Application of WWP2 protein small-molecule inhibitor in preparation of medicine for regulating cartilage lipid metabolism and lipid droplet homeostasis

Abstract

The invention relates to the field of biological medicine, in particular to novel application of a WWP2 inhibitor and a WWP2 transport blocker, in particular to application of the WWP2 inhibitor and the WWP2 transport blocker in preparation of drugs for regulating cartilage tissue lipid metabolism and application of WWP2 as a drug action target in in-vitro screening of drugs for inhibiting accumulation of TG in cartilage cells and cartilage lipid droplets. The invention not only discloses a molecular mechanism that WWP2 participates in regulating cartilage tissue lipid metabolism through interaction with ATGL protein, but also discloses expression and transport channels of WWP2 in cartilage tissue, and further provides powerful basis and support for regulation and control of cartilage tissue lipid metabolism and drug screening. Effective small molecule drugs can be quickly found through a drug screening method, especially a high-throughput drug screening method, which takes the WWP2 protein as a drug target, so that a solid foundation is provided for clinical transformation and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a small molecule inhibitor of WWP2 protein in the preparation of drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis. Background Art

[0002] The protein encoded by the WWP2 gene is a HECT-type E3 ubiquitin ligase and a major member of the NEDD4 family. 2+ / phospholipid binding C2 domain, multiple WW domains and a C-terminal HECT catalytic domain. The N-terminal C2 domain binds to membrane phospholipids in a calcium-dependent manner and mediates intracellular targeting to the plasma membrane, endosomes and multivesicular bodies. The four tandem WW domains (WW1-WW4) in the middle are mainly responsible for substrate recognition. The C-terminal HECT domain provides WWP2 with ubiquitin ligase activity, which is mainly responsible for receiving covalent ubiquitin (Ub) thioester intermediates from the E2-Ub complex and then mediating the transfer of Ub to target proteins. WWP2 has been shown to be involved in a variety of biological processes, including cell cycle, immune response, apoptosis and cell signal transduction.

[0003] Osteoarthritis (OA) is the most prevalent joint degenerative disease in the world. According to statistics, about 600 million people worldwide are suffering from OA, accounting for nearly 8% of the world's population, and the prevalence rate continues to increase with the aging of the population (Safiri S, Kolahi AA, Smith E, Hill C, Bettampadi D, Mansournia MA, et al. Global, regional and national burden of osteoarthritis 1990-2017: asystematic analysis of the Global Burden of Disease Study 2017. Ann Rheum Dis. 2020;79(6):819-28. Wong AY, Samartzis D, Maher C. The global burden of osteoarthritis: past and future perspectives. Lancet Rheumatol. 2023;5(9):e496-e7.). The core clinical symptoms of OA are joint pain and functional limitation, and the disease has a very high disability rate in the late stage. Summary of the invention

[0004] As far as OA research is concerned, there is currently no research based on chondrocyte lipid metabolism, especially the stability of lipid droplets in chondrocytes, and the deep-level molecular regulatory mechanism has not been disclosed and studied.

[0005] Based on this, the present invention further studies the lipid metabolism of chondrocytes in order to provide a basis for developing effective drugs to alleviate or reverse cartilage degeneration.

[0006] The present invention discloses the relationship and mechanism between WWP2 and cartilage lipid metabolism disorder and lipid droplet homeostasis disorder, and verifies that WWP2 participates in and regulates chondrocyte lipid metabolism and lipid droplet problems. Therefore, it is further disclosed that WWP2 inhibitors can regulate cartilage lipid metabolism and lipid droplet homeostasis, especially the accumulation of triglycerides (TG) in chondrocytes and lipid droplets, thereby further disclosing the use of WWP2 protein small molecule inhibitors in the preparation of drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis. Furthermore, the present invention also discloses that the regulation of cartilage lipid metabolism mentioned here refers to inhibiting the accumulation of TG in chondrocytes, and the regulation of lipid droplet homeostasis refers to inhibiting the accumulation of TG in cartilage lipid droplets.

