Molecular target spot capable of specifically reducing cholesterol level without interfering triglyceride metabolism and application of molecular target spot

Abstract

The invention relates to a molecular target capable of specifically reducing cholesterol level without interfering triglyceride metabolism and application of the molecular target. The molecular target is the C terminal of ANGPTL3 protein. The invention also relates to an application of the inhibitor of the C terminal of the targeted ANGPLT3 protein (Angiopoietin-like3, angiopoietin-like protein 3) in preparation of a medicine, and the medicine is used for treating hypercholesteremia, atherosclerosis and coronary heart disease, and also relates to an application of the inhibitor of the C terminal of the targeted ANGPLT3 protein (Angiopoietin-like protein 3) in preparation of the medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to molecular targets and applications thereof that specifically reduce cholesterol levels without interfering with triglyceride metabolism. Background Art

[0002] Hypercholesterolemia is the main cause of atherosclerosis and cardiovascular and cerebrovascular diseases. Clinically, the prevention and treatment of cardiovascular and cerebrovascular diseases also use cholesterol lowering as the main intervention method. Statins, PCSK9 inhibitors and other first-line clinical drugs mainly achieve the purpose of lowering blood cholesterol by increasing the expression and function of low-density lipoprotein receptors (LDLR), and have achieved significant results in the prevention and treatment of cardiovascular and cerebrovascular diseases such as coronary heart disease.

[0003] Familial hypercholesterolemia (FH) is a type of hypercholesterolemia caused by genetic mutations. On average, there is one heterozygous FH patient in every 200 to 300 people, and about one homozygous FH patient in every million people. If homozygous FH patients are not diagnosed and intervened early, they will develop serious cardiovascular and cerebrovascular diseases, and their average life expectancy is only in their teens. Heterozygous FH patients also have a 20-30 times higher incidence of coronary heart disease than ordinary people due to hypercholesterolemia. Population studies have found that more than 80% of FH patients are caused by LDLR mutations, especially homozygous FH patients whose LDLR activity is extremely low and have almost no response to statins and PCSK9 inhibitors. Currently, the most effective treatment for this type of patients is still hemodialysis. Therefore, there is an urgent need to develop new cholesterol-lowering strategies and new drugs that do not rely on LDLR.

[0004] Cholesterol and triglycerides in the blood are mainly carried by lipoprotein particles. The surface of lipoprotein is covered by a layer of phospholipid molecules, and the inside is hydrophobic cholesterol and triglyceride molecules. Endothelial cell esterase (EL) has phospholipid hydrolase activity, which mainly hydrolyzes phospholipids on lipoproteins, thereby affecting the structure of lipoprotein particles. Lipoprotein hydrolase (LPL) is a triglyceride hydrolase, which mainly catalyzes the hydrolysis of triglycerides carried by lipoproteins.

[0005] ANGPTL3 is a liver-specific secretory factor, consisting of a signal peptide, an N-terminal fragment, and a C-terminal fragment. The signal peptide guides its secretion, the N-terminal contains a functional region that can simultaneously inhibit the activity of LPL and EL, and the function of the C-terminal is unknown ( Figure 7). ANGPTL3 gene deletion mutations in the human population significantly reduce serum cholesterol and triglyceride levels without causing side effects such as fatty liver. Inhibiting the N-terminal activity of ANGPTL3 can increase the activity of EL, thereby significantly reducing serum cholesterol levels, and this process is independent of LDLR. Therefore, monoclonal antibody inhibitors of ANGPTL3 have been approved for marketing in the United States for the treatment of homozygous FH patients, and more inhibitors are still in clinical trials. However, the safety of this antibody inhibitor has not yet been reported.

[0006] Triglycerides in the blood act as an energy molecule, providing energy sources for the heart and muscles. However, if the heart and muscles take in too much fat, it will lead to lipotoxicity, causing insulin resistance, heart failure and other diseases. Lipotoxicity is also an important cause of the high incidence of diabetic cardiomyopathy (affecting about 19%-26% of diabetic patients).

[0007] LPL is synthesized and secreted by parenchymal cells of peripheral tissues such as the heart, muscle, and fat, and is transported to the capillary side to perform its functions. LPL is anchored to the surface of the vascular endothelial cell membrane through heparan sulfate proteoglycans (HSPGs) and glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), thereby catalyzing the hydrolysis and absorption of triglycerides carried by lipoproteins. LPL is a key regulatory step in the uptake of triglycerides by peripheral tissues ( Figure 8 ).

