KR20180047640A - Novel pharmaceutical composition for treating hypertension - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating and preventing hypertension, which comprises a carveollin-1 specific inhibitor or a carveollin-1 phosphorylation inhibitor as an active ingredient. The carveollin-1 specific inhibitor is antisense oligonucleotide, siRNA, shRNA or miRNA, bisphosphonate, or GGTI-286 specific for the carveollin-1 gene. The carveollin-1 phosphorylation inhibitor is imatinib, butein, mesylate, or PP2(4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d] pyrimidine).

Description

[0001] The present invention relates to novel pharmaceutical compositions for treating hypertension,

The present invention relates to a pharmaceutical composition, and more particularly, to a pharmaceutical composition for treating hypertension.

Hypertension is a chronic disease in which the blood pressure is higher than the normal range. Hypertension causes the heart to do more work to circulate the blood vessels. Blood pressure is summarized as two measures of systolic systolic blood pressure and diastolic diastolic blood pressure. The normal blood pressure at rest is 60 to 90 mmHg when relaxed to 100 to 140 mmHg when contracted, and the blood pressure is constantly 140/90 mmHg or more Hypertension is said to be.

Hypertension is divided into essential hypertension and secondary hypertension. About 90% to 95% are classified as 'essential hypertension' with no definite underlying medical cause and the remaining 5% to 10% (secondary hypertension) are classified as other health conditions affecting the kidneys, arteries, .

Hypertension is a major risk factor for stroke, myocardial infarction (heart attack), heart failure, aneurysms of the blood vessels (eg, aortic aneurysms), and an aneurysm of the ankle, which can also cause chronic renal failure. Although arterial blood pressure is slightly elevated, it can be associated with a reduction in life expectancy. Changes in diet and lifestyle can improve blood pressure control and reduce risk, and in those who are not effective or inadequate, need.

On the other hand, the surface vesicle (Caveolae) is the most abundant structure found in cells, and is the most abundant in the cells and is embedded in the plasma membrane. Surface vesicles are characterized by the expression of a protein called caveoloin. Caveolin has three isoforms, Cav-1, -2, and -3, and is characteristic for each cell. Surface vesicles are thought to be important for the signaling of many cells because of the gathering of receptors, channels, proteins and cholesterol as well as caveolin. In particular, caveolin-1 protein has recently been actively studied for its function, but it is still unclear.

On the other hand, currently available hypertension therapeutic agents include vasodilators such as diuretics, alpha or beta blockers, and calcium channel blockers, and angiotensin converting enzyme inhibitors and angiotensin II receptor blockers in addition to calcium channel blockers Especially calcium channel inhibitors that inhibit the influx of calcium by voltage-dependent Ca channel (VDCC) have been developed and used.

Prior arts related thereto include U.S. Patent No. 5,304,570, which is related to a therapeutic agent for hypertension containing an α-adrenergic agonist ibopamine and an ACE inhibitor as an active ingredient, and Korean Pat. No. 5,304,570 for prevention and / or treatment of portal hypertension 10-2006-0031614, Korean Patent Publication No. 10-2008-0075922 for dibenzene derivatives as calcium channel blockers, Korean Patent Laid-Open No. 10-2010-0014570 for first-substituted tetrahydroisoquinoline compounds, Korean Patent Laid-Open No. 10-2013-0118840 and Korean Patent Laid-Open No. 10-2013-0074834 for compositions for the treatment or prevention of hypertension, which comprise a lipase C gamma or epidermal growth factor receptor inhibitor as an active ingredient.

However, calcium channel blockers are known to have some side effects due to a somewhat lowered selectivity, and there is a problem of taking a lifetime drug because it focuses only on the lowering of the elevated blood pressure.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a more effective and improved composition for treating and preventing hypertension by identifying the control factors for controlling VDCC. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a pharmaceutical composition for the treatment and prevention of hypertension comprising a caveolin-1-specific inhibitor or a caveolin-1 phosphorylation inhibitor as an active ingredient.

