Molecular target that specifically reduces cholesterol level without interfering with triglyceride metabolism and use thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-05-22
- Publication Date
- 2026-08-13
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Figure CN2025096520_13082026_PF_FP_ABST
Abstract
Description
Molecular targets that specifically lower cholesterol levels without interfering with triglyceride metabolism and their applications Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to molecular targets that specifically lower cholesterol levels without interfering with triglyceride metabolism and their applications. Background Technology
[0002] Hypercholesterolemia is a major contributing factor to atherosclerosis and cardiovascular and cerebrovascular diseases. Lowering cholesterol is a primary intervention in the prevention and treatment of these diseases in clinical practice. First-line clinical drugs such as statins and PCSK9 inhibitors mainly lower blood cholesterol by increasing the expression and function of low-density lipoprotein receptors (LDLR), achieving significant efficacy in the prevention and treatment of coronary heart disease and other cardiovascular and cerebrovascular diseases.
[0003] Familial hypercholesterolemia (FH) is a type of hypercholesterolemia caused by genetic mutations. On average, one in every two to three hundred people has a heterozygous FH, and approximately one in a million has a homozygous FH. If homozygous FH is not diagnosed and treated early, it can lead to serious cardiovascular and cerebrovascular diseases, reducing the average lifespan to just over ten years. Heterozygous FH patients also have a 20-30 times higher incidence of coronary heart disease than the general population due to hypercholesterolemia. Population studies have found that over 80% of FH cases are caused by LDLR mutations, especially in homozygous FH patients who have extremely low LDLR activity and show almost no response to statins and PCSK9 inhibitors. Currently, the most effective treatment for these patients remains hemodialysis. Therefore, there is an urgent clinical need to develop new cholesterol-lowering strategies and drugs that are independent of LDLR.
[0004] Cholesterol and triglycerides in the blood are primarily carried by lipoprotein particles. The surface of lipoproteins is covered by a layer of phospholipid molecules, while the interior contains hydrophobic cholesterol and triglyceride molecules. Endothelial cell esterases (EL) possess phospholipid hydrolytic activity, primarily hydrolyzing the phospholipids on lipoproteins, thus affecting the structure of lipoprotein particles. Lipoprotein hydrolases (LPLs), on the other hand, are triglyceride hydrolysases, primarily catalyzing the hydrolysis of triglycerides carried by lipoproteins.
[0005] ANGPTL3 is a liver-specific secretory factor composed of a signal peptide, an N-terminal fragment, and a C-terminal fragment. The signal peptide guides its secretion, the N-terminus contains a functional region that simultaneously inhibits LPL and EL, thus suppressing their activity. The function of the C-terminus is unknown (Figure 7). In the human population, ANGPTL3 gene deletion mutations significantly reduce serum cholesterol and triglyceride levels without causing side effects such as fatty liver. Inhibiting the N-terminal activity of ANGPTL3 increases EL activity, 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 these antibody inhibitors has not been reported.
[0006] Triglycerides in the blood serve as an energy molecule, providing energy for the heart, muscles, and other organs. However, if the heart and muscles absorb too much fat, it will lead to lipotoxicity, causing diseases such as insulin resistance and heart failure. Lipotoxicity is also an important cause of the high incidence of diabetic cardiomyopathy (affecting approximately 19%-26% of diabetic patients).
[0007] LPL is synthesized and secreted by parenchymal cells in peripheral tissues such as the heart, muscle, and adipose tissue, and is transported to the capillary side to perform its function. LPL is anchored to the surface of vascular endothelial cell membranes via high-density lipoprotein-binding protein 1 (GPIHBP1), which is anchored to heparan sulfate proteoglycans (HSPGs) and glycosylphosphatidylinositol, 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. These inhibitors simultaneously increase the activity of EL and LPL, thus lowering serum triglyceride levels while also reducing serum cholesterol levels. However, existing ANGPTL3 inhibitors can lead to excessive uptake of triglycerides by tissues such as the heart and muscles, potentially causing lipotoxicity and side effects such as abnormal cardiac function.