[0008] Existing ANGPTL3 inhibitors target and inhibit the N-terminus of ANGPTL3. The inhibitors simultaneously increase the activity of EL and LPL, thereby lowering serum cholesterol levels while also lowering serum triglyceride levels. However, existing ANGPTL3 inhibitors can lead to excessive uptake of triglycerides by tissues such as the heart and muscles, which may induce lipotoxicity and cause side effects such as abnormal cardiac function.

[0009] Therefore, it is necessary to develop a target and drug that lowers serum cholesterol levels but does not lower serum triglyceride levels. Summary of the invention

[0010] The present invention first relates to the use of an inhibitor targeting the C-terminus of ANGPLT3 protein (Angiopoietin-like 3) in the preparation of a drug, wherein the inhibitor:

[0011] (1) Targeting and binding to the C-terminus of ANGPLT3 protein, and

[0012] (2) inhibiting the functional activity of the C-terminal fragment of ANGPLT3 protein;

[0013] The inhibitor is a protein level inhibitor, including but not limited to: antibodies, small molecule compounds, preferably, the small molecule compound is heparin; or

[0014] The amino acid sequence of the C-terminus of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0015] SEQ ID NO.1: CGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLY;

[0016] SEQ ID NO.2: TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKY NKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE;

[0017] The medicine is used to treat hypercholesterolemia, atherosclerosis, and coronary heart disease;

[0018] Preferably, the hypercholesterolemia is:

[0019] (1) familial hypercholesterolemia (FH); or

[0020] (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).

[0021] The present invention also relates to the use of an inhibitor of a protein targeting the C-terminus of ANGPLT3 protein (Angiopoietin-like 3) in the preparation of a preparation for lowering LDL-C (low-density lipoprotein cholesterol) levels without significantly interfering with triglyceride metabolism, wherein the inhibitor:

[0022] (1) Targeting and binding to the C-terminus of ANGPLT3 protein, and

[0023] (2) inhibiting the functional activity of the C-terminal fragment of ANGPLT3 protein;

[0024] The inhibitor is a protein level inhibitor, including but not limited to: antibodies, small molecule compounds; or

[0025] The amino acid sequence of the C-terminus of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0026] The present invention also relates to the use of the C-terminal protein of ANGPLT3 protein (Angiopoietin-like 3) in detecting the activity of a drug to be screened, wherein the detection refers to detecting the following functions of the drug to be screened:

[0027] (1) Targeting the C-terminus of ANGPLT3 protein, and

[0028] (2) inhibiting the functional activity of the C-terminal fragment of ANGPLT3 protein;

[0029] The drug to be screened is an antibody or a small molecule compound;

[0030] The drug is used to treat hypercholesterolemia, atherosclerosis or coronary heart disease; preferably, the hypercholesterolemia is:

[0031] (1) familial hypercholesterolemia (FH); or

[0032] (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).

[0033] The beneficial effects of the present invention are

[0034] 1. This study proves that inhibiting the N-terminal activity of ANGPTL3 significantly reduces blood cholesterol and triglyceride levels, but leads to lipid accumulation in the heart and causes heart failure;

[0035] 2. There are at least two forms of ANGPTL3 functioning in peripheral tissues: one is to form a complex with ANGPTL8 through its N-terminus, and then be anchored to the tissue vascular wall through GPIHBP1; the other is to be anchored to the tissue vascular wall through its C-terminus directly binding to HSPGs;

[0036] 3. Specific blocking of ANGPTL3 (C-terminus) function will be a new lipid-lowering strategy to reduce LDL-C levels without significantly interfering with triglyceride metabolism;

[0037] 4. Specific blocking of the function of ANGPTL3 (C-terminus) will be a new strategy for preventing and treating diseases such as hypercholesterolemia, atherosclerosis, and coronary heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Inhibition of ANGPTL3 activity or ANGPTL8 expression leads to cardiac lipid accumulation

[0039] 1A. Inhibitory antibody against ANGPTL3 (A3 mAb) increases cardiac triglyceride content in wild-type mice: Monoclonal antibody against the N-terminus of ANGPTL3 was injected into wild-type mice (20 μg / g), and the hearts were harvested 7 days later for cardiac lipid content analysis.