[0007] At the same time, the present invention further tracks the expression and transport of WWP2 and finds that WWP2 has obvious cytoplasmic translocation. Through further research, it is found that WWP2 mainly relies on and exports the transport protein XPO1 to transport out of the nucleus. Based on this, the present invention further discloses the use of WWP2 transport blockers in the preparation of drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis. And further, it is disclosed that the regulation of cartilage lipid metabolism described here refers to inhibiting the accumulation of TG in chondrocytes, and the regulation of lipid droplet homeostasis refers to inhibiting the accumulation of TG in cartilage lipid droplets.

[0008] In particular, the WWP2 transport blocker is an inhibitor of the nuclear export transport protein XPO1, that is, the inhibitor of the nuclear export transport protein XPO1 is used as a WWP2 transport blocker in the preparation of drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis.

[0009] At the same time, the present invention further discloses the use of WWP2 as a drug target in in vitro screening of drugs that regulate cartilage lipid metabolism and lipid droplet homeostasis. Similarly, the regulation of cartilage lipid metabolism described here refers to inhibiting the accumulation of TG in chondrocytes, and the regulation of lipid droplet homeostasis refers to inhibiting the accumulation of TG in cartilage lipid droplets. As an E3 ubiquitin ligase, we found that there is an obvious interaction between the lipid droplet surface protein adipose triacylglyceride lipase (ATGL) and WWP2. Further, we used molecular docking to simulate the interaction between the two and obtained a better interaction model. Furthermore, we used palmitic acid stimulation in C28 / I2 chondrocytes to simulate a high-fat environment, and immunofluorescence found that there was significant co-localization of lipid droplets, WWP2 protein and ATGL protein. Subsequently, we overexpressed WWP2 and ATGL in C28 / I2 cells, and Co-IP experiments confirmed that the two interacted.

[0010] ATGL is an important rate-limiting enzyme in the intracellular lipolysis process and a key enzyme in the catabolism of triglycerides into fatty acids. It dissociates fatty acids from triglycerides and converts them into free fatty acids that can provide energy. Since the strict regulation of lipolysis has important physiological significance, the key enzyme function of ATGL is finely regulated at different levels of transcription and post-transcription, and ubiquitination is also an important biological process that regulates its function.

[0011] The interaction between WWP2 and ATGL indicates that WWP2 participates in regulating lipid metabolism by interacting with ATGL. Therefore, WWP2 can be used as a drug target to screen drugs that regulate cartilage lipid metabolism and lipid droplet homeostasis, and further manifests itself in that it regulates lipid metabolism by inhibiting the accumulation of TG in chondrocytes and cartilage lipid droplets.

[0012] Based on this, we further disclosed a method for drug screening using WWP2 as a drug target.

[0013] One method is to verify whether a single candidate drug can be used as a drug for regulating cartilage tissue lipid metabolism, comprising the following steps: (1) Docking the candidate drug to the WW3 domain of the WWP2 protein (at the binding interface between WWP2 and ATGL); (2) Screening drugs that can inhibit the accumulation of TG in chondrocytes and cartilage lipid droplets based on the affinity of candidate drugs to the receptor WWP2; A molecular docking conformation search box was set up at the binding interface between WWP2 and ATGL to screen small molecules that can inhibit the binding between WWP2 and ATGL. The specific box parameters are as follows: center_x = 21.748 center_y = -5.36 center_z = -9.67 size_x = 30 size_y = 30 size_z = 30 The other is a high-throughput screening method, which still uses WWP2 as a drug target for drug screening, including the following steps: (1) Construct a library of drug compounds to be screened using marketed drug compounds; (2) Using a receptor high-throughput virtual screening system, with WWP2 as the target receptor, the molecules in the drug compound library were docked to the active sites of the target receptors; the active site here refers to the WW3 domain, which is the binding interface between WWP2 and ATGL; (3) Sort the drug compounds according to their interaction strength assessment values ​​with the target receptors; select the top-ranked drug compounds as further research objects for subsequent drug development research.