According to one aspect of the present invention there is provided a method of regulating blood pressure in a subject comprising administering to the subject a therapeutically effective amount of a caveolin-1 specific inhibitor or a caveolin-1 phosphorylation inhibitor.

In accordance with another aspect of the present invention, there is provided a method for treating cancer, comprising the steps of: i) treating a cell expressing caveolin with a test compound or a natural product; ii) measuring the degree or degree of phosphorylation of said caveolin in said cells; And iii) selecting a test compound or a natural product that significantly reduces the expression or phosphorylation degree of the caveolin as compared with a control group in which the test compound or the natural substance has not been treated, is provided .

According to another aspect of the present invention, there is provided a method for producing a caveolin-1 protein, comprising: i) mixing a caveolin-1 protein, a partner substance interacting with the caveolin-1 protein and a test compound or a natural product; And ii) selecting a test compound or a natural substance significantly inhibiting the interaction of the caveolin-1 protein and the partner substance as compared with the control group in which the test compound or the natural substance is not treated. A screening method is provided.

According to another aspect of the present invention, there is provided a method for producing a caveolin-1 protein, comprising mixing a caveolin-1 protein, ATP and a test compound or a natural product; And screening the candidate compound for hypertension treatment comprising the step of screening a test compound or natural product significantly inhibiting the phosphorylation of the caveolin-1 protein as compared with the control compound not treated with the test compound or natural product.

According to one embodiment of the present invention as described above, hypertension can be prevented and treated by controlling the calcium ion concentration in the cells. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a photograph (A) showing the results of Western blotting analysis performed to measure the degree of phosphorylation of caveolin-1 after treating cells with a storage solution according to an embodiment of the present invention, and FIG. 1 (PCav-1 / Cav-1) of the expression level of pCAV-1 (pCav-1)
FIG. 2 is a photograph showing the result of Western blotting analysis performed to measure the degree of calcium channel expression in cells after siRNA treatment for caveolin-1 expression inhibition according to an embodiment of the present invention. FIG.
3 is a graph showing the results of measuring the current density (unit: pA / pF) of the control group (A) and the siRNA-treated group (B) against the caveolin-1 gene through a patch-clamp analysis.
4 is a graph showing the results of calcium imaging over time for the control group (A) and the siRNA-treated group (C) for the caveolin-1 gene.

Definition of Terms:

As used herein, the term " caveolin-1 (Cav-1) " refers to a scaffold protein that is a major component of the caveolae plasma membrane of most cell types. Caveolin-1 acts by linking the integrin subunit with the tyrosine phosphorylase FYN, which is an early step in linking the integrin to the Ras-ERK signaling pathway and promoting cell cycle progression.

The term " caveolin-1-specific inhibitor " as used herein means a compound that specifically inhibits the expression or function of caveolin-1 protein, and the expression inhibitor includes an antisense oligonucleotide , siRNA, shRNA or miRNA, bisphosphonate, GGTI-286, ancardonate (Iguchi et al ., Anticancer Res., 26 (4B): 2977-2981, 2006).

As used herein, the term " caveolin-1 phosphorylation inhibitor " refers to a compound that specifically inhibits phosphorylation of caveolin-1 protein and includes imatinib, butein, mesylate, PP2 (4- chlorophenyl) -7- (dimethylethyl) pyrazolo [3,4-d] pyrimidine.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a pharmaceutical composition for the treatment and prevention of hypertension comprising caveolin-1-specific inhibitor or caveolin-1 phosphorylation inhibitor as an active ingredient.

In this composition, the caveolin-1-specific inhibitor may be a compound or natural product that specifically inhibits the expression or function of caveolin-1 protein, and the caveolin-1 phosphorylation inhibitor may be phosphorylated ≪ / RTI > or a natural product. Specifically, in the composition, the caveolin-1 specific inhibitor may be an antisense oligonucleotide specific for the caveolin-1 gene, siRNA, shRNA or miRNA, bisphosphonate, GGTI-286, or ancardonate, The caveolin-1 phosphorylation inhibitor may be imatinib, butein, mesylate, or PP2 (4-amino-5- (4-chlorophenyl) -7- (dimethylethyl) pyrazolo [3,4-d] pyrimidine).