[0009] Therefore, it is necessary to develop a target and drug that lowers serum cholesterol levels without lowering serum triglyceride levels. Summary of the Invention
[0010] This 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) Targets and binds to the C-terminus of the ANGPLT3 protein, and
[0012] (2) Inhibits 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 heparin; or
[0014] The C-terminal amino acid sequence 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: TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
[0017] PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE;
[0018] The aforementioned drug is used to treat hypercholesterolemia, atherosclerosis, and coronary heart disease;
[0019] Preferably, the hypercholesterolemia is as follows:
[0020] (1) Familial hypercholesterolemia (FH); or
[0021] (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).
[0022] This invention also relates to the use of an inhibitor of a protein targeting the C-terminus of ANGPLT3 (Angiopoietin-like 3) in the preparation of formulations that lower LDL-C (low-density lipoprotein cholesterol) levels without significantly interfering with triglyceride metabolism, wherein the inhibitor:
[0023] (1) Targets and binds to the C-terminus of the ANGPLT3 protein, and
[0024] (2) Inhibits the functional activity of the C-terminal fragment of ANGPLT3 protein;
[0025] The inhibitors mentioned are protein-level inhibitors, including but not limited to: antibodies, small molecule compounds; or
[0026] The C-terminal amino acid sequence of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.2.
[0027] This invention also relates to the application of the C-terminal protein of ANGPLT3 (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.
[0028] (1) Targets and binds to the C-terminus of the ANGPLT3 protein, and
[0029] (2) Inhibits the functional activity of the C-terminal fragment of ANGPLT3 protein;
[0030] The drugs to be screened are antibodies or small molecule compounds;
[0031] The drug is used to treat hypercholesterolemia, atherosclerosis, or coronary heart disease; preferably, the hypercholesterolemia is:
[0032] (1) Familial hypercholesterolemia (FH); or
[0033] (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).
[0034] The beneficial effects of this invention are as follows:
[0035] 1. This study demonstrates that inhibiting the N-terminal activity of ANGPTL3 significantly reduces blood cholesterol and triglyceride levels, but it can lead to lipid accumulation in the heart, resulting in heart failure.
[0036] 2. ANGPTL3 functions in peripheral tissues in at least two ways: one is by forming a complex with ANGPTL8 through its N-terminus, and then being anchored to the tissue vascular wall through GPIHBP1; the other is by directly binding to HSPGs through its C-terminus and being anchored to the tissue vascular wall.
[0037] 3. Specifically blocking the function of ANGPTL3 (C-terminus) would be a novel lipid-lowering strategy that reduces LDL-C levels without significantly interfering with triglyceride metabolism;
[0038] 4. Specifically blocking the function of ANGPTL3 (C-terminus) will be a new strategy for the prevention and treatment of diseases such as hypercholesterolemia, atherosclerosis, and coronary heart disease. Attached Figure Description
[0039] Figure 1. Inhibition of ANGPTL3 activity or ANGPTL8 expression leads to cardiac lipid accumulation.
[0040] 1A. Increased triglyceride content in the heart of wild-type mice by an inhibitory antibody against ANGPTL3 (A3 mAb): Wild-type mice were injected with a monoclonal antibody against the N-terminus of ANGPTL3 (20 ug / g), and hearts were collected 7 days later for analysis of cardiac lipid content.
[0041] 1B. Increased cardiac triglyceride levels in Angptl3 knockout mice (Angptl3- / -);
[0042] 1C. Increased cardiac triglyceride levels in Angptl8 knockout mice (Angptl8- / -);
[0043] TG: Triglycerides. Data are expressed as mean ± standard error. p<0.01, p<0.001.
[0044] Figure 2. Inhibition of ANGPTL3 activity or ANGPTL8 expression leads to heart failure.