[0040] 1B. Increased cardiac triglyceride content in Angptl3 knockout mice (Angptl3- / -);

[0041] 1C. Increased cardiac triglyceride content in Angptl8 knockout mice (Angptl8- / -);

[0042] TG: triglyceride. Data are expressed as mean ± standard error. **p<0.01, ***p<0.001.

[0043] Figure 2 Inhibition of ANGPTL3 activity or ANGPTL8 expression leads to heart failure

[0044] 2A. Inhibitory antibodies against ANGPTL3 (A3 mAb) induce heart failure: Monoclonal antibodies against the N-terminus of ANGPTL3 were injected into ApoE- / - hyperlipidemic mice (20 g / g, once a week), and cardiac function was detected by small animal ultrasound at 1, 3, and 5 weeks (N=7-8 / group, male, 17 weeks);

[0045] 2B. Angptl8 knockout mice (Angptl8- / -) showed heart failure under pressure load: cardiac stress was induced in Angptl8- / - and control group mice by aortic arch constriction (TAC), and cardiac function was detected by small animal ultrasound every week after surgery (N=7-8 / group, male, 11-18 weeks).

[0046] LVEF: left ventricular ejection fraction, LVFS: left ventricular fractional shortening. **p<0.01, ***p<0.001.

[0047] Figure 3 ANGPTL3 N-terminus binds to GPIHBP1 via ANGPTL8

[0048] 3A. ANGPTL3 protein levels in Angptl8- / - mice and littermate control wild-type mice (n=3 / group, female, 8-15 weeks; F: starvation, R: postprandial);

[0049] 3B. ANGPTL3 protein levels in Gpihbp1- / - mice and littermate control wild-type mice in the postprandial state (n=6 / group, male, 7-8 weeks; KO: samples from Angptl3- / - mice);

[0050] 3C, ANGPTL8 (A8) promotes the binding of ANGPTL3 N terminus (A3-N) to GPIHBP1: Purified A3-N protein or A3-N and A8 protein complex was incubated with CHO cells expressing GFP or GPIHBP1 (GP1), and then the cell lysate (Cells) and incubation medium were subjected to western blotting analysis accordingly;

[0051] 3D. A3-N and A8 complex dissociates LPL from GPIHBP1: CHO cells expressing GPIHBP1 (GP1) were incubated with LPL conditioned medium, washed, and then A3-N and A8 protein complex (A3-N / A8, 1 mg / ml) was added to the cells containing fresh medium, and then the medium (S) and cells were collected for corresponding protein immunoblot analysis (PBS, phosphate buffered saline);

[0052] 3E. Cell-bound LPL activity detection (mmol / L / hr): All experimental procedures are exactly the same as those in Figure D. After the last wash, the cells were incubated in fresh medium containing heparin (10U / ml) for 15 minutes (37°C), and the supernatant was collected for LPL activity determination. The three bars from left to right represent the same three groups of samples as in Figure D;

[0053] 3F. Schematic diagram of the dissociation of LPL from GPIHBP1 by the A3-N and A8 complexes.

[0054] FL: full-length ANGPTL3, N: N-terminal ANGPTL3, C: C-terminal ANGPTL3, PonS: Ponceau, CNX: Calnexin,

[0055] **p<0.01.

[0056] Figure 4 ANGPTL3 C-terminus (A3-C) is bound to tissues through heparan sulfate proteoglycans (HSPGs).

[0057] 4A. The C-terminus of ANGPTL3 (A3-C) bound to the cell membrane can be released into the culture medium by heparin: HepG2 cells were incubated with A3-C or GFP conditioned medium, washed, and incubated with fresh medium containing heparin (Hepa) or blank control (NC), and then the collected supernatant (S), cell lysate (Cells) and the initial conditioned medium were subjected to corresponding protein immunoblot analysis;

[0058] 4B. ANGPTL3 C-terminus binds to HepG2 cells via conserved positively charged amino acid residues: Wild-type (WT) A3-C and A3-C (MUT) conditioned media containing the indicated mutations were incubated with HepG2 cells: The media and cells were then collected and analyzed by immunoblotting. The sequence alignment of the positively charged motif at the terminal of A3-C is shown at the top, and the positively charged residues in red are mutated to alanine in mutant A3-C;

[0059] 4C. Schematic diagram of ANGPTL3 binding to tissue vascular walls: The N-terminus (N) of ANGPTL3 and part of the full-length ANGPTL3 bind to GPIHBP1 by forming a complex with ANGPTL8 (A8), and another part of the full-length ANGPTL3 can directly bind to HSPGs through its C-terminus.