[0014] The present invention not only discloses the molecular mechanism by which WWP2 participates in regulating cartilage tissue lipid metabolism through interaction with ATGL protein, but also discloses the expression and transport channel of WWP2 in cartilage tissue, thereby providing a strong basis and support for drug screening for regulating cartilage tissue lipid metabolism and lipid droplet homeostasis. Drug screening methods involving WWP2 protein as drug targets, especially high-throughput drug screening methods, can quickly find effective small molecule drugs, thereby providing a solid foundation for clinical transformation and application. Since cartilage tissue lipid metabolism and lipid droplet homeostasis are closely related to cartilage health, regulating cartilage tissue lipid metabolism can improve cartilage health, reduce cartilage destruction, alleviate subchondral bone marrow edema, inhibit osteophyte formation, and reduce the degree of synovial inflammation. Therefore, the discovery of this new use is also expected to play a role in cartilage health, especially in the treatment and prevention of cartilage inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of Oil Red O staining results of cartilage specimens in metabolic OA patients and control group.

[0016] Figure 2 Schematic diagram of the high expression of WWP2 in cartilage of metabolic osteoarthritis.

[0017] Figure 3 Schematic diagram of lipid accumulation in the high-fat diet model of WWP2 cartilage-specific knockout mice.

[0018] Figure 4This is a schematic diagram of the results of high-fat environment inducing WWP2 to be transported out of the nucleus by XPO1, where: A. When the chondrocyte lipid overload model was constructed by palmitic acid stimulation, it was found that WWP2 showed a significant increase in cytoplasmic translocation; B. After using the protein nuclear export inhibitor lmb, the cytoplasmic translocation of WWP2 caused by palmitic acid stimulation was significantly inhibited; C. The level of cytoplasmic WWP2 protein was detected by knocking down nuclear export transporters (XPO1, Exp2, Exp5, Exp7 and IPO13).

[0019] Figure 5 This is a schematic diagram of the results of WWP2 regulating chondrocyte lipid metabolism by regulating ATGL protein, where: A. WWP2 was overexpressed in the C28 / I2 chondrocyte cell line and then immunoprecipitated. The resulting protein complex was analyzed by mass spectrometry to identify proteins that interact with WWP2; B. Molecular docking simulated the interaction between WWP2 and ATGL protein; C. Palmitic acid was used to stimulate C28 / I2 chondrocytes to simulate a high-fat environment, and immunofluorescence revealed significant co-localization of lipid droplets and ATGL protein; D. Palmitic acid was used to stimulate C28 / I2 chondrocytes to simulate a high-fat environment, and immunofluorescence revealed significant co-localization of lipid droplets and WWP2 protein; E. Co-IP experiments confirmed the interaction between the two.

[0020] Figure 6 Schematic diagram of the results of screening small molecule drugs targeting WWP2 to regulate chondroitin metabolism. DETAILED DESCRIPTION

[0021] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0022] It should be noted that, unless otherwise stated, the instruments, reagents, etc. used in the following examples are all commercially available products. The experimental methods used are common experimental methods in the prior art. Example 1

[0023] Medial tibial plateau cartilage tissue samples of the knee joints of patients with osteoarthritis (MOA group) were collected from patients who were hospitalized for total knee replacement surgery at the Joint and Sports Medicine Center of Zhongda Hospital Affiliated to Southeast University, aged ≥45 years, and had abnormal lipid metabolism (high triglycerides (TG): ≥1.7mmol / L or low high-density lipoprotein cholesterol (HDL-C): <1.04mmol / L); The medial tibial plateau cartilage tissue of the knee joint in the control (NC) group was collected from patients with diabetic foot and lower limb amputation caused by injury.

