CN108187049A - The application of the albumen of functional domain containing Bromo 4 and its inhibitor in treatment fatty liver and relevant disease drug is prepared - Google Patents
The application of the albumen of functional domain containing Bromo 4 and its inhibitor in treatment fatty liver and relevant disease drug is prepared Download PDFInfo
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- CN108187049A CN108187049A CN201810022991.1A CN201810022991A CN108187049A CN 108187049 A CN108187049 A CN 108187049A CN 201810022991 A CN201810022991 A CN 201810022991A CN 108187049 A CN108187049 A CN 108187049A
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- inhibitor
- albumen
- bromo
- fatty liver
- functional domain
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
Abstract
The invention discloses the application of the albumen of functional domain containing Bromo 4 and its inhibitor in treatment fatty liver and relevant disease drug is prepared.The present invention is with different liver cells（L02 cells, HepG2 cells are experimental subjects, respectively with BRD4 inhibitor --- JQ1 and OTX015 processing, DMSO are compared, then are used（Palmitate/oleic acid）PA/OA processing, carries out oil red O stain to cell later, and coloration result is all shown：Compared with DMSO control groups, inhibitor processing group color significantly shoals.After this shows that BRD4 is suppressed, fat accumulation can be inhibited, the generation of fatty liver and relevant disease can be improved.Therefore BRD4 provides target to develop prevention, alleviation and/or the drug for the treatment of fatty liver and relevant disease.
Description
Technical field
The invention belongs to the function and application field of gene, more particularly to a kind of BRD4 is treated as drug targets in screening
The inhibitor of application and BRD4 in fatty liver medicament is preparing prevention, is alleviating and/or treatment fatty liver and relevant disease
Application in drug.
Background technology
With the continuous improvement of people's living standards, dietary structure also has changed a lot, gradually it is intended to high egg
In vain, it is high in fat, but the metaboilic level of the mankind is limited, this has resulted in continuous accumulation of the fat in liver, so as to cause fat
Fat liver.In recent years, the incidence of fatty liver increasingly increases, and morbidity group will be accompanied by steatohepatitis, liver fibrosis even
The generation of hepatic sclerosis, and hepatocellular carcinoma can be developed into.At present, it there is no lipotropic specific medicament.It is prepared so finding
Prevention, the novel targets for the drug alleviated and/or treat Fatty Liver Disease, are just particularly important.
Bromodomain-containing protein 4 (BRD4) belong to Bromodomain and extra-
Terminal (BET) family member.BRD4 is a kind of transcriptional regulation protein for being prevalent in mitotic cell, is turned by
Transcriptional regulation is played in record regulatory complex recruitment to the chromatin of acetylation, activates the transcription of downstream target gene, from
And play significant role in cell cycle regulation, tumour occur[1].BRD4 can also be important with some transcription factor (such as
P53 etc.) interaction, to influence the transcription of downstream gene.Recent studies indicate that the expression imbalance and dysfunction of BRD4
Inflammatory reaction and the occurrence and development of tumour can significantly be influenced[2][3].At present, using BET Bromodomain albumen as target,
The scientific research of the diseases such as inflammation, tumour is carried out, finds the micromolecular inhibitor that selectivity is good, safe, bioactivity is strong
Hot spot even more in hot spot.In recent years, for the research of BRD4 inhibitor there are also achievement, such as methyl-triazole-Isosorbide-5-Nitrae benzo two
Bromodomain inhibitor (+)-JQ1 of nitrogen type[4]And OTX015, they possess BRD4 very high affinity.But it closes
In effect in fatty liver of BRD4 and its inhibitor and indefinite.
Bibliography：
1.Asangani I A,Dommeti V L,Wang X,et al.Therapeutic targeting of BET
bromodomain proteins in castration-resistant prostate cancer[J].Nature,2014,
510(7504):278-82.
2.French C A,Miyoshi I,Kubonishi I,et al.BRD4-NUT Fusion Oncogene[J].
Cancer Research,2003,63(2):304-7.
3.Segura M F,Fontanalscirera B,Gazielsovran A,et al.BRD4sustains
melanoma proliferation and represents a new target for epigenetic therapy.
[J].Cancer Research, 2013,73(20):6264.
4.Filippakopoulos P,Qi J,Picaud S,et al.Selective inhibition of BET
bromodomains.[J].Nature,2010,468(7327):1067.
Invention content
The purpose of the present invention is to provide the albumen of functional domain containing Bromo 4 and its inhibitor prepare prevention, alleviate and/or
Treat the application in the drug of fatty liver and its relevant disease.
