CN110613717A

CN110613717A - Medicine for reducing blood fat

Info

Publication number: CN110613717A
Application number: CN201910410812.6A
Authority: CN
Inventors: 洪斌; 王丽; 陈晓芳; 王雪蕾; 杜郁; 杨梦夏; 张秀敏; 武彦彬; 李星星; 侍媛媛
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2019-12-27

Abstract

The invention relates to a medicament for reducing blood fat. The structure is shown in formulas (1) to (4).The invention also relates to compounds of the formulae (1) to (4)The following applications of the substance: (1) preparing a medicament for treating atherosclerotic cardiovascular disease; (2) preparing a medicament for regulating blood glucose metabolism, wherein the regulation of blood glucose metabolism refers to: reducing blood glucose, reducing glycated serum protein, reducing glycated albumin; (3) preparing a medicament for reducing liver lipid accumulation and preventing fatty liver; (4) preparation of an agent that inhibits expression of PCSK9 at the gene level and increases expression of LDLR.

Description

Medicine for reducing blood fat

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a medicament for reducing blood fat.

Background

Elevated low density lipoprotein cholesterol (LDL-C) is an important risk factor for atherosclerosis^[1]. Statins are first-line therapeutics for the prevention and treatment of atherosclerotic cardiovascular disease, acting primarily by lowering LDL-C levels^[2,3]. But about 25 percent of patients with high cardiovascular disease risk still have insignificant lipid-lowering degree after receiving sufficient statins for treatment in clinic^[4]. Some patients are intolerant to statins and have adverse reactions such as myalgia, rhabdomyolysis and the like^[5]. Therefore, a number of trials have been exploring new targets and new therapies for lowering LDL-C.

Proprotein convertase subtilisin/kexin type 9(PCSK9) is a newly discovered drug target in 2003 to lower LDL-C levels and has been developed and marketed. PCSK9 is a proprotein convertase, which is expressed in large amounts in the liver. The physiological role of PCSK9 is primarily to promote degradation of LDLR. LDL-C in the blood can be cleared in the liver by specific binding to its receptor (LDLR). In the liver, PCSK9 specifically recognizes and binds to liver cell surface LDLR epidermal growth factor a (EGF-a). Subsequently, entry of the PCSK9/LDLR complex intracellularly degrades the PCSK9 and LDLR co-entry lysosomes via the endosomal lysosomal pathway, resulting in elevated LDL-C levels. Therefore, inhibition of PCSK9 can significantly reduce LDL-C levels.

There are three main strategies currently known to inhibit PCSK 9:

1. inhibits binding of PCSK9 to LDLR at the cell surface.

2. Interfere with the maturation and secretion of PCSK 9.

3. Inhibition of PCSK9 synthesis and expression at mRNA or protein level^[10]。

Currently, two human monoclonal antibodies (mabs) are marketed as novel PCSK9 inhibitors in 2015, evocumab and Alirocumab, for the treatment of adult primary hypercholesterolemia or mixed dyslipidemia (in combination with statins or other lipid-lowering drugs)^[11,12]. They mainly applied strategy 1 to prevent PCSK9 from binding to LDLR. In addition, the small interfering RNA Inclisiran which is being developed is in phase III clinical research and is the first small interfering RNA drug for regulating blood fat, the administration mode is subcutaneous injection, and the strategy 3 is adopted to directly block the synthesis and expression of PCSK9^[13]。

The macromolecular antibody medicament and the interfering RNA need to be injected for administration, because dyslipidemia is a chronic disease, inconvenience is caused to long-term administration, and the current antibody preparation is expensive. In recent years, efforts have been made to develop small molecule PCSK9 inhibitors, but because PCSK9 protein catalyzes the surface of domain and the development of small molecules that directly inhibit protein-protein interactions is difficult, many other mechanisms of small molecule inhibitors have been developed. Berberine (BBR) was earlier reported to have PCSK9 inhibitory activity and to act by inhibiting the transcription of PCSK9^[14]. At present, a small molecular corydaline derivative CVI-LM001 enters phase I clinical research^[10]The corydaline is an acetylcholinesterase inhibitor extracted naturally, and the structure of CVI-LM001 is very similar to that of berberine. PF-06815345 developed by Perey pharmaceuticals is a new inhibitor obtained by screening and then lead optimization, and is PCSK9mRNA translation inhibitor of (1)^[15]. In addition, studies have attempted to indirectly inhibit the bonding of PCSK9 to LDLR by causing protein allosterism using a groove site near the EGF-A binding domain of PCSK9 and LDLR after bonding with a small molecule^[16]。

Reference documents:

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[4]L.Perezde Isla,R.Alonso,G.F.Watts,etal,Attainment of LDLcholesterol treatment goals in patients with familialhypercholesterolemia:5-year SAFEHEART registry follow-up,J.Am.Coll.Cardiol.67(2016),1278-1285.

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[6]Jaru Taechalertpaisarn,Bosheng Zhao,Xiaowen Liang,Small Molecule Inhibitors of the PCSK9·LDLR Interaction,J.Am.Chem.Soc.2018,140,3242-3249.

[7]van Poelgeest EP,Hodges MR,Moerland M,etal.ntisense-mediated reduction of proprotein convertase subtilisin/kexin type 9(PCSK9):a first-in-human randomized,placebo-controlled trial,Br J Clin Pharmacol,2015, 80(6):1350-1361.

[8]F.Du,Y.Hui,M.Zhang,M.F.Linton,S.Fazio,D.Fan,Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexintype 9(PCSK9)protein,J.Biol.Chem.286(2011)43054-43061.

[9]Guay,Daniel，Crane,Sheldon，Lachance,Nicolas，SMALL MOLECULE MODULATORS OF PCSK9 AND METHODS OF USE THEREOFWO2014139008(A1)。

[10]Shengtao Xu,Shanshan Luo,Zheying Zhu,etal,Small molecules as inhibitors of PCSK9:Current status and future challenges,European Journal ofMedicinal Chemistry,162(2019)212-233.

[11]Koren MJ,Sabatine MS,Giugliano RP,etal.Long-term Low-DensityLipoprotein Cholesterol-Lowering Efficacy,Persistence,and Safety ofEvolocumab in Treatment of Hypercholesterolemia:Results Up to 4Years FromtheOpen-LabelOSLER-1Extension Study[J].JAMA Cardiol,2017,2(6):598-607.

[12]Robinson JG,Farnier M,Krempf M,etal.Efficacy and safety of alirocumab in reducing lipids and cardiovascular events[J].N Engl J Med,2015,372(16):1489-1499.

[13]Nishikido T,Ray KK,Non-antibody Approaches to Proprotein Convertase Subtilisin Kexin 9Inhibition: siRNA,Antisense Oligonucleotides,Adnectins,Vaccination,and New Attempts at Small-Molecule Inhibitors Based onNew Discoveries,Front Cardiovasc Med.2018；5:199.

[14]J.Cameron,T.Ranheim,M.A.Kulseth,T.P.Leren,K.E.Berge,Berberine decreases PCSK9expression in HepG2cells,Atherosclerosis 201(2008)266-273.

[15]Allyn T.Londregan,Liuqing Wei,Jun Xiao,etal,Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9(PCSK9)Inhibitors:Hit to LeadOptimization of Systemic Agents,J.Med.Chem.2018, 61,5704-5718

[16]T.E.Barta,J.W.Bourne,K.D.Monroe,M.M.Muehlemann,A.Pandey,S.Bowers,Phenylpiperazine Proprotein Convertase Subtilisin/kexin Type 9(PCSK9)Modulators and Their Use,02March,2017.WO 2017/034997A1.

[17]Carter AA,Gomes T,Camacho X,Juurlink DN,Shah BR,Mamdani MM.Risk of incident diabetes among patients treated with statins:population basedstudy.Bmj.2013；346:f2610.

[18]R.Schulz,K.-D.Schlüter,U.Laufs,Molecular and cellular function of the proprotein convertase subtilisin/kexin type 9(PCSK9),BasicRes.Cardiol.110(2)(2015)1–19.

[19]Bin Dong,Minhao Wu,Hai Li,etal,Strong induction of PCSK9gene expression through HNF1αand SREBP2:mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters,J LipidRes.2010Jun；51(6):1486–1495.

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[21]Tao,R.,Xiong,X.,DePinho,R.A.,Deng,C.X.,and Dong,X.C.(2013)FoxO3transcription factor and Sirt6deacetylase regulate low densitylipoprotein(LDL)-cholesterol homeostasis via control of the proproteinconvertase subtilisin/kexin type 9(Pcsk9)gene expression.The Journal ofbiological chemistry 288, 29252-29259.

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Disclosure of Invention

The invention firstly relates to application of a group of compounds in preparing a medicament for reducing blood fat, wherein the compounds are shown as a formula (1) or a formula (2):

formula (1)

R is various substituted benzenes and different five-membered rings or six-membered aromatic heterocycles connected by short chains, wherein the short chains refer to

(1) Carbon chains containing 1 to 3 carbon atoms, including-CH₂-、-(CH₂)₂-or-(CH₂)₃-；

Or (2) carbon chains containing 1 to 3 carbon atoms, into which substituents such as carbonyl, ester, amide, sulfonamide and the like are introduced;

r1 and R2 are H, C1-5 alkyl containing substituents such as halogen, hydroxyl, alkoxy, amino, alkylamino and the like, and C1-5 alkyl connected with an aromatic ring;

r3 and R4 are H, C1-3 alkyl, or C1-3 alkyl containing substituents such as halogen, hydroxyl, alkoxy, amino, alkylamino, etc.;

preferably, the first and second liquid crystal materials are,

r is benzyl containing-O-CH 3 substituent on benzene ring

R1 and R2 are H, C1-5 alkyl containing hydroxyl substituent;

r3 and R4 are H and C1-3 alkyl;

or, formula (2)

R5 is various substituted benzenes and different five-membered or six-membered aromatic heterocycles connected by short chains

(1) A carbon chain containing 1 to 3 carbon atoms including-CH₂-、-(CH₂)₂-or- (CH)₂)₃-；

Or (2) introducing carbon chains containing 1-3 carbon atoms of substituents such as carbonyl, ester, acylamino, sulfonamide and the like;

r6 is hydrogen, alkyl containing 1-3 carbon atoms, or alkyl containing 1-3 carbon atoms and containing substituents such as halogen, hydroxyl, alkoxy, amino, alkylamino, etc.;

r7 is a benzene ring or an aromatic heterocycle which are substituted differently, an alkyl group with 1-3 carbon atoms and containing substituents such as halogen, hydroxyl, alkoxy, amino, alkylamino and the like, an alkyl group with 1-3 carbon atoms and substituted by an aromatic ring and the like;

preferably, the first and second liquid crystal materials are,

r5 is pyridine benzyl or furan benzyl;

r6 is hydrogen;

r7 is a benzene ring containing one methyl substituent.

