic running buffer (PBS containing a final concentration of 0.05% Tween-20 and 5% DMSO) and injected into the flow cell at various concentrations at a flow rate of 5 μl / min at 25 ° C. The sensor chip was washed with basic running buffer. Control experiments were performed with the blank (sensor chip only) and active (sensor chip with antibody only) channels in the same sensor chip. Based on the analytical curves obtained, the control signals reflecting the bulk effect of the buffers were T200 evaluation software ver. Excluded using 2.0 (GE Healthcare). Kinetic parameters and affinity constants of the interactions were calculated using a simple 1: 1 interaction model with T200 software (ver. 2.0).

17. Co-immunoprecipitation (Co- immunoprecipitations )

Anti-NLRP3 antibodies were treated with BMDMs cells or transiently transfected 293T cell lysates at 4 ° C. for 4 hours and then reacted overnight at 4 ° C. with nProtein A Sepharose-beads (GE healthcare). Samples were centrifuged at 15,000 g for 5 minutes and washed three times with RIPA buffer. Proteins co-immunoprecipitated with beads were used for immunoblot analysis.

18. Confocal  Microscopic analysis

Confocal microscopy analysis was performed as previously reported. Specifically, 293T cells were placed on coverslips in 24-well dishes and reacted overnight at 4 ° C. with anti-ASC antibody (Santa Cruz Biotechnology) and anti-NLRP3 antibody (Adipogen). Thereafter, the mixture was reacted with anti-rabbit IgG-FITC and anti-mouse IgG-Alexa 488 antibody (Sigma) at room temperature for 2 hours. Slides were mounted in fluorescence mounting medium (Vector Labs, Burlingame, Calif.). Cells were identified using an LSM710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) using Zen2010 software (Carl Zeiss).

19. Gouty Arthritis Mouse Model

Experiments on the knee joint gouty arthritis mouse model were performed as previously reported. C57BL / 6 mice (7-8 weeks old) were orally administered 0.2 ml sterile water containing β-carotene (30 mg / kg) or control (0.02% DMSO). After 1 hour, MSU crystals (100 μg in 10 μl sterile endotoxin-free PBS) or PBS alone were injected into the right knee joint of the mouse alone. After 24 hours, joint tissue homogenates were prepared and cytokine levels and MPO activity were measured.

20. Statistical Analysis

Data is expressed as means ± SEM. Data comparison between groups was performed through one-way ANOVA followed by Duncan's multiple range test. p <0.05 was determined to be significant. The graph or figure represents at least two or three independent experiments.

< Example  1> Verified by Virtual Screening NLRP3 Β-carotene as a binding molecule

We identified small molecules that bind directly to the NLRP3 inflamasome complex. To find new candidate inhibitors that directly bind to NLRP3, we performed structure-based virtual screening using molecular docking calculations. We used Glide to screen molecules that bind NLRP3 PYD. The NLRP3 PYD structure of the protein data bank (PDB) is crystallized without ligand. About 62,897 natural compounds in silico Were screened in vitro , and eleven commercially available compounds were tested in an in vitro NLRP3 action-induced IL-1β inhibition assay (FIG. 1B). We analyzed binding energy (ΔG) and Glide score scores through affinity and efficiency-based metrics. We chose β-carotene as the best candidate. β-carotene was up-ranked, which showed the best interaction with binding pocket residues in vitro and also showed a very high inhibition of NLRP3 action-induced IL-1β production. Β-carotene was thus selected as one robust candidate group (FIG. 1B).

< Example  2> In primary macrophages derived from bone marrow NLRP3 Inflamasome  Β-carotene blocks

Mouse-derived macrophages lacking the NLRP3 inflamasome component are unable to secrete active IL-1β upon stimulation of uric acid crystals. The present inventors used bone marrow-derived macrophages (BMDMs) to determine whether β-carotene can block NLRP3 inflammasome activation. We primed BMDMs with lipopolysaccharide (LPS). To rule out the possibility that β-carotene will affect the LPS-mediated signal mechanism, we added β-carotene after LPS washing. We pretreated the cells with β-carotene for 1 hour and then stimulated with MSU crystals. β-carotene inhibited MSU crystal-induced cleavage of pro-caspase-1 and pro-IL-1β, indicated by immunoblotting against caspase-1 (p10) and IL-1β in the culture supernatant (FIG. 2A ). In addition, β-carotene consistently inhibited MSU-induced IL-1β secretion in a dose-dependent manner, which was measured by enzyme-linked immunosorbent assay (ELISA) of cell culture supernatants (FIG. 2B).