As used herein, the term " natural product " means an extract derived from a substance existing in nature such as an animal, a plant or a mineral, a fraction obtained by further fractionating the extract with an organic solvent, or a compound obtained by purely isolating the extract or fraction it means.

The effective amount of the compound in the pharmaceutical composition of the present invention may vary depending on the type of affected part of the patient, the application site, the number of treatment, the treatment time, the formulation, the condition of the patient, The amount to be used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The above-mentioned daily dose may be administered once a day or two or three times a day at appropriate intervals, or intermittently administered at intervals of several days.

In the pharmaceutical composition of the present invention, the compound may be contained in an amount of 0.1 to 100% by weight based on the total weight of the composition.

The subj ective compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, solid pharmaceutical preparations or liquid pharmaceutical preparations can be used for the preparation of pharmaceutical compositions. The preparation additive may be either organic or inorganic.

Examples of the excipient include lactose, sucrose, saccharin, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals. These tablets and granules can be suitably coated with sugar (sugar coating), gelatin coating, and others as required. If necessary, preservatives, antioxidants and the like may be added.

The pharmaceutical composition of the present invention can be prepared into any of the formulations conventionally produced in the art (for example, Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), the form of the formulation is not particularly limited , Preferably an external preparation. The external preparation of the present invention may be in the form of a sheet, a liquid coating agent, a spray agent, a lotion agent, a cream agent, a papermaking agent, a powder, a penetration pad agent, a spray agent, a gel agent including a hydrogel, pasta agent, liniment agent, ointment agent, And conventional external preparations such as percutaneous absorbers may be included. These formulations are generally known prescription Seo literature ^{[Remington's Pharmaceutical Science, 15 th} Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 all Agency: is described in (Chapter 87 Blaug, Seymour).

According to one aspect of the present invention there is provided a method of regulating blood pressure in a subject comprising administering to the subject a therapeutically effective amount of a caveolin-1 specific inhibitor or a caveolin-1 phosphorylation inhibitor.

As used herein, the term " effective amount ", therapeutically, refers to an amount that can be lowered to a significant extent, especially in the normal range or close to the subject's blood pressure, when administered to a subject.

In the blood pressure control method, the caveolin-1 specific inhibitor and the caveolin-1 phosphorylation inhibitor are as described above.

In accordance with another aspect of the present invention, there is provided a method for treating cancer, comprising the steps of: i) treating a cell expressing caveolin with a test compound or a natural product; ii) measuring the degree or degree of phosphorylation of said caveolin in said cells; And iii) selecting a test compound or a natural product that significantly reduces the expression or phosphorylation degree of the caveolin as compared with a control group in which the test compound or the natural substance has not been treated, is provided .

In the above method, the degree of expression of the caveolin can be performed through Northern blot analysis, reverse transcription PCR, real-time reverse transcription PCR, DNA microarray analysis or Western blot analysis.

In this method, the phosphorylation of the caveolin-1 protein can be carried out by immunoassay using an anti-phosphorylated caveolin-1 antibody, or mass spectrometry, which includes Western blotting analysis, radioimmunoassay (RIA) , Or an enzyme-linked immunosorbent assay (ELISA).

According to another aspect of the present invention, there is provided a method for producing a caveolin-1 protein, comprising: i) mixing a caveolin-1 protein, a partner substance interacting with the caveolin-1 protein and a test compound or a natural product; And ii) selecting a test compound or a natural substance significantly inhibiting the interaction of the caveolin-1 protein and the partner substance as compared with the control group in which the test compound or the natural substance is not treated. A screening method is provided.