Above-mentioned purpose is achieved by the following technical solution：
Using different liver cells, (L02 cells, HepG2 cells are inhibited the present invention with BRD4 respectively as experimental subjects
Agent --- JQ1 and OTX015 processing, DMSO are compared, then with (palmitate/oleic acid) PA/OA processing, and cell is carried out later
Oil red O stain, coloration result are all shown：Compared with DMSO control groups, inhibitor processing group color significantly shoals.This shows
After BRD4 is suppressed, fat accumulation can be inhibited, the generation of fatty liver and relevant disease can be improved.
On this basis, it is pre- in screening as drug targets to provide the albumen of functional domain containing Bromo 4 for first aspect present invention
Application in anti-, alleviation and/or the drug for the treatment of fatty liver and its relevant disease.
Second aspect of the present invention, the inhibitor for providing the albumen of functional domain containing Bromo 4 are preparing prevention, alleviation and/or treatment
Application in fatty liver and its relevant disease drug.
Preferably, the inhibitor of the albumen of functional domain containing Bromo 4 is to inhibit BRD4 protein actives or protein level
Inhibitor or inhibit BRD4 mRNA level in-site inhibitor, inhibitory activity is reversible or irreversible.
Preferably, it is described that the inhibitor of BRD4 protein actives or protein level is inhibited to include the antibody of BRD4, inhibition
It is the protein of BRD4 protein actives or protein level, polypeptide, enzyme, native compound, synthesis compound, organic matter, inorganic
Object；It is described that the inhibitor of BRD4 protein actives or protein level is inhibited to refer to that BRD4 can be combined but do not generated when combining
The substance of biological response or the inhibitor can block, inhibit or weaken the response mediated by agonist, and can be with excitement
Agent competitive binding BRD4.
Preferably, the inhibitor of the BRD4 is JQ1 or OTX015 or its pharmaceutically acceptable salt or its solvation
Object or its metabolite.
Terms used herein " pharmaceutically acceptable salt " refers to the derivative of pharmaceutical active compounds, wherein passing through preparation
Its acid salt or basic salt modify parent compound.The example of pharmaceutically acceptable salt includes but not limited to, alkaline residue
The inorganic acid salt or acylate of (such as amine), the basic salt or organic salt of acidic residues (such as carboxylic acid), etc..It is pharmaceutically acceptable
Salt include the salt or quaternary ammonium salt of the Conventional nontoxic of parent compound that are formed by such as avirulent inorganic or organic acid.Example
Such as, these conventional non-toxic salts include thoseing the salt for being originated from inorganic acid, as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid,
Nitric acid etc.；And by organic acid prepare salt, e.g., acetic acid, propionic acid, succinic acid, hydroxyacetic acid, lactic acid, malic acid, tartaric acid,
Citric acid, fumaric acid, methanesulfonic acid, toluenesulfonic acid, salicylic acid, p-aminobenzene sulfonic acid etc..
The pharmaceutically acceptable salt of the present invention can be by conventional chemical method from containing alkaline or acidic moiety
Parent compound synthesizes.In general, this salt can by make these compounds free acid or alkali form and stoichiometry it is suitable
When alkali or acid in water or organic solvent or the mixture of the two react prepare.
Preferably, the inhibitor of the BRD4 is JQ1 or OTX015 and pharmaceutically acceptable auxiliary material.
Preferably, the pharmaceutically acceptable auxiliary material is to be commonly used in pharmaceutical field or known various auxiliary materials, including but
It is not limited to：Diluent, adhesive, antioxidant, pH adjusting agent, preservative, lubricant, disintegrant etc..
The diluent is for example：Lactose, starch, cellulose derivative, inorganic calcium salt, sorbierite etc..Described adhesive example
Such as：Starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone etc..The antioxidant is for example：Vitamin E, sulfurous acid
Hydrogen sodium, sodium sulfite, butyl anisole etc..The pH adjusting agent is for example：Hydrochloric acid, sodium hydroxide, citric acid, tartaric acid,
Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate etc..The preservative is for example：Methyl p-hydroxybenzoate, para hydroxybenzene first
Acetoacetic ester, metacresol, benzalkonium chloride etc..The lubricant is for example：Magnesium stearate, superfine silica gel powder, talcum powder etc..The disintegrant
Such as：Starch, methylcellulose, xanthans, croscarmellose sodium etc..
Preferably, the antibody of the BRD4 includes but not limited to monoclonal antibody, synthetic antibody, mostly polyclonal antibody, spy
Heterogenetic antibody, human antibody, humanized antibody, chimeric antibody, scFv (scFv) (including bispecific scFv), single-chain antibody,
Fab segments, F (ab') segment, the Fv (sdFv) of disulfide bond connection and any of above epitope binding fragments.Particularly, for this
The antibody of invention includes the immunoactive portions of immunoglobulin molecules and immunoglobulin molecules.For the immune ball of the present invention
Protein molecular can be any types (for example, IgG, IgE, IgM, IgD, IgA and IgY), the classification (example of immunoglobulin molecules
Such as, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.Preferably, antibody is people or Humanized monoclonal antibodies.