[0045] 1A. Inhibitory antibody against ANGPTL3 (A3 mAb) causes heart failure: A monoclonal antibody against the N-terminus of ANGPTL3 was injected into ApoE- / - hyperlipidemic mice (20 ug / g, once a week), and cardiac function was assessed by small animal ultrasound at weeks 1, 3, and 5 (N=7-8 / group, male, 17 weeks).
[0046] 1B. Angptl8 knockout mice (Angptl8- / -) exhibited heart failure under pressure: Angptl8- / - and control mice were subjected to cardiac pressure induction using aortic arch coarctation (TAC), and cardiac function was assessed weekly by small animal ultrasound after surgery (N=7-8 / group, male, 11-18 weeks).
[0047] LVEF: Left ventricular ejection fraction; LVFS: Left ventricular shortening fraction. p<0.01, p<0.001.
[0048] Figure 3. The N-terminus of ANGPTL3 binds to tissue GPIHBP1 via ANGPTL8.
[0049] 3A. ANGPTL3 protein levels in Angptl8- / - mice and littermate wild-type control mice (n=3 / group, female, 8–15 weeks; F: starvation, R: postprandial).
[0050] 3B. ANGPTL3 protein levels in Gpihbp1- / - mice and littermate wild-type control mice in a postprandial state (n=6 / group, male, 7-8 weeks; KO: samples from Angptl3- / - mice).
[0051] 3C, ANGPTL8 (A8) promotes the binding of the N-terminus (A3-N) of ANGPTL3 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 lysates and incubation medium were analyzed by Western blot analysis accordingly.
[0052] 3D. The A3-N and A8 complex dissociates LPL from GPIHBP1: CHO cells expressing GPIHBP1 (GP1) were incubated with LPL conditioned medium. After washing, the A3-N and A8 protein complex (A3-N / A8, 1 mg / ml) was added to the cells containing fresh medium. The medium (S) was then collected and the cells were subjected to corresponding protein immunoblotting analysis (PBS, phosphate buffer).
[0053] 3E. Cell-bound LPL activity assay (mmol / L / hr): All experimental procedures were exactly the same as in Figure D. After the final wash, the cells were incubated in fresh medium containing heparin (10 U / ml) for 15 minutes (37°C), and the supernatant was collected for LPL activity assay. The three bars from left to right represent the same three groups of samples as in Figure D.
[0054] 3F. Schematic diagram of the dissociation of LPL from GPIHBP1 by the A3-N and A8 complex.
[0055] FL: Full-length ANGPTL3, N: N-terminal ANGPTL3, C: C-terminal ANGPTL3, PonS: Ponceau S, CNX: Calnexin
[0056] p<0.01.
[0057] Figure 4. The C-terminus (A3-C) of ANGPTL3 binds to tissue via heparan sulfate proteoglycans (HSPGs).
[0058] 4A. The C-terminus (A3-C) of ANGPTL3, which is 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 then incubated with fresh medium containing heparin (Hepa) or blank control (NC). Subsequently, the collected supernatant (S), cell lysate (Cells), and the initial conditioned medium were subjected to corresponding Western blot analysis.
[0059] 4B. The C-terminus of ANGPTL3 binds to HepG2 cells via conserved positively charged amino acid residues: HepG2 cells were incubated with wild-type (WT) A3-C and A3-C containing a specified mutation (MUT) in conditioned medium; subsequently, the medium and cells were collected and analyzed by immunoblotting. Sequence alignment of the positively charged motif at the A3-C terminus is shown at the top, with the red positively charged residue mutated to alanine in the mutant A3-C;
[0060] 4C. Schematic diagram of ANGPTL3 binding to tissue vascular wall: The N-terminus (N) of ANGPTL3 and part of the full-length ANGPTL3 bind to GPIHBP1 by forming a complex with ANGPTL8 (A8), while another part of the full-length ANGPTL3 can directly bind to HSPGs through its C-terminus.