The invention also relates to application of the following compounds in preparing a medicament for reducing blood lipid, wherein the compounds are shown as the following formula (3) or formula (4):

formula (3)

Or formula (4)

The invention also relates to the following applications of the compounds shown in the formulas (1) to (4):

(1) preparing a medicament for treating atherosclerotic cardiovascular disease;

(2) preparing a medicament for regulating blood glucose metabolism, wherein the regulation of blood glucose metabolism refers to: reducing blood glucose, reducing glycated serum protein, reducing glycated albumin;

(3) preparing a medicament for reducing liver lipid accumulation and preventing fatty liver;

(4) preparing an agent that inhibits expression of PCSK9 at the gene level and increases expression of LDLR;

(5) preparing a preparation for modulating HNF1 alpha and FoxO3 transcription factors, wherein the modulation refers to:

1) promote binding of FoxO3 to the PCSK9 promoter;

or 2) attenuating binding of HNF1 α to the PCSK9 promoter;

or 3) increasing expression of FoxO3 protein;

or 4) reducing the expression of HNF1 alpha protein;

(6) a preparation was prepared that up-regulated the expression of transcription factor SREBP 2.

The invention also relates to methods of treating:

(1) hyperlipidemia;

(2) atherosclerotic cardiovascular disease;

(3) abnormal blood glucose metabolism, wherein the abnormal blood glucose metabolism refers to high blood glucose, high glycated serum protein or high glycated albumin;

(4) fatty liver;

the method comprises the following steps: administering a therapeutically effective amount of a compound of any one of formulas (1) - (4).

The invention also relates to methods of treating:

(1) hyperlipidemia and its complications that have failed statin therapy;

(2) atherosclerotic cardiovascular disease with failed statin therapy;

(3) fatty liver with failed statin therapy;

The invention also relates to methods of treating:

(1) hyperlipidemia;

(2) atherosclerotic cardiovascular disease;

(3) fatty liver;

the method comprises the following steps: administering a therapeutically effective amount of a compound of any one of formulas (1) to (4) in combination with a statin.

The invention also relates to a medicament or a pharmaceutical composition for treating the following diseases:

(1) hyperlipidemia;

(2) atherosclerotic cardiovascular disease

(4) fatty liver;

the medicament or the pharmaceutical composition comprises: a therapeutically effective amount of a compound of any one of formulae (1) to (4) and necessary pharmaceutical excipients.

(1) hyperlipidemia with statin failure;

(2) atherosclerotic cardiovascular disease with statin failure

(3) Fatty liver with failed statin therapy;

The invention also relates to a pharmaceutical combination preparation for treating the following diseases:

(1) hyperlipidemia;

(2) atherosclerotic cardiovascular disease

(3) Fatty liver;

the pharmaceutical combination preparation comprises: a therapeutically effective amount of a compound of any one of formulas (1) to (4), a statin and necessary pharmaceutical excipients.

The invention has the following beneficial effects:

we established a high-throughput screening model with human PCSK9 transcription level and post-transcription level as targets. The model is used for screening a compound library in a national new drug (microorganism) screening laboratory to obtain a plurality of active compounds, and the biological activities of positive compounds 7030B-C5 and 7031B-H9 in the active compounds are determined to be deeply researched by rescreening the active compounds at the transcription level and the protein level. In vitro experiments show that 7030B-C5 and 7031B-H9 can inhibit the expression of PCSK9 at a molecular level, thereby promoting the protein expression of LDLR and the uptake of LDL-C. 7030B-C5 is found to be capable of reducing the expression of PCSK9 protein of high-fat diet ApoE KO mice and improving the expression of LDLR, reducing LDL-C level and slowing the atherosclerosis process of the high-fat diet ApoE KO mice through in vivo pharmacodynamic studies, and the compound is unexpectedly found to have hypoglycemic activity.

PCSK9 transcriptional level inhibitors act primarily by modulating regulatory elements of the PCSK9 gene promoter portion. The human PCSK9 gene is known to be located on the short arm of chromosome 1, and as shown in fig. 1A, the proximal promoter of PCSK9 gene comprises a Sterol Regulatory Element (SRE) regulated by changes in intracellular cholesterol levels. Sterol regulatory element-binding protein-2 (SREBP-2) enhances the promoter activity of the PCSK9 gene by binding to SRE. Hepatocyte nuclear factor 1 (HNF1 α) can also transcriptionally activate the expression of PCSK9, which binds to 28bp upstream of SRE and has an enhancing effect on SREBP activation. Berberine (BBR) inhibits the transcription of the PCSK9 gene by down-regulating HNF1 α. In addition, a HINFP-binding motif is arranged between the SRE and the HNF1 alpha-binding motif, and HINFP protein is bound to the HINFP-binding motif, so that histone H4 acetylation is assisted, and PCSK9 transcription is activated. In contrast, acetylation of histones is impaired or deacetylated, which significantly reduces the activity of the PCSK9 gene promoter. An insulin-responsive element (IRE) is arranged inside the HNF1 alpha binding site, and FoxO3 protein is bound to the binding site and recruits histone deacetylase to the promoter of PCSK9, so that the transcriptional activation of HNF1 alpha on PCSK9 is blocked, and the expression of PCSK9 is negatively regulated.

Through research on action mechanism, 7030B-C5 is found to down-regulate the expression of PCSK9 by influencing the expression of FoxO3 and HNF1 alpha and further influencing the binding of FoxO3 and HNF1 alpha and a PCSK9 promoter sequence, and the action mechanism of 7030B-C5 is different from that of the existing PCSK9 small-molecule inhibitor. For example, statins induce SREBP2 to bind to the PCSK9 promoter at SRE position to activate transcription, and simultaneously up-regulate the expression of PCSK9 and LDLR. Daily administration of 20mg rosuvastatin increased PCSK9 levels in men and women by about 28% and 35%, and PCSK9 elevation might explain some patient resistance to statins^[20]. The berberine BBR can reduce the combination of SREBP2 to SRE site, reduce the combination of HNF1 alpha and HNF1 site and reduce the expression of PCSK9^[22]. While our 7030B-C5 did not significantly modulate the SREBP2 binding to SRE sites. 7030B-C5 can increase the expression of FoxO3, enhance the interaction with the PCSK9 promoter, and inhibit the expression of PCSK 9. Meanwhile, 7030B-C5 reduces the protein level of HNF1 α, reduces the binding capacity of HNF1 α to the PCSK9 promoter, and further promotes the down-regulation of PCSK 9.

In conclusion, the discovery of the invention is expected to contribute to the field of PCSK9 inhibitors, and provides a brand-new blood fat and blood sugar reducing drug.

Drawings

FIG. 1 and A: a PCSK9 promoter sequence; b: statins, the regulation of the PCSK9 promoter by the BBR through cis and trans regulatory elements.

FIG. 2 analysis of the luciferase Activity of the recombinant plasmid pGL4-PCSK9-P transient transfection

FIG. 3 analysis of psi-PCSK 9-3' UTR transient transfection luciferase Activity

FIG. 4 dose-response curves of berberine versus stably transfected cell models

FIG. 5, dose-response curve A, miR-149 of miR-149 and miR-544a on a model of stably transfected cells; b, miR-544a

FIG. 6 dose-response curves for pGL4-PCSK9-P HepG2 model positive compounds

FIG. 7 dose-response curves for positive compounds of the psi-PCSK 9-3' UTR HepG2 model

FIG. 8, Effect of different effect concentrations of Positive Compounds on PCSK9mRNA levels in HepG2 cells

FIG. 9, effect of different effect concentrations of pGL4-PCSK9-P model positive compounds on PCSK9 protein levels in HepG2 cells

FIG. 10, chemical structural formulas of compounds 7030B-C5(10A), 7031B-H9(10B), 7045B-E7(10C) and 7045B-F7(10D)

FIG. 11, 7030B-C5 Effect on HepG2 cell PCSK9 and LDLR mRNA levels

FIG. 12, effect of different concentrations of 7030B-C5 on expression of PCSK9(12A) and LDLR (12B) in HepG2 cells

FIG. 13, 7030B-C5 Effect on expression of PCSK9 and LDLR in HepG2 cells at different time points

FIG. 14, 7030B-C5 Effect on PCSK9 secreted expressed in HepG2 cell culture supernatant

FIG. 15, 7030B-C5 Effect on the uptake of DiI-LDL by HepG2 cells

FIG. 16, 7031B-H9 Effect on HepG2 cell PCSK9(16A) and LDLR (16B) mRNA levels

FIG. 17, effect of different concentrations of 7031B-H9 on expression of PCSK9 and LDLR in HepG2 cells

FIG. 18, 7031B-H9 Effect on expression of PCSK9 in HepG2 cells at different time points

FIG. 19, 7031B-H9 Effect on PCSK9 secreted expressed in HepG2 cell culture supernatant

FIG. 20, 7031B-H9 Effect on the uptake of DiI-LDL by HepG2 cells

FIG. 21, 7045B-E7 Effect on HepG2 cell PCSK9(21A) and LDLR (21B) mRNA levels

FIG. 22, 7045B-F7 Effect on HepG2 cell PCSK9(22A) and LDLR (22B) mRNA levels

FIG. 23, effect of different concentrations of 7045B-E7 on expression of PCSK9 and LDLR in HepG2 cells

FIG. 24, effect of different concentrations of 7045B-F7 on expression of PCSK9 and LDLR in HepG2 cells

FIG. 25, 7045B-E7 Effect on the uptake of DiI-LDL by HepG2 cells

FIG. 26, 7045B-F7 Effect on the uptake of DiI-LDL in HepG2 cells

FIG. 27, 7030B-C5 mice group intake changes in body weight (27A) and diet (27B)

FIG. 28, 7031B-H9 mice group intake changes in body weight (28A) and diet (28B)

FIG. 29, 7045B-E7 mice group intake changes in body weight (29A) and diet (29B)

FIG. 30, 7045B-F7 mice group intake changes in body weight (30A) and diet (30B)

FIG. 31, 7030B-C5 Effect on PCSK9(31A) and LDLR (31B) mRNA levels in the liver of C57BL/6 mice

FIG. 32, 7031B-H9 Effect on PCSK9(32A) and LDLR (32B) mRNA levels in the liver of C57BL/6 mice

FIG. 33, 7045B-E7 Effect on PCSK9(33A) and LDLR (33B) mRNA levels in the liver of C57BL/6 mice

FIG. 34, 7045B-F7 Effect on PCSK9(34A) and LDLR (34B) mRNA levels in the liver of C57BL/6 mice

FIG. 35, 7030B-C5 Effect on PCSK9 and LDLR protein levels in the liver of C57BL/6 mice

FIG. 36, 7031B-H9 Effect on PCSK9 and LDLR protein levels in the liver of C57BL/6 mice