[0024] Oil red O staining and Western blot analysis of WWP2 were performed on the medial tibial plateau cartilage tissues of the knee joints from osteoarthritis patients with metabolic syndrome (MOA group) and the control group (NC group) (diabetic foot and lower limb damage leading to amputation). The specific methods are as follows: Oil Red O staining: Prepare Oil Red O stock solution (100% isopropanol 100 mL + 0.5 g Oil Red O powder, fully dissolved, filtered, stored at 4 degrees in the dark) and Oil Red O working solution (stock solution and distilled water diluted 3:2, filtered, prepared and used immediately). EDTA-decalcified cartilage samples are used to make frozen sections. The tissue is fixed in paraformaldehyde, dehydrated with 10%, 20%, and 30% sucrose in sequence, and sliced ​​after sinking to the bottom. The slice thickness is 10 microns. After returning to room temperature, it is soaked in distilled water. First soak in 60% isopropanol for 2 minutes, then stain with Oil Red O working solution for 5 minutes, and adjust the color with 60% isopropanol. The time is controlled under the assistance of a microscope. After adjusting the ideal color, it is immediately washed with distilled water. Then, hematoxylin is counterstained within 1 minute, 1% hydrochloric acid alcohol is differentiated for 2 seconds, and running water is reversed blue. Finally, the slides are sealed with glycerol gelatin.

[0025] Western blot analysis: First, the protein extracted from the cartilage tissue samples was quantified using the BCA method to ensure that the amount of protein was consistent when loading the sample later. Then, the protein sample was mixed with the loading buffer and boiled at 100°C for 5 minutes to denature the protein and perform SDS-PAGE electrophoresis. The protein was transferred from the gel to the PVDF membrane by wet transfer (100 mA, 2 hours). After transfer, it was blocked with 5% skim milk powder at room temperature for 2 hours. Subsequently, the primary antibody was added and incubated overnight at 4°C. The membrane was washed with TBST buffer to remove the unbound primary antibody. The secondary antibody was added and incubated at room temperature for 1 hour. After washing the membrane again, the ECL chemiluminescent substrate was added, and the target protein band was detected by the chemiluminescent imaging system (Thermo Fisher).

[0026] The experimental results are as follows Figure 1 and Figure 2 As shown in , combined with the experimental results, we observed that patients with metabolic syndrome in the MOA group had obvious lipid droplet accumulation and increased WWP2 expression in the articular cartilage. Example 2

[0027] Control group mice: The control group used C57BL / 6J male mice that matched the model group mice in weight.

[0028] Method for constructing a spontaneous osteoarthritis model in mice fed a high-fat diet: After one week of adaptive feeding, 8-week-old C57BL / 6J male mice were fed with a high-fat diet with a fat energy supply ratio of 60% for 22 to 26 weeks. During the feeding period, the body weight and fasting blood glucose of the mice should be monitored regularly, and the glucose and lipid metabolism should be evaluated by measuring the Lee's index, glucose tolerance test (GTT) and blood lipid levels. After 24 weeks of feeding, the mice were killed, and the liver tissues of the mice were stained with oil red and HE to evaluate the fatty degeneration of the liver and further confirm whether the obesity model was successful. Samples of the knee joints of the lower limbs of the mice were taken for HE staining and safranin O-fast green staining to observe the degeneration of the cartilage.

[0029] Method for constructing WWP2 conditional knockout mouse model: According to the NCBI and Ensembl databases, the structure of the WWP2 gene was analyzed and the exon surrounding regions where LoxP could be inserted were identified. Flox / + model mice were prepared, and ColII-CreERT2 mice were used as Cre tool mice. Finally, WWP2-flox / flox, ColII-CreERT2 cKO inducible cartilage-specific knockout mice and WWP2-flox / flox control mice were obtained through several generations of breeding.