[0061] Figure 5. Lipid phenotypes of Angptl3- / - and Angptl8- / - mice.
[0062] 5A. Serum triglycerides and total cholesterol (Total Chol) were significantly lower in Angptl3 knockout mice (Angptl3- / -) than in control mice (n=9-10 / group, male, 8-12 weeks).
[0063] 5B. Angptl8 knockout mice (Angptl8- / -) showed significantly reduced serum triglycerides, but no significant change in total cholesterol levels (n=6 / group, male, 9-15 weeks).
[0064] NS: No significant difference. p<0.01, p<0.001.
[0065] Figure 6. Inhibition of ANGPTL8-independent ANGPTL3 significantly reduced serum total cholesterol levels but did not affect triglyceride levels.
[0066] 6A. An inhibitory antibody against ANGPTL3 (A3 mAb) significantly reduced serum cholesterol levels without affecting triglyceride levels under starvation: A monoclonal antibody against the N-terminus of ANGPTL3 was injected into wild-type mice (20 ug / g). After 4 days of starvation, the mice were starved overnight, and serum was collected for cholesterol and triglyceride content determination (N=5-6 / group, female, 9-22 weeks).
[0067] 6B. An inhibitory antibody against ANGPTL3 (A3 mAb) significantly reduced serum cholesterol levels in starved Angptl8- / - mice without affecting triglyceride levels: A monoclonal antibody against the N-terminus of ANGPTL3 was injected into Angptl8- / - mice (20 ug / g). After 4 days of starvation, the mice were starved overnight, and serum was collected for cholesterol and triglyceride content determination (N=7 / group, female, 7-16 weeks).
[0068] 6C. The inhibitory antibody against ANGPTL3 (A3 mAb) significantly reduced serum cholesterol levels in Angptl8- / - mice after feeding without affecting triglyceride levels: This data and Figure B are from the same batch of mice. The mice were starved overnight and then fed for 4 hours before serum was collected for cholesterol and triglyceride content determination.
[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). Embodiments of the present invention
[0071] Example 1: Inhibition of ANGPTL3 activity or ANGPTL8 expression leads to cardiac lipid accumulation.
[0072] I. Injection of ANGPTL3 antibody drug into wild-type mice
[0073] An inhibitory monoclonal antibody targeting the N-terminus of ANGPTL3 has been approved for the treatment of homozygous familial hypercholesterolemia, achieving significant cholesterol-lowering effects. However, the drug also significantly reduces serum triglyceride levels, and it remains unclear whether this will lead to ectopic lipid accumulation and cardiac dysfunction.
[0074] Experimental methods: The antibody was injected via tail vein, with the dosage determined based on the body weight of each mouse, at a concentration of 20 μg / g. Following injection, mice underwent three days of acclimatization training, including feeding (9:00-15:00) and fasting (15:00-9:00). On the fourth day, blood and heart samples were collected three hours after feeding to measure total cholesterol and triglyceride levels in the blood and triglyceride levels in the heart.
[0075] The results are shown in Figure 1. We found that a single injection of this antibody in wild-type mice led to triglyceride (TG) accumulation in the mouse heart (Figure 1A). Consistent with this, Angptl3 knockout mice (Angptl3...) - / - Cardiac triglyceride levels were also significantly increased (Figure 1B). ANGPTL8 expression is induced by food intake. Previous studies have found that ANGPTL8 can form a protein complex with ANGPTL3, jointly inhibiting LPL activity and regulating serum triglyceride metabolism. Angptl8 knockout mice (Angptl8 - / - The cardiac triglyceride levels were also significantly increased (Figure 1B). These data indicate that inhibiting the ability of ANGPTL3 to regulate LPL leads to lipid accumulation in the heart.