As used herein, " people " antibody include with human immunoglobulin(HIg) amino acid sequence antibody, and including from
Human immunoglobulin(HIg) library or the antibody detached from the mouse by human gene expression antibody or other animals.
Preferably, the inhibitor for inhibiting the mRNA level in-site of BRD4 can be its anti sense nucleotide sequence, siRNA, miRNA,
ShRNA, dsRNA or the protein of other mRNA level in-sites that can inhibit BRD4, polypeptide, enzyme, compound.
The dosage form of drug of the present invention can be the form of oral agents, such as tablet, capsule, pill, pulvis, granule, outstanding
Floating agent, syrup etc.；Can also be the dosage form of drug administration by injection, such as parenteral solution, powder-injection etc., by intravenous, peritonaeum, skin
Lower or intramuscular approach.All dosage forms used are all known to pharmaceutical field those of ordinary skill.
The drug of the present invention can be applied to any animal that can occur or send out fatty liver and relevant disease.These animals
Including the mankind and inhuman animal, such as pet or livestock etc..
The drug of the present invention can be applied to subject with approach known in the art, including but not limited to oral, parenteral,
Subcutaneously, intramuscular, intravenously, intraperitoneal, in liver, in cardiac muscle, in kidney, vagina, rectum, cheek is sublingual, intranasal, transdermal means etc..
Applied dose is combined the type of drug, therapeutic frequency is given by depending on the age of recipient, health and weight
Medicine approach etc..Drug can be applied with single daily dose or total daily dose is with twice daily, and three times or the separate doses of four times are applied
With.Dosage can apply it is one or many, spraying time can be with odd-numbered day to some months or longer time.
The fatty liver and relevant disease include but not limited to：Insulin resistance, metabolic syndrome, obesity, diabetes, height
Blood glucose, hyperlipemia, pure hepatic steatosis, nonalcoholic fatty liver disease, liver fibrosis, hepatic sclerosis, liver cancer etc..
The present invention is had the following advantages relative to the prior art and effect：
(1) present invention discover that the new function of BRD4 genes, i.e. BRD4 genes have the work for deteriorating fatty liver and relevant disease
With.
(2) effect based on BRD4 in fatty liver and relevant disease is deteriorated, to develop prevention, alleviation and/or treatment
The drug of fatty liver and relevant disease provides target.
(3) inhibitor of BRD4 can be used for the drug for preparing prevention, alleviating and/or treat fatty liver and relevant disease.
(4) present invention discover that the new function of BRD4 specific inhibitors JQ1 or OTX015, can be used for preparing prevention, delay
Solution and/or the drug for the treatment of fatty liver and relevant disease.
Description of the drawings
Fig. 1 is the oil red O coloration results after inhibited dose of processing 3h, 0.2/0.4mM PA/OA processing 12h of L02 cells
Figure；A is control DMSO groups, and B is 0.5 μM of JQ1 processing group, and C is 0.5 μM of OTX015 processing group.
Fig. 2 is the oil red O stain result after inhibited dose of processing 3h, 0.1/0.2mM PA/OA processing 12h of HepG2 cells
Figure；A is control group DMSO groups, and B is 0.5 μM of JQ1 processing group, and C is 0.5 μM of OTX015 processing group.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.Experiment cell line
And culture：
L02：Human liver cell system, purchased from Cell Bank of Chinese Academy of Sciences, catalog number (Cat.No.) GNHu6.
HepG2：Bel7402,
Cell is incubated in DMEM high glucose mediums (containing 10%FBS).Culture environment：37 DEG C, 5% CO2.
Experiment inhibitor：
(+)-JQ1 is purchased from sigam companies, article No.：SML1524
OTX015 is purchased from sigam companies, article No.：SML1605
Cell oil red O stain：
1. taking out cell, culture medium is sucked out, PBS is added to rinse 3 times, PBS is blotted as possible after cleaning.
The fixed 15min of 2.4% 37 DEG C of paraformaldehyde.
3. after, formaldehyde is abandoned, PBS is added in and rinses 3 times, every time 3 minutes.
4. adding in 60% isopropanol effect 30s, washed 3 times with PBS after the completion.
5. drying moisture in super-clean bench, after moisture parches completely, ware bottom is white.
6. prepare the working solution of oil red O, Red Oil：PBS=3:2 configurations.Oil red is stored at room temperature 10min after being configured,
Then it is filtered with 0.45 μM of filter, you can use.
7. liquid is made in drying post-processing, observed in time in dyeing course, reach to inhale after requirement and abandon dyeing liquor.