[0030] Cartilage tissues of cKO-induced cartilage-specific knockout mice and WWP2-flox / flox control group mice matched in weight were obtained for HE staining and safranin O fast green staining, respectively. The specific operation method is as follows: HE staining: Soak the slices in xylene twice for 10 minutes each time to remove paraffin. Soak the slices in 100% ethanol, 95% ethanol, and 70% ethanol in turn for 5 minutes each time, and then rinse with distilled water. Stain the slices in hematoxylin stain for 10 minutes, rinse the slices with distilled water to remove excess stain. Then, differentiate the slices in 1% hydrochloric acid alcohol differentiation solution for 10 seconds, and immediately rinse the slices with running water to terminate the differentiation reaction. Soak the slices in tap water for 10-15 minutes to change the color of the cell nucleus to blue-purple. Stain the slices in eosin stain for 1-2 minutes, and the specific time is adjusted according to the thickness of the slices and the freshness of the stain. Rinse the slices with distilled water to remove excess stain. Soak the slices in 70% ethanol, 95% ethanol, and anhydrous ethanol in turn for 5 minutes each time to dehydrate. Soak the slices in xylene twice for 5 minutes each time to make the slices transparent. Seal the slices with neutral gum.

[0031] Safranin O Fast Green Staining: Place the slices on a staining rack and bake them in a 65°C oven for 2 hours to prevent them from peeling off. Soak the slices in xylene three times for 5 minutes each time to dewax. Soak the slices in anhydrous ethanol, 95% ethanol, and 70% ethanol for 5 minutes each time to hydrate. Rinse the slices with distilled water. Stain the slices in fast green staining solution for 5-10 minutes. The specific time should be adjusted according to the freshness of the staining solution and the room temperature. Rinse the slices with distilled water to remove excess staining solution until the cartilage is colorless. Soak them in 1% hydrochloric acid alcohol differentiation solution for a while and rinse with tap water. Stain the slices in safranin staining solution for 15-30 seconds. The specific time should be adjusted according to the freshness of the staining solution and the room temperature. Dehydrate quickly with three cylinders of anhydrous ethanol. Make the slices transparent in xylene for 5 minutes and seal with neutral gum.

[0032] The results are as follows Figure 3 As shown, we found that the cartilage integrity of the control mice showed early spontaneous destruction and the cartilage layer became thinner, while the cartilage integrity and cartilage thickness of the cKO mice (WWP2 conditional knockout mice) were significantly better than those of the control group. At the same time, high-fat diet feeding led to obvious lipid accumulation in the oil red O staining of the cartilage tissue sections of the control mice, but the lipid accumulation observed in the cartilage tissue sections of the cKO mice was significantly reduced. Example 3

[0033] It is generally believed in the prior art that WWP2 is highly expressed in the nucleus. In this example, we constructed a lipid overload model of chondrocytes by stimulating with palmitic acid, and stimulated chondrocytes with 200 μM palmitic acid for 24 hours to simulate the high-fat environment of chondrocytes. Figure 4 A, We found that WWP2 showed a significant increase in cytoplasmic translocation in this model.

[0034] Furthermore, we added an inhibitor of LMB protein nuclear export to the culture system and observed the WWP2 content outside the nucleus by immunofluorescence. Figure 4 As shown in B, combined Figure 4 B shows that the cytoplasmic translocation of WWP2 induced by palmitic acid stimulation was significantly inhibited after the addition of protein nuclear export inhibitor.

[0035] In order to further identify the nuclear export transporter that transports WWP2 out of the nucleus, we knocked down common nuclear export transporters (XPO1, Exp2, Exp5, Exp7 and IPO13) by: Select cells in the logarithmic growth phase and plant them in a 24-well plate one day in advance. Inoculate 1×10^5 cells per well and transfect when the cell density reaches about 70%. Add 0.67μg siRNA (XPO1, Exp2, Exp5, Exp7, IPO13 siRNA and siRNA control) to serum-free diluent (serum-free DMEM) and mix thoroughly to make RNA diluent. The final volume is 25μl. Take 1μl Entranster-R4000, add 24μl serum-free diluent, mix thoroughly to make transfection reagent diluent. After standing at room temperature for 5 minutes, mix the transfection reagent diluent and RNA diluent thoroughly. Stand at room temperature for 15 minutes to prepare a transfection complex. Aspirate the cell culture medium and add 350μl fresh serum-free medium to each well of cells. Aspirate the transfection mixture and drip 150μl into the cells drop by drop in each well. Shake the well plate to mix well and place in the incubator for culture. 4-8 hours after transfection, discard the transfection medium and replace with fresh serum-containing medium and continue culturing. 24-48 hours after transfection, harvest the cells.