[0076] II. Injection of ANGPTL3 antibody into ApoE gene knockout mice
[0077] Excessive lipid accumulation can lead to lipotoxicity, a significant contributing factor to diabetic cardiomyopathy. ApoE knockout mice are a commonly used mouse model of hyperlipidemia, effectively mimicking patients with familial hypercholesterolemia.
[0078] Experimental methods: Each mouse was injected weekly with 20 μg / g of the antibody via the tail vein, based on its body weight. Blood lipid levels were measured weekly from the tail vein. Simultaneously, mouse echocardiography was performed under anesthesia using a Vevo3100 imaging system equipped with an MX550D sensor to assess cardiac function.
[0079] The results are shown in Figure 2. We found that in ApoE knockout mice, injection of the ANGPTL3 antibody drug resulted in decreased cardiac function as early as week 3, and by week 7, cardiac function was significantly lower than that of the control group (Figure 2A). To further verify this result, we also analyzed Angptl8 knockout mice. Because these mice had normal blood lipid levels, no obvious cardiac dysfunction was observed under baseline conditions. However, when we subjected the mouse hearts to stress using aortic arch coarctation (TAC), Angptl8... - / -The cardiac function of the mice was significantly lower than that of the control group (Figure 2B). This result indicates that inhibiting the activity of ANGPTL3 or ANGPTL8 not only leads to lipid accumulation in the heart, but also causes or accelerates the occurrence of heart failure under stress.
[0080] Example 2: ANGPTL3 exists in two binding forms in peripheral tissues.
[0081] I. ANGPTL3 is a liver-expressed secretory factor that primarily functions in peripheral tissues. To further investigate the physiological functions of ANGPTL3, we systematically analyzed how ANGPTL3 is recruited to peripheral tissues.
[0082] Experimental methods:
[0083] ANGPTL8- / - and GPIHBP1- / - mice underwent three days of feeding / fasting acclimatization training. On the fourth day, three hours after feeding, blood, heart, and white adipose tissue samples were rapidly collected to detect ANGPTL3 protein levels in mouse serum, heart, and white adipose tissue (Figure 3A, B). To confirm the binding of ANGPTL3 to ANGPTL8 and GPIHBP1,
[0084] Construction of GPIHBP1 or GFP expression plasmids: The cDNA fragments of mouse Gpihbp1 and 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) were amplified by PCR and purified, and then ligated to the EcoRI and BamHI restriction sites of the pLVX-IRES-Puro plasmid (Clontech, 632183) by enzyme digestion and ligation. The constructed plasmids were transformed into competent DH5α cells for amplification culture to obtain a large number of target plasmids.
[0085] A3-N: The human Angptl3-N fusion plasmid with a Flag tag at the C-terminus was expressed by transient transfection into Hek293 cells. (The Flag tag, with the base sequence GATTACAAGGACGACGATGACAAG, was ligated to the C-terminus of human Angptl3-N (the cDNA sequence of human Angptl3-N is shown in SEQ ID NO. 6) by PCR. After amplification and purification, it was ligated to the EcoRI and BamHI restriction sites of the pLVX-IRES-Puro plasmid by enzyme digestion. The constructed plasmid was transformed into competent DH5α cells for amplification culture to obtain a large number of target plasmids. Serum-free culture medium was collected and purified using M2 anti-Flag resin. The protein purity was assessed by Coomassie staining.
[0086] A3-N & A8 complex: 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 were co-transfected into HEK293 cells. (The Strep tag, with the base sequence TGGAGCCACCCGCAGTTCGAAAAA, was ligated to the C-terminus of the human Angptl8 plasmid (the mouse Angptl8 cDNA sequence is shown in SEQ ID NO.5) by PCR. After amplification and purification, the plasmid was ligated to the EcoRI and BamHI restriction sites of the pLVX-IRES-Puro plasmid by enzyme digestion. The constructed plasmid was transformed into competent DH5α cells for amplification culture to obtain a large number of target plasmids.) Serum-free culture medium was collected and purified by Strep-Tactin Resin according to the instructions. The protein purity was assessed by Coomassie staining.