In the above method, wherein the partner material is PDGFRα, PDGFRβ, EGFR, the insulin receptor, TGFβRI, TrkA / p75NTR, hedge hump (Hedgehog) receptor, estrogen receptor, androgen receptor, H-Ras, Gα _q / Gα _o / Gα _s, (A, b, c, d, d, e, g, and n), adenylcyclase / PLCβ2, Trp1 / IP3R / G _q11 , c-Src, Lyn, Csk, GRK1, GRK2, GRK5, COX-2, PLD / PKCα, PKA, PKC, beta] / Shc / Fyn uPAR / integrin beta, eNOS, nNOS, Flotillin 1, 2 / Cav-2, 190-kDa pY, 30-kDa pY, HSP56 / cyc40 / cycA, Filamin, Grb7, Striatin / SG2NA / , Cholesterol, fatty acids, GM1 or GD3.

In this method, the interaction of the caveolin-1 protein and the partner material may be determined by immunoprecipitation analysis, GST pulldown assay, yeast two-hybrid assay, protein microarray, surface plasmon resonance assay, or Fluorescence resonance energy transfer analysis (FRET).

According to another aspect of the present invention, there is provided a method for the production of a composition comprising: i) mixing a caveolin-1 protein, ATP and a test compound or a natural product; And ii) selecting a test compound or a natural substance that significantly inhibits the phosphorylation of the caveolin-1 protein as compared with the control group in which the test compound or the natural substance is not treated, is provided .

In this method, the phosphorylation of the caveolin-1 protein can be measured by immunoassay, mass spectrometry or luciferase analysis using an anti-phosphorylated caveolin-1 antibody. The immunoassay may be Western blotting analysis, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA), wherein the luciferase assay further comprises mixing luciferin and luciferase in step i above for phosphorylation, As an indirect method of measuring the concentration of ATP, bioluminescence by luciferase is dependent on the concentration of ATP. When the phosphorylation of caveolin-1 is inhibited by a test compound or a natural substance, the concentration of ATP in the reaction solution will be high. Therefore, the degree of bioluminescence due to luciferase will be increased in proportion to the degree of inhibition of phosphorylation of caveolin- Can be measured by using a luminometer to measure the degree of inhibition of phosphorylation of caveolin-1.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Animals and Cells

Male Sprague-Dawley (SD) rats (10-11 weeks old) were used in the experiment. All experiments were carried out in accordance with the US National Institutes of Health guidelines, and the study was approved by Konkuk University Animal Experiment Management Committee. Rats removed the blood by carotid artery incision after inhalation of carbon dioxide. The mesenteric artery was then isolated and isolated into single cells. The composition for single cell separation was prepared as previously described (Kim et al . Eur. J. Pharmacol . 483 : 117-126, 2004) Respectively.

Example 2: Solutions and Chemicals

Experiments were performed with isotonic solution (pH 7.4) containing 115 mM of NaCl, 5 mM of 4- (2-hydroxyethyl) -1-piperazine-ethanesulphonic acid (HEPES), 11 mM of glucose and 65 mM of sucrose, ) Were used. In order to record VDCC currents by cell expansion in single cells, a hypotonic solution was prepared in which 60 mM sucrose was removed from isotonic solution. To record the VDCC current, an isotonic solution containing 10 mM BaCl ₂ was used. 100 mM of cesium methanesulfonic acid (CH ₃ O ₃ SCs), 40 mM of CsCl, 1 mM of MgCl ₂ , 5 mM of MgATP, 0.05 mM of HEPES, 5 mM and 0.05 mM of EGTA were contained for the recording of whole cell patches And added to the pipette solution titrated to pH 7.2 with CsOH. All chemicals were purchased from Sigma Chemical Co., USA unless otherwise noted.