FIG. 37, 7045B-E7 Effect on PCSK9 and LDLR protein levels in the liver of C57BL/6 mice

FIG. 38, 7045B-F7 Effect on PCSK9 and LDLR protein levels in the liver of C57BL/6 mice

FIG. 39 weight Change course of ApoE KO mice in control group (Chow), model group (HFD) and 7030B-C5 administration groups

FIG. 40 Effect of 7030B-C5 on ApoE KO mouse body weight 12 weeks after administration

FIG. 41, 7030B-C5 Effect on the serum lipid levels (total cholesterol: 41A, LDL-C: 41B, HDL-C: 41C, triglyceride: 41D) in ApoE KO mice

FIG. 42 and 7030B-C5 Effect on the serum blood glucose levels (blood glucose: 42A, glycated serum protein: 42B, glycated albumin: 42C) of ApoE KO mice

FIG. 43, 7030B-C5 Effect on ApoE KO mouse aortic plaque formation

FIG. 44, 7030B-C5 Effect on ApoE KO mouse Heart outflow tract plaque formation

FIG. 45, 7030B-C5 Effect on ALT and AST in ApoE KO mouse serum

FIG. 46, 7030B-C5 Effect on ApoE KO mouse liver lipid accumulation

FIG. 47, 7030B-C5 Effect on PCSK9 and LDLR mRNA levels in the liver of ApoE KO mice

FIG. 48, 7030B-C5 Effect on PCSK9 and LDLR protein levels in the liver of ApoE KO mice

FIG. 49, 7030B-C5 Effect on ApoE KO mouse secreted PCSK9

FIGS. 50, 7030B-C5 Regulation of luciferase Activity on pGL4-PCSK9-P (50A) and psi-PCSK 9-3' UTR (50B) reporter plasmids

FIG. 51, results of cleavage and identification of report plasmid of PCSK9 upstream promoter region with different lengths

FIG. 52 luciferase Activity of different recombinant plasmids

FIGS. 53, 7030B-C5 Effect on luciferase Activity of different recombinant plasmids

FIG. 54, schematic representation of different recombinant luciferase plasmids

FIGS. 55, 7030B-C5 Effect on luciferase Activity of different recombinant plasmids

FIG. 56, 7030B-C5 Effect on HepG2 cell HNF1 α, FoxO3 and HINFP expression

FIGS. 57, 7030B-C5 Effect on HepG2 cell SREBP2 and Sp1 expression

FIG. 58, 7030B-C5 Effect on the expression levels of PCSK9 in HepG2 cells after FoxO3siRNA action

FIG. 59, 7030B-C5 Effect on expression levels of PCSK9 in HepG2 cells after HNF 1. alpha. siRNA action

FIG. 60, 7030B-C5 Effect on the expression level of PCSK9 in HepG2 cells after action of HINFP siRNA

FIG. 61, 7030B-C5 Effect on the expression level of PCSK9 in HepG2 cells after SREBP2siRNA action

FIG. 62, 7030B-C5 Effect on expression levels of PCSK9 in HepG2 cells after Sp1siRNA action

FIG. 63, 7030B-C5 Effect on HNF1 α, FoxO3 transcription factor binding Activity to PCSK9 promoter

FIG. 64, 7030B-C5 Effect on PI3K/AKT Signaling pathway

FIGS. 65, 7030B-C5 Effect on PEPCK (65A), G6Pase (65B), MTP (65C) and ApoC-III (65D) mRNA in HepG2 cells

FIG. 66 Effect of Positive Compounds on sugar consumption by L6 myocyte

FIG. 67, chemical structures of CVI-LM001 and berberine

Detailed Description

Experimental Material

1. Plasmids

Cloning vector pEASY Blunt Zero, available from Beijing Okinawa Total gold.

Eukaryotic expression vector pGL4.17, purchased from Promega, USA.

The dual-luciferase expression vector psiCHECK was purchased from Promega, USA.

The recombinant reporter gene plasmid pGL4-PCSK9-P is constructed.

The recombinant reporter gene plasmid pGL4-PCSK9-D1/D2/D3/D4/D5/D6/D7 is constructed in the work.

The dual-luciferase recombinant reporter plasmid psi-PCSK 9-3' UTR is constructed in the work.

The recombinant double reporter gene plasmid pc-IRE-L is stored in the laboratory.

2. Strains and cell lines

Coli DH 5a, purchased from total gold, beijing.

Coli Trans-T1, purchased from holojin, beijing.

Human liver cancer cell HepG2(ATCC, HB-8065), stored in this laboratory.

Human kidney cells HEK-293T (ATCC, CRL-11268) were stored in the laboratory.

The luciferase reporter gene stably transfects a cell strain pGL4-PCSK9-P HepG2, and the work is constructed.

The dual-luciferase reporter gene stably expresses a cell strain psi-PCSK 9-3' UTR HepG2 cell, and the work is constructed.

3. Experimental animals and feeds

Healthy 8 week-old male C57BL/6 mice, purchased from Beijing sbefu Biotechnology, Inc.

Healthy 8-week-old male ApoE knockout (ApoE knockout, ApoE KO) mice, purchased from beijing waukang biotechnology gmbh.

Common feed, provided by the animal house of the institute.

High fat diet (0.15% cholesterol added, 20% lard oil) purchased from Beijing Huafukang biotech GmbH.

4. Small RNA

(1)MicroRNA(miRNA)

miRNA in this work was purchased from sharp bo, guangzhou.

miRNAmimic：

The miRNA simulacrum synthesized by a chemical method can simulate the high-level expression of mature miRNA in cells so as to enhance the regulation and control function of endogenous miRNA and carry out the gain study of cell functions.

miRNAinhibitor：

The miRNA inhibitor synthesized by a chemical method can inhibit the miRNA action by specific combination with mature miRNA molecules, can weaken the gene regulation and control action caused by endogenous miRNA in cells, and can be used for miRNA function deletion research.

miRNA mimic and inhibitor negative control：

C.elegans miRNA is selected, bioinformatics analysis shows that the miRNA has minimum homology with human, mouse and rat genomes and all miRNA in a miRBase database, and the miRNA is suitable for miRNA experiment negative control of human, mouse and rat.

(2) Small interfering RNA (siRNA)

siRNAs for HNF1 α, FoxO3, HINFP, SREBP2 and Sp1 in this work were purchased from Invitrogen, USA.

5. PCR primer

Primers for constructing promoter and 3' -UTR reporter plasmid of PCSK9

FP＝Forward primer，RP＝Reverse primer

AAGCTT: hind III cleavage site;GCGGCCGC: not I cleavage site;CTCGAG: xho I cleavage site.

6. Data analysis

All cell experiments had at least 3 biological replicates and the experimental data are expressed as mean + -SEM and statistically analyzed using Student's t-test and one-way ANOVA, with P <0.05 considered a significant difference. Databases such as miRBase (http:// www.mirbase.org /), targetScan (http:// www.targetscan.org /) and miRanda (http:// www.microrna.org /) are used for miRNA sequence analysis and target prediction. Data analysis and mapping were performed using GraphPad Prism 5 software.

Example 1 construction of human PCSK9 Gene expression Modulator screening model

1. Acquisition of upstream and downstream regulatory sequences of human PCSK9 gene

The target fragment, namely the upstream promoter (-2112 to-1 bp, total 2112bp, A of transcription initiation site ATG as +1) and the downstream 3' UTR region sequence (total 1269bp), of PCSK9 is amplified by a PCR method by using human genome DNA extracted from human HepG2 cells as a template. The amplified product was detected by agarose gel electrophoresis, showing a uniform band, consistent with the expected fragment size.

2. Construction of recombinant plasmid for reporter Gene

And (3) purifying and recovering the PCR product obtained by amplification, Cloning the PCR product into a pEasy-blast Zero Cloning Vector to obtain recombinant plasmids pT-PCSK9-P and pT-PCSK 9-3' UTR, transforming Trans-T1 competent cells, and picking a transformant. After the transformant extracts plasmids, pT-PCSK9-P is subjected to enzyme digestion identification by Xho I/Hind III, pT-PCSK 9-3' UTR is subjected to enzyme digestion identification by Xho I/Not I, and recombinant plasmids are subjected to sequencing analysis.

After the sequencing is correct, Xho I and Hind III respectively carry out double digestion on recombinant plasmids pT-PCSK9-P and pGL4-Basic plasmids, and a target gene fragment is connected with the linear pGL4-Basic plasmids after double digestion, so that the upstream regulatory sequence of the human PCSK9 gene is directionally inserted into the upstream of the luciferase reporter gene of the pGL4-Basic vector, and the recombinant plasmid pGL4-PCSK9-P is constructed.

The recombinant plasmid pT-PCSK9-3 'UTR is recovered by Xho I/Not I enzyme digestion and is connected to a psiCHECK vector which is also subjected to enzyme digestion to obtain a recombinant plasmid psi-PCSK 9-3' UTR, and a DNA band which is consistent with the size of an expected fragment can be seen by enzyme digestion identification.

3. Recombinant plasmids pGL4-PCSK9-P and psi-PCSK 9-3' UTR transiently transfected HepG2 cells

PureYield for transfection grade reporter gene recombinant plasmid^TMPlasmid Midiprep System (Promega) kit extraction. HepG2 cells were transiently transfected with the recombinant plasmid pGL4-PCSK9-P using pGL4-Basic as a negative control, and luciferase activity was measured. For example, pGL4-PCSK9-P luciferase expression activity was significantly increased. Berberine (BBR) acts on SREBP2 and HNF1 alpha to regulate the transcription of PCSK9, so BBR is selected as a positive compound to perform primary evaluation on the constructed recombinant plasmid pGL4-PCSK 9-P. As shown in figure 2, after BBR action on transiently transfected cells, pGL4-PCSK9-P luciferase activity was significantly down-regulated, consistent with literature reports. Therefore, the pGL4-PCSK9-P luciferase reporter plasmid can be used for constructing a high-throughput screening model of the down-regulation agent of the PCSK9 small-molecule compound.

Similarly, the recombinant plasmid psi-PCSK 9-3' UTR was transiently transfected into HepG2 cells and luciferase activity was measured, as shown in FIG. 3, with reduced fluorescence compared to psiCHECK plasmid without the inserted regulatory sequences. Due to the sequence insertion at the downstream of the luciferase gene, the stability of the mRNA of the luciferase gene is affected, so that the fluorescence value is reduced. This result preliminarily demonstrated the success of the psi-PCSK 9-3' UTR plasmid construction. Based on the results of transient transfection, we next constructed further stable transgenic cell lines.

4. Establishment of stably expressing cell lines

(1) Determination of optimal resistance selection concentration

Due to factors such as cell type, source, passage times, culture conditions, and the manufacturer and potency of the antibiotic G418, preliminary experiments are required to determine the optimal G418 screening concentration for a particular cell prior to stable transfection. And (3) allowing G418 solutions with a series of concentrations to act on HepG2 cells for 10-14 days, and finishing the culture when the survival states of the cells are obviously different under the action of G418 with different concentrations. The final concentration of HepG2 cell anti-G418 used in this work was determined by analysis to be 700 μ G/ml.