[0087] CHO cells were transfected with GPIHBP1 or GFP expression plasmids. After 60 hours, the cells were incubated with 1 μg / mL A3-N and A3-N&A8 complex at 37°C for 15 min, and then the cells were collected for immunoblotting analysis. Simultaneously, CHO cells were transfected with GPIHBP1 or GFP expression plasmids. After 60 hours, the cells were incubated with LPL medium at 37°C for 10 min. After washing three times with PBS-CM, the cells were incubated with 1 μg / mL A3-N and A3-N&A8 complex at 37°C for 15 min, and then the medium and cells were collected for immunoblotting analysis (Figures 3C, D). To determine the binding mode of the C-terminus of ANGPTL3, HepG2 cells were incubated with the C-terminal form of A3 protein (human ANGPTL3-C-terminus, 241-455Aa) at 37°C for 10 min, washed three times with PBS, and then incubated with buffer or 10 U / mL Heparin in DMEM at 37°C for 15 min. Cells were then collected for immunoblotting analysis. Simultaneously, HepG2 cells were incubated with A3-C-WT protein (human ANGPTL3-C-terminus, 241-455Aa) and A3-C-MUT protein (human ANGPTL3-C-terminus, 241-455Aa containing mutants R421A, K423A, K425A, R428A, R429A, R430A) at 37°C for 10 min, and cells were collected for immunoblotting analysis (Figure 4A, 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 (SEQ ID NO.7) of the A3-C-MUT protein is as follows:
[0090] TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPAAASAPEAAAGLSWKSQNGRLYSIKSTKMLIHPTDSESFE.
[0091] The results are shown in Figure 3. Using the heart and white adipose tissue (epiWAT) as two representative peripheral tissues, we found that food intake significantly increased the binding of the full-length, N-terminal, and C-terminal ANGPTL3 in these tissues (Figure 3A). We also found that the N-terminal binding in all peripheral tissues was dependent on ANGPTL8 expression, while the full-length ANGPTL3 was only partially dependent on ANGPTL8, and the C-terminal binding to peripheral tissues was essentially independent of ANGPTL8 (Figure 3A). Furthermore, we found that the binding of all N-terminal and part of the full-length ANGPTL3 to peripheral tissues was dependent on GPIHBP1, while the C-terminal binding to peripheral tissues was completely independent of GPIHBP1 (Figure 3B).
[0092] II. Investigating how ANGPTL3 binds to ANGPTL8 and GPIHBP1
[0093] To further investigate how ANGPTL3 binds to ANGPTL8 and GPIHBP1, we prepared recombinant ANGPTL3 N-terminus (A3-N) protein and the A3-N / ANGPTL8 (A8) complex protein, respectively. By incubating these proteins with CHO cells expressing GFP (control) and GPIHBP1, we found that A8 promotes the binding of A3-N to GPIHBP1 (Figure 3C). Simultaneously, the A3-N / A8 protein complex also released LPL bound to GPIHBP1 into the supernatant (Figure 3D), indicating that this protein complex has the ability to inhibit LPL. Furthermore, we examined the activity of residual LPL on cells under different treatment conditions and found that the A3-N / A8 protein complex significantly inhibited LPL activity on cells (Figure 3E). Based on this, we propose the mechanism of action of the A3-N / A8 protein complex in inhibiting LPL activity. As shown in Figure 3F, the A3-N / A8 protein complex inactivates LPL by dissociating LPL from GPIHBP1.
[0094] III. A3 functions in two forms in peripheral tissues.
[0095] 1. The C-terminus (A3-C) of ANGPTL3, which is bound to the cell membrane, can be released into the culture medium by heparin.