[0060] Figure 5 .Lipid phenotypes of Angptl3- / - mice and Angptl8- / - mice.

[0061] 5A. Serum triglycerides and total cholesterol (Total Chol) in Angptl3 knockout mice (Angptl3- / -) were significantly lower than those in control mice (n=9-10 / group, male, 8-12 weeks);

[0062] 5B. Serum triglyceride levels in Angptl8 knockout mice (Angptl8- / -) were significantly reduced, but total cholesterol (Total Chol) levels did not change significantly (n=6 / group, male, 9-15 weeks);

[0063] NS: no significant difference, **p<0.01, ***p<0.001.

[0064] Figure 6 .Inhibition of ANGPTL8-independent ANGPTL3 significantly reduced serum total cholesterol levels but did not affect triglyceride levels.

[0065] 6A. Inhibitory antibody against ANGPTL3 (A3 mAb) significantly reduces serum cholesterol level under starvation without affecting triglyceride level: Monoclonal antibody against the N-terminus of ANGPTL3 was injected into wild-type mice (20 g / g). Four days later, the mice were starved overnight and serum was collected for determination of cholesterol and triglyceride content (N=5-6 / group, female, 9-22 weeks);

[0066] 6B. Inhibitory antibody against ANGPTL3 (A3 mAb) significantly reduced serum cholesterol level in Angptl8- / - mice under starvation without affecting triglyceride level: Monoclonal antibody against the N-terminus of ANGPTL3 was injected into Angptl8- / - mice (20 g / g), and mice were starved overnight 4 days later, and serum was collected for determination of cholesterol and triglyceride content (N=7 / group, female, 7-16 weeks);

[0067] 6C. Inhibitory antibody against ANGPTL3 (A3 mAb) significantly reduced serum cholesterol levels in Angptl8- / - mice after feeding without affecting triglyceride levels: The data were from the same batch of mice as those in Figure B. The mice were fed for 4 hours after being starved overnight, and then serum was collected for determination of cholesterol and triglyceride levels;

[0068] 6D. Inhibition of ANGPTL3 activity significantly reduced the levels of serum apolipoproteins ApoB-100 and ApoB-48.

[0069] Figure 7 .Schematic diagram of the ANGPTL3 protein structure (SS: signal peptide).

[0070] Figure 8 .Schematic diagram of LPL-catalyzed tissue triglyceride absorption (LPL: lipoprotein hydrolase, TG: triglyceride, FFA: free fatty acid). DETAILED DESCRIPTION

[0071] Example 1: Inhibition of ANGPTL3 activity or ANGPTL8 expression leads to cardiac lipid accumulation

[0072] 1. Injection of ANGPTL3 antibody drug into wild-type mice

[0073] Inhibitory monoclonal antibodies targeting the N-terminus of ANGPTL3 have been approved for the treatment of homozygous familial hypercholesterolemia and have achieved significant cholesterol-lowering effects. However, the drug also significantly reduces serum triglyceride levels. Whether it will lead to ectopic accumulation of lipids and abnormal cardiac function is unclear.

[0074] Experimental method: The antibody was injected through the tail vein, and the amount of antibody was injected according to the weight of each mouse, with a concentration of 20ug / g. After injection, the mice were subjected to three days of feeding (9:00-15:00) / fasting (15:00-9:00) adaptation training. On the fourth day, blood and heart were collected 3 hours after feeding to measure the total cholesterol and triglyceride levels in the blood and the triglyceride level in the heart.