Next, the inventors confirmed whether β-carotene can inhibit NLRP3 inflamasome activation by other activators such as ATP and nigericin, which act through a mechanism different from uric acid. Β-carotene in BMDMs inhibited ATP-induced cleavage of caspase-1 (p10) and IL-1β in pro-caspase-1 and pro-IL-1β, respectively (FIG. 2C). In addition, β-carotene inhibited ATP-induced IL-1β secretion (FIG. 2D). In addition, β-carotene in BMDMs inhibited nigericin-induced cleavage of pro-caspase-1 and pro-IL-1β (FIG. 2E and FIG. 2F). The results show that β-carotene inhibits NLRP3 inflammasome activation induced by several forms of activators including MSU, ATP and nigericin.

< Example  3> NLRP3 With PYD  Β-carotene that binds directly

We have identified a mechanism by which β-carotene inhibits NLRP3 inflammasome activation. We overexpressed each component with the iGLuc luciferase reporter gene in response to inflammasome activation to reconstruct the NLRP3 inflammasome complex in 293T cells. When we transfected 293T cells with caspase-1 or ASC and caspase-1, β-carotene did not inhibit the expression of iGLuc luciferase (FIGS. 3B and 3C). However, when 293T cells were transfected with NLRP3, ASC and caspase-1, β-carotene inhibited the expression of iGLuc luciferase induced by three components (FIG. 3A). The inhibitory effect of β-carotene was observed only in the presence of NLRP3, which supports that NLRP3, but not ASC or caspase-1, is the target of β-carotene.

Since the PYD of NLRP3 participates in binding to ASC, the inventors confirmed that β-carotene blocked NLRP3-ASC binding and binds to NLRP3 PYD. Co-immunoprecipitation with anti-NLRP3 antibody and immunoblotting with anti-ASC antibody showed that β-carotene in BMDMs inhibited MSU crystal-induced NLRP3 and ASC binding (FIG. 4A). In addition, when NLRP3 and ASC were expressed externally in 293T cells, β-carotene also inhibited this interaction (FIG. 4B). ASCs undergo oligomerization in combination with NLRP3, and we have determined that β-carotene affects ASC oligomerization. Exogenous expression of NLRP3 and ASC in 293T cells showed ASC oligomerization, confirmed by confocal microscopic analysis of particle formation. On the other hand, β-carotene inhibited ASC oligomerization in 293T cells transfected with NLRP3 and ASC (FIG. 4C). The results indicate that β-carotene binds to the PYD of NLRP3, blocking the assembly of the NLRP3 inflammasome complex.

Next, the inventors confirmed that β-carotene can directly bind to NLRP3, thereby blocking the activation of NLRP3 inflamasome. NLRP3 inflammasome assembly is critically dependent on the PYD / PYD interaction between NLRP3 and ASC. The present inventors analyzed the surface plasmon resonance (SPR) of recombinant NLRP3 PYD to confirm the binding of β-carotene and NLRP3 PYD. We found that β-carotene binds directly to PYD of NLRP3 in a dose dependent manner (FIG. 5A). The parameters of interaction kinetics and affinity constants were calculated based on a simple 1: 1 interaction model using the Biacore T200 software (FIG. 5A). We found that β-carotene did not bind to ASC (FIG. 5B). The results support that the target of β-carotene is PYD of NLRP3, not ASC.

< Example  4> NLRP3 With PYD  Ala-69, Val-72, Trp-73, Tyr-84 and Glu-91, essential for the binding of β-carotene

In order to determine whether the binding of β-carotene and NLRP3's PYD is Ala 69 and Val 72 dependent, we applied β- Carotene bound. SPR analysis showed little β-carotene bound to the A69K-V72K-PYD variant of NLRP3 (FIGS. 6B and 6C). Consistent with the above results, the binding of β-carotene and PYD of NLRP3 was dependent on Ala 69 and Val 72 (FIG. 6A). We transfected HEK 293T cells with plasmids encoding pro-caspase-1, ASC and mutant NLRP3. We mutated five amino acid residues that form β-carotene binding pockets in human NLRP3 with lysines or arginines and confirmed the binding of β-carotene and mutant proteins (FIG. 7A). In 293T cells expressing wild type NLRP3, β-carotene inhibited inflammasome activation-induced luminescence. A69K, V72K, W73K, Y84R and E91R mutations were not sensitive to β-carotene-induced luminescence inhibition (FIG. 7A). IL-1β secretion between wild-type and mutant NLRP3-transfected cells was similar, but IL-1β was not reduced (FIG. 7B). In addition, when we exogenously expressed NLRP3, ASC and caspase-1 in 293T cells, β-carotene inhibited the cleavage of caspase-1 in cells with NLRP3 wild type, but did not inhibit NLRP3 mutation (FIG. 7C). We found that β-carotene inhibited ASC oligomerization in 293T cells transfected with wild type NLRP3 and ASC, but not 293T cells transfected with mutant NLRP3 and ASC (FIG. 8).