[0096] HepG2 cells were incubated with A3-C or GFP conditioned medium and washed. The cells were then incubated with fresh medium containing heparin (Hepa) or blank control (NC). Subsequently, the collected supernatant (S), cell lysate (Cells), and the initial conditioned medium were subjected to corresponding Western blot analysis.
[0097] The results are shown in Figure 4A. The C-terminal (A3-C) sequence of ANGPTL3 in Figure 4A 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 through other mechanisms. We found that A3-C bound to cells can be inhibited by heparin (Figure 4A). Heparin is strongly negatively charged. This result suggests that A3-C may bind to negatively charged heparan sulfate proteoglycans (HSPGs) in peripheral tissues through its positively charged group.
[0098] 2. The C-terminus of ANGPTL3 binds to HepG2 cells via conserved positively charged amino acid residues.
[0099] HepG2 cells were incubated with conditioned media containing wild-type (WT) A3-C and A3-C containing a specified mutation (MUT). The media and cells were then collected and analyzed by immunoblotting. The results are shown in Figure 4B. The C-terminal (A3-C) sequence of ANGPTL3 in Figure 4B is SEQ ID NO.1. The sequence alignment of the positively charged motif at the A3-C terminal is shown at the top, with the red positively charged residue mutated to alanine in the mutant A3-C.
[0100] We found a highly conserved positively charged group in the C-terminal region of ANGPTL3. If the six positively charged amino acids in this group are mutated to uncharged alanine, the binding of A3-C to the cell membrane is completely inhibited (Figure 4B).
[0101] Based on this, we propose a model for the function of ANGPTL3 in peripheral tissues. All A3-N atoms are anchored to GPIHBP1 via A8. Some full-length A3 atoms can bind to GPIHBP1 by forming a complex with A8 through N-terminus removal, while free full-length A3 atoms can directly bind to HSPGs through their positively charged C-terminal groups (Figure 4C). These results indicate that A3 functions in peripheral tissues in two ways: one is through forming a complex with A8, and the other is through direct binding to HSPGs.
[0102] Example 3: Inhibiting ANGPTL8-independent ANGPTL3-specific LDL-C reduction without affecting triglyceride metabolism.
[0103] I. Experimental Methods
[0104] 1. Construction of Angptl3 gene knockout mice
[0105] Angptl3- / - mice (Jicui Yaokang, strain number T006230) were used to obtain Cas9 mRNA and gRNA through in vitro transcription. Cas9 mRNA and gRNA were microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. PCR amplification and sequencing confirmed that the F0 generation mice (chimeras) were positive. These mice were then mated with C57BL / 6J mice to obtain F1 generation mice. F1 generation mice were self-crossed to obtain Angptl3- / - mice, with the exon 1-6 region of the Angptl3 gene knocked out.
[0106] 2. Construction of Angptl8 gene knockout mice
[0107] Angptl3- / - mice (Nanmo Biotechnology, strain number NM-KO-190241) were used to obtain Cas9 mRNA and gRNA through in vitro transcription. Cas9 mRNA and gRNA were microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. PCR amplification and sequencing confirmed that the F0 generation mice (chimeras) were positive. These mice were then mated with C57BL / 6J mice to obtain F1 generation mice. F1 generation mice were self-crossed to obtain Angptl8- / - mice, with the exon 1-4 region of the Angptl8 gene knocked out.
[0108] 3. Detection of serum triglyceride and total cholesterol levels
[0109] After blood collection, the blood was allowed to stand at room temperature for 60 minutes, then centrifuged at 3500g at 4℃ for 10 minutes to collect serum. Serum triglyceride and total cholesterol levels were detected using an enzyme assay kit (Shanghai Kehua Bioengineering Co., Ltd.). 5 μL of serum was added to each well. Reagents R1 and R2 from the kit were mixed in the correct proportions, and 150 μL of the mixture was added to each well. The mixture was incubated at 37℃ for 15 minutes, and values were recorded at wavelengths of 546 nm and 660 nm.