(2) Selection of stably transfected cell lines

Using Lipofectamine^TM2000(Invitrogen) by transfecting the recombinant plasmids pGL4-PCSK9-P and psi-PCSK9-3 'UTR into HepG2 cells, respectively (the recombinant plasmid psi-PCSK 9-3' UTR is co-transfected with pcDNA3.1 into HepG2 cells). After about 15 days of treatment with 700. mu.g/ml G418, G418 resistant cell clones developed. The resulting cell clones were expanded and assayed for luciferase expression activity. Clones with higher activity were further subjected to monoclonality.

And (3) performing monoclonality on the HepG2 cells with high luciferase expression obtained by resistance screening, transferring the cells to a 24-pore plate, a 12-pore plate and a 6-pore plate in sequence after monoclonality is formed, performing expanded culture, detecting the luciferase expression activity of the cells every 3-5 generations, and screening monoclonal cell strains with normal cell cycle and high luciferase expression activity capable of being stably passaged for more than 20 generations. Through screening, monoclonal cell strains stably transfected with pGL4-PCSK9-P or psi-PCSK9-3 'UTR are successfully obtained and named as pGL4-PCSK9-P HepG2 and psi-PCSK 9-3' UTR HepG2 respectively and used for establishing a screening model.

5. Establishment, optimization and evaluation of PCSK 9expression regulator stable transfection cell model

(1) Establishment, optimization and evaluation of transcription level PCSK9 gene expression regulator screening model

1) Effect of DMSO concentration on luciferase expression Activity of cells

In the screening of the compounds, samples are generally dissolved in dimethyl sulfoxide (DMSO), but high concentration of DMSO generates cytotoxicity, so that the influence of DMSO on luciferase expression activity of stably transfected cells is determined in the construction and optimization of a screening model. The study examined the effect of 0.01% -2% DMSO on luciferase expression activity. Within the concentration range of 0.01-0.25%, DMSO does not have obvious influence on cell activity, namely the luciferase expression activity of pGL4-PCSK9-P HepG2 stable transfection cell strains is not obviously reduced, so that DMSO selection is less than 0.25%. Thus, the final screening conditions were determined to be: at 5X 10⁵The cells were seeded at a density of one/ml and the compound screening was carried out at a concentration of 25. mu.g/ml, using a final concentration of 0.25% DMSO solvent, and a negative control for the screening model was established at this concentration.

2) Dose-effect relationship of berberine in stable transfection cell model

When the positive compound berberine (BBR) reported in the previous literature is used for acting on the screening model, the result shows that the luciferase activity value is reduced along with the increase of the concentration of the BBR within a certain concentration range and is concentration-dependent and IC₅₀27.95. mu.M (FIG. 4), indicating that BBR can effectively act on the constructed pGL4-PCSK9-P model, thereby demonstrating the feasibility of the constructed stably transfected cells for drug screening.

6. Effect of drug action time on luciferase expression Activity of cells

To determine the appropriate time for the compound to act, 16h, 18h, 20h and 24h were chosen to measure luciferase expression activity in the cells. The results show that the luciferase activity of the pGL4-PCSK9-P HepG2 stably transfected cell strain is slowly reduced from 16-24 h after the compound acts on the stably transfected cells, and the expression is relatively stable. Finally, the time for the compound to act on pGL4-PCSK9-P HepG2 stably transfected cells is determined to be 24 hours by combining the growth cycle of the cells and the experimental time schedule.

7. Evaluation of stably transfected cell selection model

The parameters currently widely used for evaluating high throughput screening models are: signal/background ratio (S/B), signal/noise ratio (S/N), coefficient of variation of signal background (CV), and Z' factor. The Z' factor is a characteristic parameter for evaluating a high-throughput screening model and can be used for evaluating the stability and reliability of the model. The calculation formula of each parameter is as follows:

BBR is used for evaluating multiple high-throughput screening indexes, and results show that all parameters meet the requirements of high-throughput screening (table 1.3), which shows that the model established in the research is stable and reliable and can be used for high-throughput screening.

TABLE 1.3 evaluation of pGL4-PCSK9-P stably transfected HepG2 model

8. Establishment, optimization and evaluation of post-transcriptional PCSK9 gene expression regulator screening model

(1) Prediction of effect of microRNA on expression activity of PCSK9 gene mRNA 3' UTR luciferase reporter gene in HepG2 cells

Currently, there are few studies on the post-transcriptional level of PCSK9, and no positive compounds on PCSK 93' UTR have been reported in the literature.

mirnas are widely present in eukaryotes, and are endogenous single-stranded non-coding RNAs with a size of about 22 nucleotides, and most of mirnas mediate degradation or translational inhibition of target gene mrnas by matching complete or incomplete bases with a specific region on the 3' UTR of the target gene mRNA, thereby regulating expression of the target gene at a post-transcriptional level. Bioinformatics methods predict the possible presence of miRNA sites of action on the 3' UTR of human PCSK9 gene mRNA. And (3) comprehensively analyzing the prediction result, and selecting 12 miRNAs with higher scores and stronger conservation for analysis in the next experiment.

According to miRNA predicted by software and possibly using PCSK9 gene mRNA 3' UTR as an action target, HepG2 cells are transiently transfected by using psi-PCSK9-3 ' UTR reporter gene plasmid, and the inhibition effect of 14 miRNAs on the expression activity of PCSK9mRNA 3' UTR luciferase reporter gene is investigated. The luciferase reporter gene analysis result shows that 100nM miR-224, miR-149 and miR-544a has a certain inhibition effect on luciferase gene expression activity in HepG2 cells transiently transfected with psi-PCSK 9-3' UTR reporter gene plasmid.

The miR-149(3 ' -cccucaCUUCUGUGCCUCGGUCu-5 ') and miR-544a (3 ' -cuUGAACGAUUUUUACGUCUUa-5 ') with better activity performance on a model are taken to further act on psi-PCSK9-3 ' UTR HepG2 stably transfected cells. Luciferase expression activity was detected 48 hours after transient transfection of psi-PCSK 9-3' UTR HepG2 cells with a series of concentrations of miR-149 and miR-544a, respectively. The results are shown in figure 5, and both miR-149 and miR-544a act on the PCSK9mRNA 3' UTR in a concentration-dependent manner, so that the luciferase activity is reduced.

(2) Evaluation of stably transfected cell selection model

Since no small molecule compound which can mediate and inhibit the stability of PCSK9mRNA through PCSK9mRNA 3' UTR has been proved for a while at present, miR-149 obtained by screening is used as a positive control primarily, and parameters of a screening model are evaluated according to the change of luciferase activity expressed by cells. The evaluation result shows that S/B, S/N, CV percent meets the requirement of high-throughput drug screening, and the Z' factor is slightly lower than 0.5. Since miR-149 is not the optimal positive control of the model, the obtained positive compound is used for reevaluating the drug screening model which is established by the work and aims at the human PCSK9 gene mRNA stability as the target.

TABLE 1.4 psi-PCSK 9-3' UTR stably transfected HepG2 model evaluation

Example 2 high throughput screening of PCSK9 Gene expression modulators

1. Obtaining primary screening and secondary screening results aiming at different screening models

The high-throughput drug screening model established by the research and aiming at the transcription level and post-transcription level of the PCSK9 gene as a target point is applied to screen compounds of 5,200 samples of 7 series of samples 7001B-7065B in a national new drug (microorganism) screening compound sample library. Individual compounds were dissolved in 100% DMSO at a concentration of 10mg/ml and a final concentration of 25. mu.g/ml was used for screening. The down-regulation of 50% was taken as a primary screening positive. Through primary screening, secondary screening and verification,for the pGL4-PCSK9-P model, we 20 positive compounds were obtained (typical compounds are shown in Table 1.5 below)The screening positive rate is 0.38%.Against pGL4- PCSK 9-3' UTR model, 7 positive compounds were finally obtained (typical compounds are shown in Table 1.6 below)The positive rate was 0.13%. And the positive compound is diluted in a gradient way, a dose-effect relation curve is drawn (figures 6-7), and the half inhibitory concentration IC is calculated₅₀。

TABLE 1.5 pGL4-PCSK9-P model rescreening of typical positive compounds identified

TABLE 1.6 pGL4-PCSK 9-3' UTR model rescreens positive compounds identified

We used the screened positive compound 7018B-E6 to re-evaluate each screening parameter of the high-throughput drug screening model targeting human PCSK9 gene mRNA stability. The evaluation result shows that the Z' factor is improved, the related parameters meet the requirement of high-throughput screening, the model is stable and reliable, the sensitivity is high, the specificity is strong, and the method can be applied to large-scale compound screening tests, as shown in Table 1.7.

TABLE 1.7 psi-PCSK 9-3' UTR stably transfected HepG2 model evaluation

2. Effect of Positive Compounds on the expression level of hepatocyte PCSK9 Gene

Changes in luciferase expression activity after positive compound action were detected on pGL4-PCSK9-P HepG2 cells and psi-PCSK9-3 'UTR HepG2 cells, respectively, and as can be seen from FIGS. 6 and 7, positive compounds dose-dependently down-regulate luciferase expression activity to different degrees on pGL4-PCSK9-PHepG2 and psi-PCSK 9-3' UTR HepG2 cells, thereby affecting the transcription of the PCSK9 gene. To determine the effect of positive compounds on the target gene PCSK9, we performed further activity determination at the cellular level for positive compounds obtained from model screening, and detected changes in PCSK9mRNA levels in cells using Real-Time PCR method, and protein level changes in PCSK9 and downstream target gene LDLR by western blot method. Results as shown in figures 8 and 9, positive compounds obtained from the screening were all able to reduce PCSK9mRNA levels and protein levels to varying degrees. Since PCSK9 can regulate the degradation of LDLR in hepatocytes at the post-translational level. Therefore, we also detect the change level of the LDLR, and find that some compounds, although reducing the level of PCSK9, cannot effectively increase the expression of LDLR, and cannot become a candidate drug or lead compound for the treatment of new cardiovascular diseases.

Through cell level activity determination, pGL4-PCSK9-P model positive compounds 7030B-C5, 7031B-H9, 7045B-E7, 7045B-F7 and psi-PCSK 9-3' UTR model positive compounds 7018-E6 and 7018B-F6 have good inhibitory activity on the expression of PCSK9, and when the concentration of the compounds is lower than 5 mu M, the compounds still have extremely strong inhibitory effect on the expression of PCSK9, can obviously improve the expression level of LDLR, and is worthy of deep research.