[0075] The results are as follows Figure 1As shown, we found that a single injection of the antibody in wild-type mice resulted in accumulation of triglycerides (TG) in the mouse heart ( Figure 1 A). In line with this, Angptl3 knockout mice (Angptl3 - / - ) Heart triglyceride levels also increased significantly ( Figure 1 B). The expression of ANGPTL8 is induced by food intake. Previous studies have found that ANGPTL8 can form a protein complex with ANGPTL3 to jointly inhibit the activity of LPL and regulate serum triglyceride metabolism. - / - ) Heart triglyceride levels also increased significantly ( Figure 1 B). These data suggest that inhibition of ANGPTL3's ability to regulate LPL leads to cardiac lipid accumulation.

[0076] 2. Injection of ANGPTL3 antibody drug into ApoE knockout mice

[0077] Excessive accumulation of lipids can lead to lipotoxicity, which is an important cause of diabetic cardiomyopathy. ApoE gene knockout mice are a commonly used hyperlipidemia mouse model that can better simulate familial hypercholesterolemia patients.

[0078] Experimental method: Each mouse was injected with 20ug / g of the antibody through the tail vein according to its body weight every week, and blood lipid levels were measured from the tail vein every week. At the same time, the Vevo3100 imaging system equipped with the MX550D sensor was used to perform mouse echocardiography under anesthesia to detect the heart function of the mice.

[0079] The results are as follows Figure 2 As shown in Figure 2, we found that the heart function of ApoE knockout mice injected with ANGPTL3 antibody drug decreased in the third week, and by the seventh week, the heart function was significantly lower than that of the control group ( Figure 2 A). To further verify this result, we also analyzed Angptl8 knockout mice. Because the blood lipid levels of these mice were normal, no obvious abnormalities in cardiac function were observed under background conditions. However, when we used aortic arch constriction (TAC) to perform a pressure stress on the mouse hearts, Angptl8 - / - The heart function of mice was significantly lower than that of control mice ( Figure 2 B). This result suggests that inhibiting the activity of ANGPTL3 or ANGPTL8 not only leads to cardiac lipid accumulation, but also causes or accelerates the occurrence of heart failure under stress conditions.

[0080] Example 2: ANGPTL3 exists in two binding forms in peripheral tissues

[0081] 1. ANGPTL3 is a liver-expressed secretory factor that mainly functions in peripheral tissues. To further study the physiological function of ANGPTL3, we systematically analyzed how ANGPTL3 is recruited to peripheral tissues.

[0082] Experimental methods:

[0083] ANGPTL8- / - mice and GPIHBP1- / - mice were trained to adapt to eating / fasting for three days. On the fourth day, three hours after eating, blood, heart, and white adipose tissue samples were quickly collected to detect ANGPTL3 protein levels in mouse serum, heart, and white adipose tissue. Figure 3 A, B). To confirm the binding of ANGPTL3 to ANGPTL8 and GPIHBP1,

[0084] Construction of GPIHBP1 or GFP expression plasmid: The cDNA fragment of mouse Gpihbp1 / cDNA fragment of GFP (the cDNA sequence of mouse GPIHBP1 is shown in SEQ ID NO.3, and the cDNA sequence of GFP is shown in SEQ ID NO.4) was PCR amplified and purified, and then ligated to the EcoRⅠ and BamHⅠ restriction sites of pLVX-IRES-Puro plasmid (Clontech, 632183) by restriction digestion and enzyme ligation. The constructed plasmid was transformed into competent DH5α for expansion culture to obtain a large amount of target plasmid.

[0085] A3-N: Human Angptl3-N fusion plasmid with a Flag tag at the C-terminus was expressed in Hek293 cells by transient transfection (the Flag tag with the base sequence of GATTACAAGGACGACGATGACAAG was connected to the C-terminus of human Angptl3-N (the cDNA sequence of human Angptl3-N is shown in SEQ ID NO.6) by PCR, amplified and purified, and then ligated to the EcoRI and BamHI restriction sites of the pLVX-IRES-Puro plasmid by enzyme ligation, and the constructed plasmid was transformed into competent DH5α for expansion culture to obtain a large amount of target plasmid), serum-free culture medium was collected and purified using M2 anti-Flag resin, and protein purity was evaluated by Coomassie staining;

[0086] A3-N&A8 complex: co-transfect HEK293 cells with a human Angptl3-N fusion plasmid with a Flag at the C-terminus and a human Angptl8 plasmid with a Strep tag at the C-terminus (the Strep tag with a base sequence of TGGAGCCACCCGCAGTTCGAAAAA was connected to the C-terminus of human Angptl8 (the mouse ANGPTL8 cDNA sequence is shown in SEQ ID NO.5) by PCR, amplified and purified, and then ligated to the EcoRⅠ and BamHⅠ restriction sites of the pLVX-IRES-Puro plasmid by enzyme digestion and ligation. The constructed plasmid was transformed into competent DH5α for expansion culture to obtain a large amount of target plasmid), the serum-free culture medium was collected and purified with Strep-Tactin Resin according to the instructions, and the protein purity was evaluated by Coomassie staining.