[0110] The results are shown in Figure 5. Using the gene knockout mouse model, we found that Angptl3 gene knockout mice (Angptl3 - / - ) significantly reduced serum triglyceride and total cholesterol levels (Figure 5A), while Angptl8 knockout mice (Angptl8 - / -It only significantly reduced total cholesterol levels (Figure 5B). This result indicates that ANGPTL3 (N-terminus), which forms a complex with ANGPTL8 in peripheral tissues, mainly regulates serum triglyceride metabolism, while ANGPTL8-independent ANGPTL3 (C-terminus) mainly regulates serum cholesterol metabolism.
[0111] ANGPTL8 expression is induced by feeding and is almost non-existent under starvation. To further verify this hypothesis, we injected ANGPTL3 inhibitory antibodies into mice that had been starved overnight. The results are shown in Figure 6. We found that under these conditions, inhibiting ANGPTL3 significantly reduced serum cholesterol levels but had no significant effect on serum triglyceride levels (Figure 6A). Furthermore, we investigated this in Angptl8 knockout mice (Angptl8...). - / - Inhibiting ANGPTL3 activity, we found that regardless of whether the patient was in a starvation state or a post-meal state, inhibiting ANGPTL3 activity only significantly reduced serum cholesterol levels, while having no significant effect on serum triglyceride levels (Figure 6B, C). Simultaneously, inhibiting ANGPTL3 activity significantly reduced the levels of serum apolipoproteins ApoB-100 and ApoB-48, indicating a significant reduction in low-density lipoprotein (LDL) levels (Figure 6D).
[0112] In summary, the results show that ANGPTL3 targeting HSPGs can specifically reduce serum LDL-C without significantly affecting triglyceride metabolism. Since ANGPTL3 binds to HSPGs through its C-terminal fragment, targeting and inhibiting the C-terminal function of ANGPTL3 would be a novel strategy for developing strategies to specifically reduce serum LDL-C.
[0113] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends 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 is: (1) Targets and binds to the C-terminus of the ANGPLT3 protein, and (2) Inhibits the functional activity of the C-terminal fragment of ANGPLT3 protein; The inhibitors mentioned are protein-level inhibitors; The C-terminal amino acid sequence of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.2; The drug is used to treat hypercholesterolemia, atherosclerosis, or coronary heart disease.
2. The use according to claim 1, characterized in that, The inhibitors include at least one of antibodies and small molecule compounds.
3. The use according to claim 1, characterized in that, The small molecule compound mentioned is heparin.
4. The use according to claim 3, characterized in that, The hypercholesterolemia mentioned above 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 drug 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 the C-terminus of ANGPLT3 protein (Angiopoietin-like 3) in the preparation of formulations that lower LDL-C (low-density lipoprotein cholesterol) levels without interfering with triglyceride metabolism, wherein the inhibitor: (1) Targets and binds to the C-terminus of the ANGPLT3 protein, and (2) Inhibits the functional activity of the C-terminal fragment of ANGPLT3 protein; The inhibitors mentioned are protein-level inhibitors, including but not limited to: antibodies and small molecule compounds; The C-terminal amino acid sequence of the ANGPLT3 protein is shown in SEQ ID NO.1 or SEQ ID NO.
2.
8. The 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 the drug to be screened refers to detecting the following functions of the drug to be screened. (1) Targets and binds to the C-terminus of the ANGPLT3 protein, and (2) Inhibits the functional activity of the C-terminal fragment of ANGPLT3 protein; The drugs to be screened are antibodies or small molecule compounds.
9. The application according to claim 8, characterized in that, The drug is used to treat hypercholesterolemia, atherosclerosis, or coronary heart disease.
10. The application according to claim 8, characterized in that, The hypercholesterolemia mentioned above is: (1) Familial hypercholesterolemia (FH); or (2) Hypercholesterolemia in patients with low expression or function of low-density lipoprotein receptor (LDLR).