8. being washed 3 times with PBS, PBS is added to infiltrate observation of taking pictures.
【Embodiment 1】The influence that L02 cytolipins are accumulated in the processing of BRD4 inhibitor
L02 cells are divided into three tissue cultures and support, and after cell is adherent, are separately added into DMSO, 0.5 μM of JQ1,0.5 μM of OTX015
After handling 3h, 12h is handled with 0.2/0.4mM PAOA, carries out oil red O stain.
As shown in Figure 1, compared with control group DMSO groups, inhibitor processing group color significantly shoals coloration result.The result
Illustrate, through BRD4 inhibitor treated L02 cells, lipid accumulation is significantly inhibited.【Embodiment 2】The processing of BRD4 inhibitor
Influence to the accumulation of HepG2 cytolipins
HepG2 cells are divided into three tissue cultures and support, after cell is adherent, be separately added into DMSO, 0.5 μM of JQ1,0.5 μM
OTX015 handles 3h, after then handling 12h with 0.1/0.2mM PA/OA, carries out oil red O stain.
As shown in Fig. 2, compared with control group DMSO groups, inhibitor processing group color significantly shoals coloration result.The result
Illustrate, through BRD4 inhibitor treated HepG2 cells, lipid accumulation is significantly inhibited.
The above results show, after the expression of BRD4 is suppressed, can significantly improve the liver lipids heap of PA/OA stimulation inductions
Product illustrates that BRD4 gene pairs deteriorates fatty liver and the occurrence and development of relevant disease and has remarkable effect.BRD4 can be used for screening pre-
Drug that is anti-, alleviating and/or treat fatty liver and relevant disease, inhibitor such as JQ1, OTX015 of BRD4 can be used for preparing pre-
Drug that is anti-, alleviating and/or treat fatty liver and relevant disease.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. the albumen of functional domain containing Bromo 4 prepares prevention, alleviation and/or treatment fatty liver and its phase as drug targets in screening
Application in the drug of related disorders.
2. application according to claim 1, which is characterized in that prevention, alleviation and/or the treatment fatty liver and its phase
Related disorders drug is the drug that the albumen of functional domain containing Bromo 4 is inhibited to express.
3. the inhibitor of the albumen of functional domain containing Bromo 4 is preparing prevention, alleviating and/or is treating fatty liver and its relevant disease medicine
Application in object.
4. application according to claim 3, which is characterized in that the inhibitor of the Bromo functional domains albumen 4 is to inhibit
The mRNA of the inhibitor or inhibition Bromo functional domains albumen 4 of Bromo functional domain albumen april protein activity or protein level
Horizontal inhibitor, inhibitory activity are reversible or irreversible.
5. application according to claim 4, which is characterized in that the inhibition Bromo functional domain albumen april protein activity or
The antibody of the inhibitor of protein level including Bromo functional domains albumen 4, inhibit Bromo functional domain albumen april protein activity or
Protein, polypeptide, enzyme, native compound, synthesis compound, organic matter, the inorganic matter of protein level；The inhibition Bromo
The inhibitor of functional domain albumen april protein activity or protein level refers to that Bromo functional domains albumen 4 can be combined but is combining
When do not generate the substance of biological response or the inhibitor and can block, inhibit or weaken the response mediated by agonist, and
It can be with agonist competitive binding Bromo functional domains albumen 4.
6. application according to claim 4, which is characterized in that the inhibitor of the Bromo functional domains albumen 4 be JQ1 or
OTX015 or its pharmaceutically acceptable salt or its solvate or its metabolite.
7. application according to claim 4, which is characterized in that the mRNA level in-site of the inhibition Bromo functional domains albumen 4
Inhibitor is its anti sense nucleotide sequence, siRNA, miRNA, shRNA, dsRNA or other can inhibit Bromo functional domain eggs
The protein of white 4 mRNA level in-site, polypeptide, enzyme, compound.
8. claim 1-7 any one of them applications, which is characterized in that fatty liver and its relevant disease include but not limited to：
Pure hepatic steatosis, nonalcoholic fatty liver disease, liver fibrosis, hepatic sclerosis, liver cancer etc..
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| CN201810022991.1A CN108187049A (en) | 2018-01-10 | 2018-01-10 | The application of the albumen of functional domain containing Bromo 4 and its inhibitor in treatment fatty liver and relevant disease drug is prepared |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103119160A (en) * | 2010-05-14 | 2013-05-22 | 达那-法伯癌症研究所 | Compositions and methods for modulating metabolism |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103119160A (en) * | 2010-05-14 | 2013-05-22 | 达那-法伯癌症研究所 | Compositions and methods for modulating metabolism |
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Application publication date: 20180622 |
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