3. In vitro pharmacodynamic study of positive compounds 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7

Next, we performed in vitro pharmacodynamic evaluations on HepG2 cells for compounds 7030B-C5, 7031B-H9, 7045B-E7, 7045B-F7 that were positive among them.

The molecular formula of the compound 7030B-C5 is C₁₇H₂₁N₅O₄Molecular weight 359.38, structure shown in FIG. 10A.

The molecular formula of the compound 7031B-H9 is C₂₀H₁₈N₄Molecular weight 314.38, the structure of which is shown in FIG. 10B.

The molecular formula of the compound 7045B-E7 is C₁₆H₁₅N₃O, molecular weight 265.31, the structure of which is shown in FIG. 10C.

The molecular formula of the compound 7045B-F7 is C₁₆H₁₄N₃OCl, molecular weight 299.75, the structure of which is shown in FIG. 10D.

After a series of concentrations of 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 act on pGL4-PCSK9-P HepG2 cells for 24 hours respectively, luciferase expression activity is detected, and a dose-response relationship curve is obtained. The 4 positive compounds 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 (figure 10) all reduced the luciferase expression activity in a dose-dependent manner within a certain concentration range. IC of 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 calculated by GraphPad Prism 5 software₅₀The values were 1.61. mu.M, 6.25. mu.M, 2.97. mu.M, and 3.01. mu.M, in that order.

4. Evaluation of the cytotoxicity of Positive Compounds

To exclude possible cytotoxicity of the compounds, the MTT method was first used to examine the effect of PCSK 9-positive compounds on the survival and growth of HepG2 cells at different concentrations. The results show that the cell survival rates of the control groups are more than 90 percent when the concentrations of 7030B-C5, 7031B-H9 and 7045B-E7 are between 0.5 and 100 mu M, and the growth and survival of the cells are hardly influenced. The cell survival rate of the compound 7045B-F7 was over 90% of that of the control group at the concentration of 0.5-50. mu.M, and when the compound reaches the higher concentration of 100. mu.M, the compound showed slight toxicity, and the cell survival rate was reduced to 88%. As the concentration of the compound selected for this study was below 50 μ M, the effect of 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-E7 on HepG2 cell growth was negligible in this concentration range.

Example 3 Effect of Positive Compounds on PCSK9 and LDLR expression levels and function in HepG2 cells

1. 7030B-C5 Effect on expression levels and function of PCSK9 and LDLR in HepG2 cells

(1)7030B-C5 Effect on PCSK9 and LDLR mRNA levels in HepG2 cells

The study on the dose-effect relationship of the compound on a PCSK9 gene expression regulator screening model shows that in a certain range, the influence of 7030B-C5 on the luciferase expression activity of cells is in negative correlation, and the transcription inhibition effect of the compound on target genes is demonstrated at a molecular level.

To further examine the biological activity of 7030B-C5, we used the Real-Time PCR method to study the effect of positive compounds on PCSK9mRNA in HepG2 cells. After adding 2.5. mu.M, 5. mu.M, 12.5. mu.M and 25. mu.M 7030B-C to HepG2 for 524 hours, total cellular RNA was extracted and reverse-transcribed into cDNA, and real-time PCR experiments were performed. Results fig. 11 shows that 7030B-C5 significantly reduced PCSK9mRNA levels and up-regulated LDLR mRNA levels in HepG2 cells over a range of concentrations and was dose-dependent.

(2) Effects of 7030B-C5 on PCSK9 and LDLR protein levels in HepG2 cells

The compound 7030B-C5 with different concentrations is applied to HepG2 cells, and total cell protein is extracted after 24 hours to carry out a western blot experiment. The results show that HepG2 cell PCSK9 protein expression levels were negatively correlated with 7030B-C5 working concentration, and protein levels were down-regulated to 0.17 at 2.5 μ M.

We further examined the effect of compound 7030B-C5 on PCSK 9expression at different time points. It can be seen that 7030B-C5 can also reduce the expression of PCSK9 in a time-dependent manner.

Since PCSK9 can regulate the degradation of LDLR in hepatocytes at the post-translational level. Meanwhile, we also detected the changed level of the downstream target gene LDLR of PCSK9, and 7030B-C5 was able to up-regulate the expression of LDLR dose-dependently and time-dependently. In the concentration range of 2.5-50 μ M, 7030B-C5 significantly increased the LDLR protein level of HepG2 cells, which was 2.9 times higher than that of the control group at 25 μ M (fig. 12). In time point studies, we found that LDLR protein expression increased significantly after 12 hours of action time (fig. 13).

(3) Effect of 7030B-C5 on secreted PCSK9 protein levels

The PCSK9 protein is processed and modified by the endoplasmic reticulum and Golgi apparatus, and is finally secreted into blood to become mature PCSK9 protein. We collected the culture supernatant and total cellular protein separately for examination and observed a significant reduction in PCSK9 protein expression in HepG cells treated with 7030B-C5 for 24 hours (fig. 14).

(4) Effect of 7030B-C5 on LDL uptake Activity of HepG2 cells

The change of DiI-LDL uptake of HepG2 cells after the compound effect is detected by flow cytometry shows that 7030B-C5 can obviously increase the DiI-LDL uptake of LDLR, when the 7030B-C5 effect concentration is 12.5 mu M, the DiI-HDL uptake of HepG2 of liver cells is 1.34 times of that of a control (figure 15), and the effect of the compound 7030B-C5 is confirmed at a cell level.

2. 7031B-H9 Effect on expression levels and function of PCSK9 and LDLR in HepG2 cells

(1)7031B-H9 Effect on PCSK9 and LDLR mRNA levels in HepG2 cells

After adding 2.5. mu.M, 5. mu.M, 12.5. mu.M and 25. mu.M 7031B-H to HepG2 for 924 hours, total cellular RNA was extracted and reverse-transcribed into cDNA, and real-time PCR experiments were performed. As shown in fig. 16, 7031B-H9 significantly reduced PCSK9mRNA levels and up-regulated LDLR mRNA levels in HepG2 cells over a range of concentrations and was dose-dependent.

(2)7031B-H9 Effect on PCSK9 and LDLR protein levels in HepG2 cells

To examine the effect of 7031B-H9 on PCSK9 and LDLR protein expression in cells, HepG2 cells were treated with different concentrations of 7031B-H9, respectively, for 24 hours. The results show that with increasing concentrations of compound 7031B-H9, PCSK9 protein expression levels gradually decreased and LDLR expression gradually increased (fig. 17).

We further examined the effect of compound 7031B-H9 on PCSK 9expression at different time points. From fig. 18), 7031B-H9 can also decrease expression of PCSK9 time-dependently. The level of LDLR protein changes slowly with increasing duration of action, and the level of LDLR expression increases only after 24 hours of action.

(3) Effects of 7031B-H9 on secreted PCSK9 protein levels

We performed Western Blot analysis of culture supernatants and total cell proteins after exposing different concentrations of 7031B-H9 to HepG2 cells for 24 hours, and found that PCSK9 protein expression was concentration-dependent decreased in both HepG2 cell culture supernatants and cell lysates (fig. 19).

(4)7031B-H9 Effect on LDL uptake Activity of HepG2 cells

The change of DiI-LDL uptake of HepG2 cells after the compound effect is detected by flow cytometry shows that 7031B-H9 can obviously increase the DiI-LDL uptake of LDLR, when the 7031B-H9 effect concentration is 12.5 mu M, the DiI-HDL uptake of HepG2 of liver cells is 1.41 times of that of a control (figure 20), and the effect of the compound 7031B-H9 is confirmed at a cell level.

3. 7045B-E7, 7045B-F7 effects on PCSK9 and LDLR expression levels and function in HepG2 cells

Since compounds 7045B-E7, 7045B-F7 are structurally similar, based on a single basic parent nucleus, differing only in substituents, these two compounds are analyzed together.

(1) Effects of 7045B-E7, 7045B-F7 on PCSK9 and LDLR mRNA levels in HepG2 cells

After 2.5. mu.M, 5. mu.M, 12.5. mu.M and 25. mu.M 7045B-E7 and 7045B-F7, the expression of PCSK9 is obviously reduced, but the expression of LDLR in HepG2 cells is not subjected to concentration-dependent up-regulation. Interestingly, we found that LDLR was up-regulated at 2.5. mu.M, and suspected that the presence of 7045B-E7 and 7045B-F7 exerted only a regulatory effect on LDLR over a range of concentrations.

We gradually diluted the concentrations of compounds 7045B-E7 and 7045B-F7 from 5. mu.M to a series of concentrations, acted on HepG2 cells for 24 hours, extracted the total RNA of the cells and reverse transcribed to cDNA, and carried out real-time PCR experiments. Results as shown in fig. 21 and 22, after 7045B-E7, 7045B-F7 treatment, the mRNA level of PCSK9 in HepG2 cells was significantly reduced and dose-dependent; LDLR mRNA levels did not show a trend with increasing compound concentration.

(2) Effects of 7045B-E7, 7045B-F7 on PCSK9 and LDLR protein levels in HepG2 cells

To examine the effect of 7045B-E7, 7045B-F7 on PCSK9 and LDLR protein expression in cells, HepG2 cells were acted on at different concentrations of 7045B-E7, 7045B-F7, respectively, for 24 hours.

As can be seen in figure 23, PCSK9 protein expression levels gradually decreased with increasing concentrations of compound 7045B-E7. When the concentration of 7045B-E7 is 0.05-0.5 mu M, the LDLR expression increases in a concentration-dependent manner, when the concentration of the compound is 0.5 mu M, the LDLR expression level reaches the highest level, and then, the LDLR expression level gradually decreases with the increase of the compound concentration.

As can be seen in figure 24, PCSK9 protein expression levels in HepG2 cells were negatively correlated with increased concentrations of compound 7045B-F7, with PCSK9 protein expression levels decreasing with increasing compound concentrations. The expression of LDLR was slightly up-regulated with increasing compound concentration, and reached the highest level at 0.5. mu.M, and then gradually decreased with increasing compound concentration.

Since the LDLR mRNA levels did not vary dose-dependently with compound concentration after 7045B-E7, 7045B-F7 action on HepG2 cells, however, as the target gene downstream of PCSK9, LDLR protein levels showed dose-dependent changes, 7045B-E7, 7045B-F7 were preliminarily presumed to be PCSK9 target-specific compounds.

(3)7045B-E7, 7045B-F7 Effect on LDL uptake Activity in HepG2 cells

The change of DiI-LDL uptake of HepG2 cells after the compound was applied was examined by flow cytometry. The results are shown in figure 25, 7045B-E7 can significantly increase the uptake of DiI-LDL by LDLR, and when the 7045B-E7 action concentration is 5 mu M, the DiI-HDL uptake of HepG2 of the liver cell is 1.22 times that of the control. 7045B-F7 at an action concentration of 5. mu.M, the DiI-HDL uptake of hepatocyte HepG2 was 1.16-fold that of the control (FIG. 26).