[0036] Thus, cells with different nuclear export transporters knocked down were obtained. The extranuclear WWP2 content was further observed by immunofluorescence. The results are as follows Figure 4 As shown in C, we found that WWP2 content was significantly reduced in cells where XPO1 was knocked down, indicating that XPO1 is a nuclear export protein for WWP2 to be transported out of the nucleus. Example 4

[0037] To explore the molecular mechanism by which WWP2 regulates cartilage lipid metabolism and inflammation, we overexpressed WWP2 in the C28 / I2 chondrocyte cell line, immunoprecipitated it, and performed mass spectrometry analysis on the resulting protein complex.

[0038] The experimental method is as follows: The target protein WWP2 is obtained by immunoprecipitation, and then separated and purified by SDS-PAGE. The purified protein is identified using liquid chromatography-mass spectrometry (LC-MS / MS), which can identify all currently known proteins.

[0039] The results are as follows Figure 5 As shown in A, we found that there was an obvious interaction between the lipid droplet surface protein adiposetriacylglyceride lipase (ATGL) and WWP2, suggesting that ATGL may be one of the substrates of WWP2.

[0040] Further, in this example, we used molecular docking to simulate the interaction between protein fat triglyceride lipase and WWP2 in the Autodock system, and the results were as follows: Figure 5 As shown in B, it can be seen that the two can form a good interaction model.

[0041] Furthermore, we used palmitic acid stimulation in C28 / I2 chondrocytes to simulate a high-fat environment and performed immunofluorescence detection. The specific experimental method was: C28 / I2 chondrocytes were inoculated onto pre-treated cell slides, and chondrocytes were stimulated with palmitic acid at a concentration of 200 μM for 24 hours. When the cells grew to 70% full, they were incubated with 4% paraformaldehyde for 10 minutes for fixation. After fixation, they were rinsed 3 times with TBS buffer precooled at 4°C for 5 minutes each time. 0.3% Triton X-100 prepared in TBS buffer was added, incubated at room temperature for 10 minutes, and then rinsed 3 times with TBS for 5 minutes each time. The samples were completely covered with 5% goat serum and incubated in a 37°C constant temperature and humidity incubator for 30 minutes. The blocking solution was removed, and the primary antibody working solution prepared in TBS buffer was added directly to the sample. The sample needed to be completely covered. After incubation at 4°C overnight, the sample was placed at room temperature and reheated for 15 minutes. The antibody working solution was removed and washed 3 times with TBST buffer for 5 minutes each time. Add fluorescent secondary antibody working solution to the sample, cover the sample completely, protect from light, incubate at 37°C for 1 hour, remove the secondary antibody working solution, wash 3 times with TBST buffer, 5 minutes each time. Add 1 µg / mL DAPI working solution to the sample, prepare DAPI solution with 0.01M pH7.2 TBS buffer, protect from light, incubate at room temperature for 30 minutes, wash 3 times, 5 minutes each time.

[0042] The results are as follows Figure 5 As shown in C and 5D, immunofluorescence revealed significant co-localization of lipid droplets with WWP2 protein (5C) and ATGL protein (5D).

[0043] We then overexpressed WWP2 and ATGL in C28 / I2 cells and performed Co-IP experiments. The specific method was as follows: Wash the cells twice with pre-cooled PBS, then add pre-cooled RIPA Buffer, scrape the cells, incubate the collected cells on ice for 5 minutes, and ultrasonicate the samples in an ice bath. Centrifuge at 4°C, 14000g for 15 minutes and collect the supernatant. Mix the protein A / G magnetic beads containing the protective solution and take them out, wash the magnetic beads with the equilibration solution. Pre-wash the cell lysate or pre-treated samples with protein A / G magnetic beads to remove non-specifically bound proteins. Add WWP2 antibody to the pre-washed samples and incubate overnight on a shaker at 4°C. Add protein A / G magnetic beads and continue incubation to allow them to bind to the antigen-antibody complex, thereby precipitating the complex from the solution. Wash multiple times with pre-cooled RIPA buffer, centrifuge to remove the supernatant after each wash, and retain the precipitate. Add elution buffer and detect by Western Blot.