Example 3: Western Blotting

Mesenteric arterial smooth muscle cells (RMASMC) were isolated from the rats and cultured in 3 to 10 passages. The cells were used for Western analysis and cultured in RPMI 1640 containing 10% FBS, 1% penicillin-streptomycin DMEM. For all experiments, cells were incubated with 3 to 10 passages, followed by incubation at 80% saturation versus the total dish area, followed by starvation in DMEM without FBS for 12-24 hours. Two hours before the experiment, the culture medium was replaced with fresh medium. The cells were then washed three times with physiological saline (PBS) and the cells were disrupted using RIPA buffer (TNT Research, LTD., KOREA). Samples were loaded on 8% or 14% SDS-polyacrylamide non-reducing gel and electrophoresed to separate by protein size and transferred to PVDF membrane (Millpore, USA). Each of the blots was subjected to immunohistochemistry using anti-Caveolin-1 antibody (1: 1000; cell signaling), anti-pCaveolin-1 antibody (1: (1: 2,000; Cell Signaling) using an anti-rabbit IgG secondary antibody with anti-rabbit anti-human IgG secondary antibody (1.2: 1 channel antibody (1: 500; Alomone Lab.) And anti-GAPDH antibody ) Analysis. Increased phosphorylation signals were observed using Las-4000 (Fuji Film). As a result of Western blot analysis, it was found that phosphorylation of caveolin-1 was increased by cell membrane expansion. In particular, it was found that phosphorylation was increased with time.

Example 4: Small interfering RNA

From the results of Example 3, siRNA capable of inhibiting the expression of caveolin-1 was constructed to examine the effect of caveolin-1 on the expression of calcium channel, and then the RMASMC was transfected with Cav 2.1 The degree of expression of the channel was measured by Western blot analysis.

Specifically, cells were starved in the same manner as in Example 3 above. Before the start of the experiment, the solution contained in the culture dish was removed and 1.6 ml of DMEM medium containing 10% FBS was added to a 60 mm culture dish. Two solutions were prepared. The first solution composition was 198 ml DMEM, 2 ml Caveolin-1 siRNA (100 mM) (L-093600-02, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and the second solution composition was 193 ml DMEM, 7 ml DhamaFECT (T-2002-02, Thermo Scientific, Waltham, Massachusetts, USA). Both solutions were mixed at room temperature for 5 minutes each. Solutions 1 and 2 were then mixed for 30 minutes at room temperature. Was added to a 60 mm culture dish containing 1.6 ml of the above-mentioned medium. After 2 days, Western blot analysis was carried out in the same manner as described in Example 3 above.

As a result, as shown in Fig. 2, it was confirmed that Cav2.1 channel expression was decreased in cells transfected with caveolin-1 siRNA. Suggesting that it can inhibit the expression of calcium channel by inhibiting the expression of caveolin-1, thereby ultimately suppressing an increase in intracellular calcium concentration.

Example 5: Electrophysiological record

Mesenteric arterial smooth muscle cells (RMASMC) were isolated from the rats and whole cell patch-clamp experiments were performed. EPC8 patch-clamp amplifier (Heka, Germany) was used as the equipment. The data was low-pass filtered at 1 kHz and digitized by a program at a sampling rate of 1-10 kHz and stored in a computer. Voltage pulse generation was also controlled by software. The patch pipette was made from borosilicate capillaries (Clark Electromedical Instruments, Pangbourne, UK) using a puller (PP-83, Narishige, Japan). When filled with pipette solution, a patch pipette with 2-3 MΩ resistance was used. All experiments were performed at room temperature. A commercially available water bath (RC-11, WPI, USA) and a silver / silver chloride (Ag / AgCl) pellet electrode (WPI, USA) were used.

As a result of the patch-clamp analysis, as shown in FIG. 3, the VDCC was increased by the storage solution in the control cells that were not treated with electrophysiological recording, whereas the VDCC itself was decreased in the caveolin-1 siRNA treated group, Of the total number of patients.

Example 6 Calcium Imaging Experiment

Single cells were isolated from the control group and caveolin-1 siRNA transfected RMASMC cells in Example 5, and then mixed with Fura2-AM (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a concentration of 1: 1000. After mixing for 30 minutes, the cells were placed on a fluorescence microscope. Then, the remaining Fura2-AM which did not enter the cells was washed with NT using a perfusion device. The intracellular calcium concentration was measured using a Lambda DG-4 instrument (Sutter Instrument Company, Novato, CA, USA).

As a result, as shown in FIG. 4, intracellular calcium was increased by the 70 mM KCl solution in the control group (left), and the increase of intracellular calcium by 70 mM KCl was further increased by the storage solution, In the siRNA treatment group (right), it was confirmed that intracellular calcium increase was suppressed.