Example 4 pharmacodynamic evaluation of Positive Compounds in C57BL/6 mice

A good animal model is an important means for researching the treatment effect of the medicament. In this experiment, positive compounds 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 were first evaluated for in vivo activity in C57BL/6 mice.

1. Positive compound acute toxicity evaluation

Acute toxicity test is one of the research contents for evaluating the safety of drugs, and is used for evaluating the toxicity response of animals after single or multiple (2 times at intervals of 6-8 hours) cumulative administration within 24 hours, wherein the toxicity response comprises general behavior and appearance changes, gross morphological changes and death effects. By observing the poisoning performance, the toxic effect strength and the death condition, the toxic effect characteristics of the medicine are preliminarily evaluated, the safety factor of the medicine is calculated, and meanwhile, a reference is provided for the selection of the subsequent pharmacological test dose.

First, we evaluated the acute toxicity of four positive compounds 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 at a dose of 1000 mg/kg. Healthy 8-week-old C57BL/6 male mice were used and divided into 4 groups of 5 mice each, and after administration of 1000mg/kg of 7030B-C5, 7031B-H9, 7045B-E7, 7045B-F7 at a time, the reaction was immediately observed, and 10 minutes, 15 minutes, 30 minutes, 1 hour, and 2 hours after the start of the observation. The rest of the days are observed once, and the weight and the food intake condition of the mice are observed simultaneously for seven days. We observed that mice in 7031B-H9 (FIG. 28), 7045B-E7 (FIG. 29), and 7045B-F7 (FIG. 30) administration groups all survived 24 hours after administration. Mice showed variable reductions in body weight and dietary intake at 24 hours post-dose and returned to normal 48 hours post-dose, with normal other characteristics and behavior. This is probably due to transient loss of appetite in mice after large doses, resulting in weight loss and reduced diet. This gives half the lethal dose LD of 7031B-H9, 7045B-E7, 7045B-F7₅₀Are all more than 1000 mg/kg.

However, 1000mg/kg 7030B-C5 administration group miceAll deaths indicated that the mice failed to tolerate the 1000mg/kg dose of 7030B-C5. Next, we performed acute toxicity tests with a reduction in 7030B-C5 concentration. We used healthy 8-week-old C57BL/6 male mice divided into 3 groups of 5 mice each, and observed responses immediately after a single administration of different doses of 7030B-C5(100, 300, 500mg/kg), beginning 10 minutes, 15 minutes, 30 minutes, 1 hour, and 2 hours. The rest of the observation is carried out once every day for seven days. We observed that all mice in the 500mg/kg, 1000mg/kg group died 24 hours after administration, 2 mice in the 300mg/kg group remained, and all mice in the 100mg/kg group survived. And mice showed variable reductions in body weight and dietary intake at 24 hours post-dose and returned to normal 48 hours post-dose with normal other characteristics and behavior (figure 27). By calculation, half the lethal dose LD of 7030B-C5₅₀≈300mg/kg。

2. Effect of Positive Compounds on liver PCSK9mRNA levels in C57BL/6 mice

The influence of four positive compounds 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 on PCSK9 and LDLR is proved at a cellular level, healthy 8-week-old C57BL/6 male mice are adopted, randomly divided into 9 groups (the grouping conditions are shown in a table 1.8), and are subjected to intragastric administration, ordinary diet feeding, free drinking water, room temperature (22 +/-2) DEG C, humidity of 55-65% and illumination/darkness 12-hour circulation for continuous feeding for 4 weeks in order to further examine the in-vivo pharmacodynamics of the positive compounds.

TABLE 1.8 grouping of pharmacodynamic evaluations of Positive Compounds on C57BL/6 mice

We first extracted total RNA from liver tissue and examined the levels of PCSK9 and LDLR in mouse liver tissue, with the results shown in figures 31 to 34. The liver tissue of mice in the 4 positive compound administration groups had a reduced level of PCSK9mRNA and also a reduced level of LDLR mRNA compared to the control group.

3. Effect of Positive Compounds on liver PCSK9 protein levels in C57BL/6 mice

The expression of the levels of PCSK9 and LDLR protein in the liver samples of mice in the control group and the administration group was measured by the western blot method, and the results are shown in fig. 35 to 38 as the expression of the relevant genes in the liver tissue of C57BL/6 mice. Compared with the control group, the 7030B-C5 and 7031B-H9 obviously reduce the expression of PCSK9 protein in the liver of the C57BL/6 mouse and obviously increase the level of LDLR protein. 7045B-E7 and 7045B-F7 significantly reduced the expression of PCSK9 protein in the liver of C57BL/6 mice, and also reduced the level of LDLR protein.

Example 5 pharmacodynamic evaluation of 7030B-C5 on ApoE KO mice

The good animal model is an important means for researching the pathological process of atherosclerosis and the prevention and treatment effect of the medicine. ApoE KO mice lack ApoE genes, and obvious AS symptoms can appear after being fed with high-fat feed for several months, so the animal model is adopted in the experiment to evaluate the activity of preventing and treating AS of the positive compound.

Healthy 8-week-old ApoE KO male mice were selected for the experiment, and ApoE KO mice were fed with normal diet for control group (Chow), and high fat diet containing 0.15% cholesterol and 20% lard for model group (HFD) and 7030B-C5 administration group (10mg/kg, 30mg/kg) continuously while stomach-drenching administration for 12 weeks. During the period, blood is taken from the orbit every 4 weeks, and the blood lipid index in the serum is monitored to judge the molding condition.

(1)7030B-C5 Effect on ApoE KO mouse body weight

After ApoE KO mice were continuously fed high fat diet, the body weight of the model group (HFD) was significantly increased compared to the control group (Chow), and the body weight of the animals was gradually decreased compared to the HFD group after administration of 7030B-C5 dry prognosis (fig. 39). When administered for 12 weeks, the animals in the administered group were significantly reduced in body weight compared to the HFD group and returned to levels comparable to the Chow group with no statistical difference compared to the Chow group (fig. 40).

(2)7030B-C5 Effect on the serum lipid level in ApoE KO mice

Total Cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and Triglyceride (TG) are important indexes for measuring the lipid level in vivo, and the blood lipid content can reflect the condition of lipid metabolism in vivo.

After ApoE KO mice are fed with high-fat feed, the serum TC and LDL-C contents of the mice in the HFD group are obviously increased compared with those in the Chow group, which indicates that the modeling of the atherosclerosis high-fat model is successful. After 7030B-C5 treatment, serum TC and LDL-C were both reduced, while HDL-C levels were increased. In addition, TG was significantly reduced in the 7030B-C5-administered group compared to the HFD group (fig. 41). The results preliminarily show that 7030B-C5 is helpful for promoting the removal of excess cholesterol in the plasma of high-fat apoE KO mice, and has good lipid-lowering effect.

(3)7030B-C5 Effect on serum blood glucose indicators in ApoE KO mice

Blood glucose (Glu) is an important component of the body, and the blood glucose content can reflect the carbohydrate metabolism in the body, and is one of the most important examination items for diagnosing diabetes. Glu reflects the immediate blood glucose level of the body. Glycated Serum Proteins (GSPs) are the products of the non-enzymatic reaction of blood glucose with plasma proteins (approximately 70% albumin). GSP reflects the mean blood glucose concentration 1-3 weeks before blood sampling. Glycated Albumin (GA) is quantified on the basis of GSP, and is represented by the percentage of glycated serum albumin to total serum protein, thus removing the effect of the level of glycated serum albumin on the assay results and being more accurate than GSP. Since the half-life of albumin is 17-20 days, GA reflects the average blood glucose level 2-3 weeks before blood collection. The glucose metabolism related index monitoring has good significance for the diagnosis of clinical diabetic patients and the adjustment of treatment schemes.

After ApoE KO mice are fed with high-fat feed, the serum Glu content of the mice in the HFD group is obviously increased compared with that in the Chow group, and the serum Glu is obviously reduced after the treatment of 7030B-C5. In addition, the 7030B-C5-administered group also showed a decrease in GSP and GA compared to the HFD group (FIG. 42). The results preliminarily show that 7030B-C5 also has good effect on blood sugar regulation of high-fat diet ApoE KO mice.

(4)7030B-C5 Effect on ApoE KO mouse aortic plaques

Aortic plaque increase is an important marker for atherosclerotic disease, and the plaque change in the aorta of mice before and after 7030B-C5 administration was analyzed in this experiment using the classical oil red O staining (ORO staining). We calculated the area of the plaque after ORO staining the aorta obtained by separation from the aortic root section. The results are shown in fig. 43, where the HFD group showed a significant increase in plaque area compared to Chow group, indicating that high fat diet promoted atherosclerotic plaque formation and successful modeling. Compared with the HFD group, after the 7030B-C5 administration, the area of the hardened plaque in the aorta is reduced, particularly the atherosclerotic plaque in the high-dose 30mg/kg administration group is obviously reduced, and the results show that 7030B-C5 has a better effect of inhibiting the formation of the atherosclerotic plaque and can reduce the accumulation of aortic lipid, so that the study and the development of the study and the development.

(5)7030B-C5 Effect on ApoE KO mouse Heart outflow plaque

In addition to the aortic vessel wall, the aortic root cardiac outflow tract is also a site where lipid plaque is heavily deposited. By ORO staining on cardiac outflow tract sections, the results are shown in FIG. 44, and a large amount of plaques appear in the cardiac outflow tract part of high-fat-fed ApoE KO mice, which is obviously higher than that of the control group, thereby indicating that the modeling is successful. The plaque area at the aortic root was significantly reduced in the 7030B-C5 dosed group compared to the HFD group. These results further demonstrate that 7030B-C5 can significantly improve the development of atherosclerosis.

(6)7030B-C5 Effect on ApoE KO mouse serum transaminase levels

Liver function examination is an examination project reflecting liver physiological functions, the ratio of aspartate Aminotransferase (AST) to alanine Aminotransferase (ALT) is the most widely clinical biochemical index reflecting liver cell injury, and serum transaminase detection shows that the transaminase level of HFD is higher than that of Chow group, and slightly lower than that of the Chow group after 7030B-C5 intervention, which indicates that 7030B-C5 can slightly improve the liver function of ApoE KO mice to a certain extent (FIG. 45).

(7)7030B-C5 Effect on lipid accumulation in the liver of ApoE KO mice

Frozen sections of liver groups from ApoE KO mice were ORO stained to observe the effect of 7030B-C5 on lipid accumulation in the liver. The results are shown in FIG. 46, where staining was lighter in the Chow group and abundant staining in the HFD group, indicating a higher lipid content. Compared with the HFD group, ORO staining of the 7030B-C5 administration group is remarkably reduced, which shows that 7030B-C5 can reduce liver lipid accumulation and is helpful for preventing fatty liver formation.