< Example  5> mouse windbag  Inhibition of Uric Acid-Induced Inflammasome Activation by Oral β-Carotene in Inflammation Model

We have used a mouse airbag inflammation model in vivo to identify β-carotene inhibitory effects on NLRP3 inflamasomes. The present inventors formed an air bag on the back of the mouse, and then orally administered 30 mg / kg β-carotene to the mouse. After one hour, we injected MSU crystals into the air sacs to activate the NLRP3 inflammasomes. Injecting MSU crystals, cleaved caspase-1 and IL-1β were released into the air sap exudates, indicating in vivo activation of NLRP3 inflammasomes by MSU crystals (FIG. 9). Oral administration of β-carotene inhibited MSU crystal-induced cleavage of pro-caspase-1 and pro-IL-1β in air bag exudates (FIG. 9A). In addition, compared to mouse-derived air sap effluent injected with only MSU crystals, mouse-derived effusion with β-carotene showed lower caspase-1 activity (FIG. 9B). In addition, oral administration of β-carotene inhibited MSU crystal-induced release of IL-1β and IL-18 in air bag exudates, measured by ELISA (FIGS. 9C and 9D). In comparison, neither MSU crystals nor β-carotene altered TNF-α levels in air bag exudates, which supports that TNF-α is not critical for uric acid-induced inflammation (FIG. 9E). The results confirm the inhibitory effect of β-carotene on NLRP3 inflamasome induced by uric acid crystals. We have identified whether inhibition of MSU crystal induced NLRP3 inflammasome activation by β-carotene attenuates the inflammatory response. β-carotene reduced neutrophil invasion in air pocket tissue and exudate, which was shown by histological testing and myeloperoxidase activity, respectively (FIGS. 9F and 9G). The results indicate that β-carotene treatment inhibits NLRP3 inflammasome activation, thereby alleviating the inflammatory response induced by uric acid.

< Example  6> Oral administration of β-carotene in mice NLRP3 Inflamasome  Inhibition of uric acid-induced acute gout by blocking activation

We have determined whether oral administration of β-carotene suppresses the inflammatory symptoms of gout in an acute gout mouse model. We injected MSU crystals into the hind paws of mice, which increased paw thickness and neutrophil invasion of paw tissue and increased cytokine levels in paw tissue homogenate (FIG. 10). Oral administration of β-carotene reduced foot thickness to normal levels (FIGS. 10A and 10B). β-carotene blocked MSU-induced recruitment of neutrophils into paw tissue, which was shown by histological testing of paw tissue and analysis of myeloperoxidase activity in paw tissue homogenate (FIGS. 10C and 10D). . The results indicated that oral administration of β-carotene alleviated the inflammatory symptoms of uric acid induced acute gout in mice.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Binding sites of beta-carotene to NLRP3 PYD domain and screening          method for therapeutic agent of gout using same <130> ADP-2018-0128 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 112 <212> PRT <213> Human <400> 1 Met Lys Met Ala Ser Thr Arg Cys Lys Leu Ala Arg Tyr Leu Glu Asp   1 5 10 15 Leu Glu Asp Val Asp Leu Lys Lys Phe Lys Met His Leu Glu Asp Tyr              20 25 30 Pro Pro Gln Lys Gly Cys Ile Pro Leu Pro Arg Gly Gln Thr Glu Lys          35 40 45 Ala Asp His Val Asp Leu Ala Thr Leu Met Ile Asp Phe Asn Gly Glu      50 55 60 Glu Lys Ala Trp Ala Met Ala Val Trp Ile Phe Ala Ala Ile Asn Arg  65 70 75 80 Arg Asp Leu Tyr Glu Lys Ala Lys Arg Asp Glu Pro Lys Trp Gly Ser                  85 90 95 Asp Asn Ala Arg Val Ser Asn Pro Thr Val Ile Cys Gln Glu Asp Ser             100 105 110

Claims (8)

Reacting the NLRP3 pyrine domain (PYD) with the candidate compound; And selecting candidate compounds that directly bind to Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 residues of the NLRP3 pyrine domain (PYD) in the reacted reactant. The candidate compound blocks the binding of NLRP3 PYD and ASC PYD to inhibit NLRP3 inflammasome activation. delete delete The method of claim 1, wherein the selected candidate compound is β-carotene. Reacting the NLRP3 pyrine domain (PYD) with the candidate compound; And selecting candidate compounds that directly bind to Ala-69, Val-72, Trp-73, Tyr-84, and Glu-91 residues of the NLRP3 pyrine domain (PYD) in the reacted reactant. The candidate compound blocks the binding of NLRP3 PYD and ASC PYD to inhibit NLRP3 inflammasome activation. delete delete The method of claim 5, wherein the selected candidate compound is β-carotene.

Images