[0087] CHO cells were transfected with GPIHBP1 or GFP expression plasmids. After 60 hours, the cells were incubated with 1ug / mL A3-N and A3-N&A8 complex at 37°C for 15min, and then the cells were collected for immunoblot analysis. CHO cells were transfected with GPIHBP1 or GFP expression plasmids. After 60 hours, the cells were incubated with LPL medium at 37°C for 10min. After washing three times with PBS-CM, the cells were incubated with 1ug / mL A3-N and A3-N&A8 complex at 37°C for 15min, and then the medium and cells were collected for immunoblot analysis ( Figure 3 C, D). To determine the binding mode of the C-terminus of ANGPTL3, the C-terminal form of A3 protein (human ANGPTL3-C-terminus, 241-455Aa) was incubated with HepG2 cells at 37°C for 10 min, then washed three times with PBS, and then incubated with buffer or 10U / mL Heparin in DMEM at 37°C for 15 min, and the cells were collected for immunoblot analysis; at the same time, A3-C-WT protein (human ANGPTL3-C-terminus, 241-455Aa) and A3-C-MUT protein (human ANGPTL3-C-terminus, 241-455Aa containing mutations R421A, K423A, K425A, R428A, R429A, R430A) were incubated with HepG2 cells at 37°C for 10 min, and the cells were collected for immunoblot analysis ( Figure 4 A, B).

[0088] The amino acid sequence of the A3-C-WT protein (i.e., the C-terminal form of the A3 protein) is shown in SEQ ID NO.2.

[0089] The amino acid sequence of the A3-C-MUT protein (SEQ ID NO.7) is:

[0090] TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN

[0091] ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAIT

[0092] GNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPAAASAPEAAAGLSWKSQNGRLYSIKSTKMLIHPTDSESFE.

[0093] The results are as follows Figure 3 As shown, using heart and epiWAT as two representative peripheral tissues, we found that feeding significantly increased the binding of the full length, N-terminus, and C-terminus of ANGPTL3 in these tissues ( Figure 3 A). We also found that all N-terminals that bind to peripheral tissues are dependent on the expression of ANGPTL8, while full-length ANGPTL3 is only partially dependent on ANGPTL8, and its C-terminal fragments are basically independent of ANGPTL8 in binding to peripheral tissues ( Figure 3 A). We also found that the binding of all N-terminal and part of full-length ANGPTL3 to peripheral tissues depends on GPIHBP1, while the binding of its C-terminal to peripheral tissues is completely independent of GPIHBP1 ( Figure 3 B).

[0094] 2. Exploring how ANGPTL3 binds to ANGPTL8 and GPIHBP1

[0095] To further investigate how ANGPTL3 binds to ANGPTL8 and GPIHBP1, we prepared ANGPTL3 N-terminal (A3-N) recombinant protein and A3-N and ANGPTL8 (A8) complex protein. By incubating these proteins with CHO cells expressing GFP (control) and GPIHBP1, we found that A8 could promote the binding of A3-N to GPIHBP1 ( Figure 3 C). At the same time, the A3-N / A8 protein complex can also release LPL bound to GPIHBP1 into the supernatant ( Figure 3 D), indicating that the protein complex has the ability to inhibit LPL. We further detected the activity of residual LPL on cells under different treatment conditions, and found that the A3-N / A8 protein complex significantly inhibited the activity of LPL on cells ( Figure 3E) Based on this, we propose that the A3-N / A8 protein complex inhibits LPL activity. Figure 3 As shown in F, the A3-N / A8 protein complex inactivates LPL by dissociating LPL from GPIHBP1.