[0044] The experimental results are as follows Figure 5 As shown in E, this experiment confirmed the interaction between WWP2 and ATGL.

[0045] These experimental results revealed that WWP2 can regulate cartilage tissue lipid metabolism by interacting with ATGL. Example 5

[0046] Small molecule drug screening targeting WWP2: (1) Constructing a drug compound library to be screened using drug compounds that have been marketed; in this example, we use a drug compound library that has been marketed by the FDA as a drug compound library; (2) Using the receptor high-throughput virtual screening system, with WWP2 as the target receptor and the three-dimensional structure information of the target receptor, the molecules in the drug compound library were docked to the active sites of the target receptor respectively; the interaction strength between each drug compound and the receptor was evaluated respectively. (3) Sort the drug compounds according to their interaction strength assessment values ​​with the target receptors; select the top-ranked drug compounds as further research objects for subsequent drug development research.

[0047] The results are as follows Figure 6 As shown, according to Figure 6 The results shown in the table show that the interaction strength (binding score -7 or above) between the 30 FDA-approved drug compounds and the active site WW3 domain of the target receptor WWP2 can meet the requirement of inhibiting the binding of WWP2 to ATGL. Therefore, these drug compounds are effective target compounds that can achieve the effect of regulating lipid metabolism and lipid droplet homeostasis, and can be used as candidate compounds for subsequent in vitro activity tests, and further developed into clinical drugs for regulating lipid metabolism and lipid droplet homeostasis.

[0048] The above is a specific implementation of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered to be within the protection scope of the present invention.

Claims

1. Use of small molecule inhibitors of WWP2 protein in the preparation of drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis.

2. Use of WWP2 transport blockers in the preparation of drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis.

3. The use according to claim 1 or 2, characterized in that: The regulation of cartilage lipid metabolism refers to inhibiting the accumulation of TG in chondrocytes, and the regulation of lipid droplet homeostasis refers to inhibiting the accumulation of TG in lipid droplets in cartilage.

4. The use according to claim 2, characterized in that: The WWP2 transport blocker is an inhibitor of the nuclear export transport protein XP01.

5. Application of WWP2 as a drug target in in vitro screening of drugs that regulate cartilage lipid metabolism and lipid droplet homeostasis.

6. The use according to claim 5, characterized in that: The regulation of cartilage lipid metabolism refers to inhibiting the accumulation of TG in chondrocytes, and the regulation of lipid droplet homeostasis refers to inhibiting the accumulation of TG in lipid droplets in cartilage; the active binding site in WWP2 used for drug screening is the WW3 domain.

7. A method for screening drugs for regulating cartilage lipid metabolism and lipid droplet homeostasis, characterized in that: Drug screening using WWP2 as a drug target includes the following steps: (1) Docking the candidate drug to the WW3 domain of the receptor WWP2; (2) Screening drugs that can inhibit the accumulation of TG in chondrocytes and cartilage lipid droplets based on the affinity of candidate drugs to the receptor WWP2; The drug candidate has a group that docks with the WW3 domain.

8. A method for screening drugs for regulating cartilage tissue lipid metabolism, characterized in that: Drug screening using WWP2 as a drug target includes the following steps: (1) Construct a library of drug compounds to be screened using marketed drug compounds; (2) Using the receptor high-throughput virtual screening system, with WWP2 as the target receptor, the molecules in the drug compound library were docked to the active sites of the target receptor respectively; (3) Sort the drug compounds according to their interaction strength assessment values ​​with the target receptors; select the top-ranked drug compounds as further research objects for subsequent drug development research.

9. The method according to claim 7 or 8, characterized in that: The regulation of cartilage lipid metabolism refers to inhibiting the accumulation of TG in chondrocytes, and the regulation of lipid droplet homeostasis refers to inhibiting the accumulation of TG in lipid droplets in cartilage.

Images