(8)7030B-C5 Effect on PCSK9 and LDLR expression in ApoE KO mouse liver

To investigate whether PCSK 9expression levels in the liver of ApoE KO mice were affected by 7030B-C5, we performed total RNA extraction and protein extraction on liver tissue, respectively, and analyzed mRNA and protein levels of liver PCSK9 and LDLR by qPCR and western blot methods.

Expression at both the mRNA and protein levels of PCSK9 in the liver of ApoE KO mice was reduced following 7030B-C5 administration compared to the HFD group. 7030B-C5 significantly reduced the expression of PCSK9 when administered at 10 mg/kg. At the same time, LDLR protein expression was significantly increased in the liver of ApoE KO mice (FIGS. 47 and 48). This result is consistent with the increased LDLR expression observed in the liver of C57BL/6 mice.

Taken together, these results indicate that 7030B-C5 can effectively modulate the expression of PCSK9 and LDLR in a mouse model of diet-induced hyperlipidemia.

(9) Effect of 7030B-C5 on expression of ApoE KO mouse secreted PCSK9

PCSK9 binds to and internalizes the receptor in plasma after being secreted from hepatocytes into the blood circulation, forming the PCSK9-LDLR complex which brings the LDLR into close proximity to and degrades in lysosomes. Thus, modulation of circulating PCSK9 levels is of greater clinical significance. We collected mouse sera and analyzed the serum for levels of PCSK9 using ELISA methods. As shown in fig. 49, PCSK9 levels in HFD group were significantly increased compared to Chow group, and PCSK9 levels were decreased by 45% after 7030B-C5 intervention, further indicating that 7030B-C5 can effectively regulate PCSK9 levels in ApoE KO mice.

Example 6, 7030B-C5 molecular mechanism of action Studies of PCSK9 inhibition mediated

1. Effect of 7030B-C5 on transcriptional Activity of the PCSK9 Gene promoter

To verify that the inhibition of PCSK9 gene expression by 7030B-C5 acted on the transcriptional level, we treated previously constructed pGL4-PCSK9-P HepG2 stably transfected cells and psi-PCSK 9-3' UTR HepG2 stably transfected cells with 2 μ M7030B-C5 for 24 hours, respectively, and then detected luciferase activity. The results are shown in figure 50, 7030B-C5 was able to significantly down-regulate the luciferase activity of pGL4-PCSK9-P, with no effect on the luciferase activity of the psi-PCSK 9-3' UTR. This result preliminarily suggests that 7030B-C5 regulates expression of PCSK9 by affecting transcription of PCSK9, and does not mediate inhibition of expression of PCSK9 by affecting stability of PCSK9 mRNA.

2. Construction of recombinant plasmid of PCSK9 promoter region segmentation sequence

To further explore the molecular mechanism of 7030B-C5-mediated PCSK9 down-regulation, we examined the effect of 7030B-C5 on the transcriptional activity of the PCSK9 promoter in HepG2 cells. The reference reports that the known upstream regulatory elements of the PCSK9 gene are mainly concentrated within-440 bp (taking A of an initiation codon ATG as +1, the same below), SRE is mainly provided, the expression of the SRE is regulated by a transcription factor SREBP2, and an HNF1 alpha binding site is arranged at the upstream 28bp of the SRE, so that the activation of the SREBP2 is enhanced. It was subsequently reported that an H1NFP-binding motif is present between SRE and HNF1 α -binding motif, to which the H1NFP protein can bind, and plays a crucial role in the basic transcription and cholesterol-regulated transcription of the PCSK9 promoter. In addition, an IRE element is also present within the HNF1 α binding site, to which the FoxO3 protein binds, negatively regulating PCSK9 transcription.

Based on the distribution of homeopathic acting element sites, we divided the PCSK9 gene promoter region into seven segments. PCSK9-P segmented domian of different lengths was amplified by PCR using different 5 'forward primers and a common 3' downstream primer. Different lengths of amplified fragments were inserted upstream of pGL4-basic luciferase reporter gene using Xho I/Hind III restriction sites, recombinant luciferase reporter gene plasmids pGL4-PCSK 9-D1-D7 are obtained and named as pGL4-PCSK9-D1(-1711bp to-1 bp), pGL4-PCSK9-D2(-1214bp to-1 bp), pGL4-PCSK9-D3(-709bp to-1 bp), pGL4-PCSK9-D4(-440bp to-1 bp), pGL4-PCSK9-D5(-392bp to-1 bp), pGL4-PCSK9-D6(-351bp to-1 bp) and pGL4-PCSK9-D7(-335bp to-1 bp), and enzyme digestion identification is carried out (figure 51).

The 7 recombinant plasmids are respectively transiently transfected into HepG2 cells, the respective luciferase activities are measured, pGL4-Basic is used as a negative control, and the luciferase expression activities of pGL4-PCSK 9-D1-D7 are obviously improved, which indicates that the plasmids are successfully constructed. As can be seen from FIG. 52, the luciferase activities of the recombinant plasmids containing different promoter lengths were different after transient transfection of human hepatoma cells HepG 2. Compared with pGL4-PCSK9-P, the luciferase activity of pGL4-PCSK 9-D1-D5 is gradually enhanced, and the luciferase activity of pGL4-PCSK 9-D6-D7 is sharply reduced, which indicates that important elements for the transcriptional regulation of the PCSK9 gene exist between-392 bp and-351 bp, namely HNF1a recognition site (HNF1), IRE and HINFP binding site (HINFP binding site, HINFP-bs), and other regulation elements which are not discovered/reported may also exist. The above results are substantially consistent with the plasmid transfection results reported by Jeong et al (Jeong HJ, Lee HS, Kim KS, Kim YK, Yoon D, ParkSW. Sterol-dependent regulation of protein conversion sub-tilisin/key type9expression by solvent-dependent element binding protein-2.Journal of lipid research.2008; 49: 399-.

3. Influence of 7030B-C5 on PCSK9 promoter region segmentation sequence

To determine the possible response region of 7030B-C5 in the PCSK9 promoter region, pGL4-PCSK9-P and pGL4-PCSK 9-D1-D7 were transiently transfected into HepG2 cells, and 2. mu.M 7030B-C5 was added to continue the action for 24 hours to detect luciferase activity. The results show that 7030B-C5 significantly reduced the luciferase activity of the PCSK9 promoter D1-D5domain plasmid, while 7030B-C5 restored the inhibitory effect on the PCSK9 promoter to some extent when 7030B-C5 acted on D6 and D7domain, indicating that 7030B-C5 might act at a position of base sequence between-392 bp to-351 bp (FIG. 53).

4. 7030B-C5 Effect on PCSK9 promoter cis-responsive element mutation reporter plasmid

The previous literature reports that the binding sites of HNF1 alpha, FoxO3 and HINFP are determined on 41bp bases between-392 bp to-352 bp of the PCSK9 promoter. Thus, we mutated the core base sequences of HNF1/IRE site and HINFP-bs site on D5domain, respectively (FIG. 54), and then treated with 7030B-C5.

First, a PCR fragment containing a mutation was prepared by amplification of TransStart FastFastPfu DNA Supermix using Fast Multi site Mutagenesis System (all-purpose gold) kit with pGL4-PCSK9-D5 as a template. After the sequencing is correct, the DNA fragment is subjected to double digestion by Xho I and Hind III and is connected to a pGL4-Basic vector which is also subjected to digestion to construct mutant recombinant plasmids pGL4-D5-HNF1/IRE-mu and pGL 4-D5-HINFP-bs-mu.

The mutant luciferase reporter plasmid constructed above and pGL4-PCSK9-D5/D6 plasmid are transfected into HepG2 cells, and after 7030B-C5 acts for 24 hours, luciferase activity is detected. As shown in FIG. 55, 7030B-C5 was able to significantly down-regulate the luciferase activity of D5domain, and 7030B-C5 was found to have reduced down-regulation of luciferase activity when the HNF 1. alpha. and the HINFP binding site of D5domain were mutated (pGL4-D5-HNF1/IRE-mu, pGL 4-D5-HINFP-bs-mu).

The above results indicate that three transcription factors, HNF1 α, FoxO3 and HINFP, may mediate the inhibitory effect of 7030B-C5 on the level of PCSK9 transcription.

5. 7030B-C5 on the effects of three transcription factors HNF1 alpha, FoxO3 and HINFP

To investigate whether 7030B-C5 affects the expression of PCSK9 via HNF1 α, FoxO3 and HINFP transcription factors, we performed the following experiments.

(1)7030B-C5 Effect on HNF1 alpha, FoxO3 and HINFP Gene expression levels

First we assessed the effect of 7030B-C5 on HNF1 α, FoxO3 and HINFP protein levels. After 24 hours of treatment with a series of concentrations of 7030B-C5 added to HepG2 cells, respectively, total cell protein was extracted for protein expression studies. As shown in FIG. 56, 7030B-C5 dose-dependently reduced the protein expression level of HNF1 α and significantly up-regulated the expression of FoxO3 in the concentration range of 2.5-25 μ M. However, the levels of HINFP protein were not significantly altered following treatment with 7030B-C5.

In addition, the 7030B-C5 also has down-regulation effect on the PCSK9 promoter D6 and D7domain through luciferase activity detection. SREBP2 and Sp1 proteins are known to bind to this region, SREBP2 activates transcription of PCSK9 upon binding to SRE, upstream of which there is a series of Sp1 binding sites. The literature reports that Sp1 does not play a critical role in the transcriptional activation of PCSK 9. To investigate whether 7030B-C5 also regulated the expression of PCSK9 by acting on SREBP2 and Sp1, we studied the expression of SREBP2 and Sp 1. The results are shown in figure 57, where the protein levels of SREBP2 and Sp1 were significantly up-regulated 24 hours after HepG2 cells were treated with 7030B-C5, indicating that 7030B-C5 did not inhibit the transcription of PCSK9 by affecting the expression of SREBP2 and Sp 1.

These results preliminarily suggest that it is likely that HNF1 α and FoxO3 transcription factors are involved in 7030B-C5-mediated PCSK9 down-regulation.

(2) Effect of HNF1 α, FoxO3 and HINFP siRNAs on intracellular PCSK 9expression levels

To further confirm that the downregulation of PCSK 9expression by 7030B-C5 was dependent on HNF1 α and FoxO3, but not on the HINFP transcription factor, we performed interference experiments with siRNA addition. After 48 hours of interference by transfection of HNF1 alpha, FoxO3 or HINFPsiRNA in cells respectively, the cells were further acted for 24 hours with or without 7030B-C5 (12.5. mu.M), and the change in the expression level of PCSK9 was studied by extracting the total cell protein.

As shown in figures 58-60, siRNA (siHNF1 α, siFoxO3, siHINFP) transfection significantly reduced the levels of HNF1 α, FoxO3 and HINFP proteins compared to siRNA negative control transfected cells (siNC).