[0096] 3. A3 functions in two forms in peripheral tissues

[0097] 1. The C-terminus (A3-C) of ANGPTL3 bound to the cell membrane can be released into the culture medium by heparin

[0098] HepG2 cells were incubated with A3-C or GFP conditioned medium, washed, and then incubated with fresh medium containing heparin (Hepa) or blank control (NC). The collected supernatant (S), cell lysate (Cells) and initial conditioned medium were then subjected to corresponding protein immunoblot analysis.

[0099] The results are as follows Figure 4 As shown in A, Figure 4 The C-terminus (A3-C) sequence of ANGPTL3 in A is SEQ ID NO.2. The binding of the C-terminus (A3-C) of ANGPTL3 to peripheral tissues is independent of A8 and GPIHBP1, suggesting that it may bind to peripheral tissues in other ways. We found that A3-C binding to cells can be inhibited by heparin ( Figure 4 A). Heparin is highly negatively charged. This result suggests that A3-C may bind to negatively charged heparan sulfate proteoglycans (HSPGs) in peripheral tissues through its positively charged groups.

[0100] 2. ANGPTL3 C-terminus binds to HepG2 cells through conserved positively charged amino acid residues

[0101] Wild-type (WT) A3-C and A3-C (MUT) conditioned media containing the indicated mutations were incubated with HepG2 cells: the media and cells were then collected and analyzed by immunoblotting. Figure 4 As shown in B, Figure 4 The C-terminal (A3-C) sequence of ANGPTL3 in B is SEQ ID NO. 1, and the sequence alignment of the positively charged motif at the terminal of A3-C is shown at the top, and the positively charged residues in red are mutated to alanine in the mutant A3-C.

[0102] We found that there is a very conserved positively charged group in the C-terminal region of ANGPTL3. If the six positively charged amino acids in this group are mutated into uncharged alanine, the binding of A3-C to the cell membrane is completely inhibited ( Figure 4 B).

[0103] Based on this, we propose a model for the function of ANGPTL3 in peripheral tissues. All A3-Ns are anchored to GPIHBP1 through A8, and some full-length A3s can bind to GPIHBP1 by forming a complex with A8 after removing the N-terminus, while free full-length A3s can directly bind to HSPGs through its C-terminal positively charged group ( Figure 4 C) These results suggest that A3 functions in two forms in peripheral tissues, one by forming a complex with A8 and the other by directly binding to HSPGs.

[0104] Example 3: Inhibition of ANGPTL8-independent ANGPTL3 specifically lowers LDL-C without affecting triglyceride metabolism

[0105] 1. Experimental Methods

[0106] 1. Construction of Angptl3 knockout mice

[0107] Angptl3- / - mice (Jicui Yaokang, strain number T006230) were used to obtain Cas9 mRNA and gRNA by in vitro transcription; Cas9 mRNA and gRNA were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 mice. F0 mice (chimeras) that were positive for PCR amplification and sequencing were mated with C57BL / 6J mice to obtain F1 mice, and F1 mice were self-fertilized to obtain Angptl3- / - mice, knocking out the exon1-6 region of the Angptl3 gene.

[0108] 2. Construction of Angptl8 knockout mice

[0109] Angptl3- / - mice (Shanghai Model Organisms, strain number NM-KO-190241) were used to obtain Cas9 mRNA and gRNA by in vitro transcription; Cas9 mRNA and gRNA were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 mice. F0 mice (chimeras) that were positive for PCR amplification and sequencing were mated with C57BL / 6J mice to obtain F1 mice, and F1 mice were self-bred to obtain Angptl8- / - mice, knocking out the exon1-4 region of the Angptl8 gene.

[0110] 3. Detection of serum triglyceride and total cholesterol levels

[0111] After the blood was collected, it was placed at room temperature for 60 minutes, and then centrifuged at 3500g for 10 minutes at 4°C to collect serum. The levels of triglycerides and total cholesterol in serum were detected using an enzymatic kit (Shanghai Kehua Bioengineering Co., Ltd.). 5ul of serum was added to the enzyme-labeled wells, and R1 and R2 reagents in the kit were mixed in proportion, and 150ul was added to each well and mixed with serum. The mixture was reacted at 37°C for 15 minutes, and the values ​​were read at wavelengths of 546nm and 660nm.