As shown in the above results, it was confirmed that the inhibition of the expression of the caveolin-1 gene suppressed the increase of intracellular calcium concentration in the storage solution. These results demonstrate that treatment and prevention of hypertension is possible by inhibiting the function including expression or phosphorylation of the caveolin-1 gene.

The experimental results are shown as mean ㅁ standard deviation. Paired or independent Student t-tests were tested for significance as appropriate and P <0.05 was considered significant.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (12)

A pharmaceutical composition for the treatment and prevention of hypertension comprising caveolin-1-specific inhibitor or caveolin-1 phosphorylation inhibitor as an active ingredient. The method according to claim 1,
Wherein the caveolin-1-specific inhibitor is an antisense oligonucleotide, siRNA, shRNA or miRNA, bisphosphonate, GGTI-286, or ancardonate specific for the caveolin-1 gene. The method according to claim 1,
Wherein the caveolin-1 phosphorylation inhibitor is imatinib, butein, mesylate, or PP2 (4-amino-5- (4-chlorophenyl) -7- (dimethylethyl) pyrazolo [3,4-d] pyrimidine). Comprising administering to a subject a therapeutically effective amount of a caveolin-1 specific inhibitor or a caveolin-1 phosphorylation inhibitor. i) treating the cell expressing caveolin with a test compound or a natural product; ii)
Measuring the degree or degree of phosphorylation of the caveolin in the cell; And
iii) selecting a test compound or a natural substance which significantly reduces the expression or phosphorylation degree of the caveolin as compared with the control group in which the test compound or the natural substance is not treated. 6. The method of claim 5,
Wherein the degree of expression of the caveolin is performed through Northern blot analysis, reverse transcription PCR, real-time reverse transcription PCR, DNA microarray analysis or Western blot analysis. 6. The method of claim 5,
Wherein the phosphorylation of the caveolin-1 protein is carried out by immunoassay using an anti-phosphorylated caveolin-1 antibody, or by mass spectrometry. i) mixing a caveolin-1 protein, a partner substance interacting with the caveolin-1 protein and a test compound or natural product; And
ii) screening for a candidate compound for hypertension treatment comprising the step of screening a test compound or a natural substance which significantly inhibited the interaction of the caveolin-1 protein and the partner substance as compared with the control group in which the test compound or the natural substance was not treated Way. 9. The method of claim 8,
The partner material is selected from the group consisting of PDGFRα, PDGFRβ, EGFR, insulin receptor, TGFβRI, TrkA / p75NTR, Hedgehog receptor, estrogen receptor, androgen receptor, H-Ras, Gα _q / Gα _o / Gα _s , PKC ?, Integrin / cortactin / Src, Integrin (?,?) / Shc / 2, Trp1 / IP3R / G _q11 , c-Src, Lyn, Csk, GRK1, GRK2, GRK5, COX- Coninxin, cholesterol, fatty acids, and lipids, such as Fyn uPAR / integrin? 1, eNOS, nNOS, Flotillin 1, 2 / Cav-2, 190-kDa pY, 30-kDa pY, HSP56 / cyc40 / cycA, Filamin, Grb7, Striatin / SG2NA / GM1 or GD3. 10. The method of claim 9,
The interaction of the caveolin-1 protein and the partner material can be determined by immunoprecipitation analysis, GST pulldown assay, yeast two-hybrid assay, protein microarray, surface plasmon resonance assay, or fluorescence resonance energy transfer assay (FRET). &Lt; / RTI &gt; i) mixing the caveolin-1 protein, ATP and the test compound or natural product; And ii)
Selecting a test compound or a natural substance that significantly inhibits phosphorylation of the caveolin-1 protein as compared with a control group in which the test compound or the natural substance is not treated. 12. The method of claim 11,
Wherein the phosphorylation of the caveolin-1 protein is measured by immunoassay, mass spectrometry or luciferase analysis using an anti-phosphorylated caveolin-1 antibody.

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