Silencing FoxO3 increased protein expression of PCSK9 compared to siNC group. After 7030B-C5 treatment, the inhibition of PCSK9 protein in FoxO3 silenced cells disappeared.

The abundance of HNF1 α protein in siHNF1 α transfected cells was greatly reduced, while PCSK9 protein expression was also significantly reduced. When HNF1 alpha is knocked down, the inhibition effect of 7030B-C5 on PCSK9 protein is disappeared.

After the HINFP is silenced by siRNA, the expression of PCSK9 is significantly inhibited due to the lack of HINFP. In contrast, 7030B-C5 still reduced the expression of PCSK9 when HINFP was knocked down.

The above data preliminarily indicate that HNF1 α and FoxO3 proteins play a key role in 7030B-C5-mediated inhibition of PCSK9 gene expression.

To further confirm that the down-regulation of PCSK 9expression by 7030B-C5 did not involve SREBP2 and Sp1, we performed interference experiments using siRNA. After transfection of SREBP2 or Sp1siRNA interference in cells for 48 hours, respectively, the cells were treated with 7030B-C5 (12.5. mu.M) for 24 hours and total cell protein was extracted and studied by Western blot.

When 7030B-C5(12.5 mu M) is added for treatment, SREBP2 expression is up-regulated, PCSK 9expression is down-regulated, and LDLR expression is obviously up-regulated, and the result preliminarily shows that 7030B-C5 regulates LDLR expression by up-regulating SREBP2 expression, and is not related to the down-regulation of PCSK9 expression. When the siSREBP2 is added, SREBP2 expression is inhibited, and the expression of PCSK9 and LDLR is reduced remarkably. The siRNA interfered the expression of SREBP2, thus inhibiting the up-regulation effect of 7030B-C5 on LDLR (FIG. 61).

The existing literature reports that Sp1 has no main effect on the transcription of PCSK9, but has an important effect on the transcriptional activation of LDLR. When 7030B-C5(12.5 mu M) is added for treatment, Sp1 expression is up-regulated, PCSK 9expression is down-regulated, and LDLR expression is obviously up-regulated, and the result preliminarily shows that 7030B-C5 regulates LDLR expression by up-regulating Sp1 expression, and is not related to the down-regulation of PCSK9 expression. When siSp1 was added, Sp1 expression was suppressed while LDLR expression was significantly reduced, whereas PCSK9 protein levels were not significantly changed (fig. 62).

The above results indicate that the expression inhibition of PCSK9 by 7030B-C5 does not involve SREBP2 and Sp1 proteins, but regulates gene transcription of LDLR by SREBP2 and Sp 1.

(3) Effect of 7030B-C5 on the binding Activity of HNF1 α, FoxO3 transcription factors to PCSK9 promoter

By usingThe Plus enzyme Chromatin IP Kit was used for Chromatin Immunoprecipitation (ChIP) experiments. We treated HepG2 cells for 24 hours at 7030B-C5 (12.5. mu.M)Fixation was carried out at room temperature for 10 minutes in 1% formaldehyde at the final concentration. Chromatin was cleaved to a length of about 150-900bp by sonication. Chromatin extracts were subsequently immunoprecipitated overnight at 4 ℃ with HNF1 α, FoxO3 or control IgG. Proteins in the immunoprecipitation complex were separated from chromatin by A-agarose beads, followed by obtaining precipitated DNA. The binding activity of HNF1 alpha, FoxO3 transcription factors to the PCSK9 promoter was analyzed by agarose gel electrophoresis and real-time PCR, respectively.

As shown in figure 63, the formation of HNF1 α/PCSK9 promoter complex in cells was significantly reduced following 7030B-C5. Meanwhile, 7030B-C5 significantly increased the binding of FoxO3 to the PCSK9 promoter. These data indicate that 7030B-C5 can promote the interaction of FoxO3 with the PCSK9 promoter and attenuate PCSK9 promoter binding capacity of HNF1 α, thereby reducing PCSK9 transcription in HepG2 cells, which in turn reduces PCSK9 expression.

(4)7030B-C5 affect protein expression levels of PCSK9 through AKT signaling pathway

The transcriptional activity of FoxO3 is affected by phosphorylation, dephosphorylation modifications. FoxO3 can be phosphorylated by a variety of kinases. Among them, protein kinase b (akt) plays an important role in the phosphorylation process of FoxO 3. Activated Akt phosphorylates FoxO3, and after phosphorylation of FoxO3 transcription factor, nuclear rejection occurs and the transcription factor is retained in cytoplasm, and the transcription activity is inhibited.

To further elucidate the regulation mechanism of 7030B-C5 on PCSK9, we treated HepG2 cells with 030B-C5 and examined the expression of p-FoxO3, p-AKT/AKT in the cells. As shown in figure 64, 7030B-C5 significantly up-regulated FoxO3 expression, while down-regulated p-AKT/AKT and p-FoxO3/FoxO3 levels. The above results indicate that 7030B-C5 can inhibit phosphorylation of FoxO3 by inhibiting the PI3K/AKT signaling pathway, resulting in increased expression of FoxO3, thereby inhibiting PCSK9 expression.

Example 7, 7030B-C5 Regulation of sugar metabolism

By analyzing the sequencing result of transcriptome of 7030B-C5 after ApoE KO mice, 7030B-C5 was found to be involved in the pathways that mediate glucose metabolism, such as pentose phosphate pathway, glucagon signaling pathway, insulin secretion, insulin resistance, PI3K-Akt signaling pathway, glycolysis/gluconeogenesis, and the like. And in the biochemical index test of the mouse of 7030B-C5 administered with ApoE KO, the 7030B-C5 is found to reduce the serum glucose level significantly. Therefore, we performed the detection analysis of the glycolysis/gluconeogenesis pathway related genes.

(1)7030B-C5 mediate sugar metabolism by regulating gluconeogenesis

Phosphoenolpyruvate Carboxykinase (PEPCK) is widely present in animals, plants, microorganisms and cells, catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, a key enzyme that regulates the gluconeogenic pathway.

Glucose 6phosphatase (G6 Pase) is a phosphatase that hydrolyzes phosphate compounds. Glucose is released into the blood in the liver by hydrolysis of glucose-6-phosphate and is also a key enzyme in the gluconeogenic pathway.

In addition, Microsomal Triglyceride transfer protein (MTP), a protective protein present in the lumen of endoplasmic reticulum that catalyzes lipid transfer to apoB molecules, plays an important role in the assembly and synthesis of Very Low Density Lipoprotein (VLDL), Apolipoprotein C-III (Apolipoprotein C-III), an Apolipoprotein that is a component exchange of High-density lipoprotein (HDL) with Triglyceride-rich (TG) particles (e.g., VLDL, chylomicron, etc.), it has been found that elevated plasma apoC-III levels cause hydrolysis and clearance disorders of TG particles, leading to accumulation of plasma VLDL, chylomicron, thereby promoting development.

We treated HepG2 cells with 7030B-C5 for 24 hours, then extracted total cellular RNA and performed real-time PCR detection. The results are shown in fig. 65, 7030B-C5 significantly down-regulated G6Pase, MTP and ApoC-III mRNA levels and presented a dose-dependence, which can also significantly reduce PEPCK mRNA levels in the low concentration range.

The above results demonstrate that 7030B-C5 regulates hepatic glucose metabolism by decreasing the expression of G6Pase and PEPCK, and that hepatic lipid metabolism can be regulated by decreasing the expression of MTP and ApoC-III. 7030B-C5 has effects of regulating glycolipid metabolism.

(2)7030B-C5 mediate sugar metabolism by regulating glycolysis

Glycolysis is a common phase that all organisms must pass through for glucose catabolism. Under anaerobic conditions, glucose or glycogen is decomposed into lactic acid by glycolysis reaction while a small amount of ATP is produced. Metformin (Metformin) is known to promote glycolysis and further exert a hypoglycemic effect. Therefore, we further explored the effect of 7030B-C5 on glycolysis of L6 myotubular cells. As a result, as shown in fig. 66, Metformin was able to significantly increase the level of lactate production of L6 myocyte, which was about 1.9 times that of the control group, significantly promoting intracellular sugar consumption. Meanwhile, the positive compound 7030B-C5 also significantly increased the level of lactate production by L6 myocyte, significantly promoted intracellular sugar consumption, and promoted glycolysis, which was about 1.6 times that of the control group (fig. 66). Furthermore, we found that 7031B-H9 also significantly promoted intracellular sugar consumption.

Thus, 7030B-C5 and 7031B-H9 show the effect of reducing blood sugar by promoting glycolysis.

Example 8 screening and validation of structurally similar Compounds

According to the screening results obtained in examples 2-5, the compounds 7030B-C5 and 7031B-H9 can reduce the expression level of PCSK9 in the liver of C57BL/6 and ApoE KO mice to a certain extent, and simultaneously effectively improve the expression of LDLR.

Next, compounds having the following structures similar to 7030B-C5 and 7031B-H9 were obtained by screening existing compounds

TABLE, 7030B-C5 structural analogs and prescreening activities

TABLE, 7030B-H9 structural analogs and prescreening activities

Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of the present invention.

Claims

1. The application of a group of compounds in preparing a medicament for reducing blood lipid is disclosed, wherein the compounds are shown as the following formula (1) or formula (2):

formula (1)Formula (2)

In formula (1):

(1) Carbon chains containing 1 to 3 carbon atoms, including-CH₂-、-(CH₂)₂-or- (CH)₂)₃-；

preferably, the first and second liquid crystal materials are,

r is benzyl containing-O-CH 3 substituent on benzene ring

R1 and R2 are H, C1-5 alkyl containing hydroxyl substituent;

r3 and R4 are H and C1-3 alkyl;

in formula (2):

preferably, the first and second liquid crystal materials are,

r5 is pyridine benzyl or furan benzyl;

r6 is hydrogen;

r7 is a benzene ring containing one methyl substituent.

2. The use according to claim 1, wherein the compound is represented by the following formula (3) or formula (4),

formula (3)Formula (4)

3. The following uses of compounds represented by formulae (1) to (4):

(1) preparing a medicament for treating atherosclerotic cardiovascular disease;

1) promote binding of FoxO3 to the PCSK9 promoter;

or 2) attenuating binding of HNF1 α to the PCSK9 promoter;

or 3) increasing expression of FoxO3 protein;

or 4) reducing the expression of HNF1 alpha protein;

4. A medicament or pharmaceutical composition for the treatment of:

(1) hyperlipidemia;

(2) atherosclerotic cardiovascular disease

(4) fatty liver;

5. A medicament or pharmaceutical composition for the treatment of:

(1) hyperlipidemia with statin failure;

(2) atherosclerotic cardiovascular disease with statin failure

(3) Fatty liver with failed statin therapy;

6. The medicine combination preparation for treating the following diseases:

(1) hyperlipidemia;

(2) atherosclerotic cardiovascular disease

(3) Fatty liver;