[0112] The results are as follows Figure 5 As shown, using a knockout mouse model, we found that Angptl3 knockout mice (Angptl3 - / - ) significantly reduced serum triglyceride and total cholesterol levels ( Figure 5 A), while Angptl8 knockout mice (Angptl8 - / - ) only significantly reduced total cholesterol levels ( Figure 5 B). This result indicates that ANGPTL3 (N-terminus) that forms a complex with ANGPTL8 in peripheral tissues mainly regulates serum triglyceride metabolism, while ANGPTL8-independent ANGPTL3 (C-terminus) mainly regulates serum cholesterol metabolism.

[0113] The expression level of ANGPTL8 is induced by food intake and is almost not expressed in the starved state. To further verify the above hypothesis, we injected ANGPTL3 inhibitory antibodies into mice starved overnight. Figure 6 As shown in Figure 2, we found that inhibition of ANGPTL3 only significantly reduced serum cholesterol levels, but had no significant effect on serum triglyceride levels ( Figure 6 A). We further investigated the relationship between the expression of Angptl8 gene in Angptl8 knockout mice (Angptl8 - / - ) and found that inhibiting ANGPTL3 activity only significantly reduced serum cholesterol levels, but had no significant effect on serum triglyceride levels, whether in a hungry state or in a fed state. Figure 6 B, C). At the same time, inhibition of ANGPTL3 activity significantly reduced the levels of serum apolipoprotein ApoB-100 and ApoB-48, indicating that the content of low-density lipoprotein LDL was significantly reduced ( Figure 6 D).

[0114] In summary, the above results show that targeting HSPGs-binding ANGPTL3 can specifically reduce serum LDL-C without significantly affecting triglyceride metabolism. ANGPTL3 binds to HSPGs through its C-terminal fragment, so targeted inhibition of ANGPTL3 C-terminal function will be a new strategy for developing specific lowering of serum LDL-C.

[0115] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of an inhibitor targeting the C-terminus of ANGPLT3 protein (Angiopoietin-like 3) in the preparation of a drug, wherein the inhibitor: (1) Targeting and binding to the C-terminus of ANGPLT3 protein, and (2) inhibiting the functional activity of the C-terminal fragment of ANGPLT3 protein; The inhibitor is an inhibitor at the protein level; The amino acid sequence of the C-terminus of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.2; The medicine is used for treating hypercholesterolemia, atherosclerosis or coronary heart disease.

2. The use according to claim 1, characterized in that The inhibitor comprises at least one of an antibody and a small molecule compound.

3. The use according to claim 1, characterized in that The small molecule compound is heparin.

4. The use according to claim 3, characterized in that The hypercholesterolemia is: (1) familial hypercholesterolemia (FH); or (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).

5. The use according to claim 3, characterized in that: The medicine also includes pharmaceutically acceptable excipients and carriers.

6. The use according to claim 3, characterized in that: The dosage form of the drug includes at least one of granules, tablets, pills, capsules and injections.

7. Use of an inhibitor of a protein targeting the C-terminus of ANGPLT3 protein (Angiopoietin-like 3) in the preparation of a preparation for lowering LDL-C (low-density lipoprotein cholesterol) levels without interfering with triglyceride metabolism, wherein the inhibitor: (1) Targeting and binding to the C-terminus of ANGPLT3 protein, and (2) inhibiting the functional activity of the C-terminal fragment of ANGPLT3 protein; The inhibitors are inhibitors at the protein level, including but not limited to: antibodies, small molecule compounds; The amino acid sequence of the C-terminus of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.

2.

8. Application of the C-terminus of ANGPLT3 protein (Angiopoietin-like 3) in detecting the activity of a drug to be screened, wherein the detection of the activity of a drug to be screened refers to the detection of the following functions of the drug to be screened: (1) Targeting the C-terminus of ANGPLT3 protein, and (2) inhibiting the functional activity of the C-terminal fragment of ANGPLT3 protein; The drug to be screened is an antibody or a small molecule compound.

9. The use according to claim 8, characterized in that: The medicine is used for treating hypercholesterolemia, atherosclerosis or coronary heart disease.

10. The use according to claim 8, characterized in that: The hypercholesterolemia is: (1) familial hypercholesterolemia (FH); or (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).

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