CN109550051B

CN109550051B - Application of histone demethylase KDM6A inhibitor in preparation of obesity treatment drug

Info

Publication number: CN109550051B
Application number: CN201910098757.1A
Authority: CN
Inventors: 王旭; 陈万涛; 韦严
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine; XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine; XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2021-04-06
Anticipated expiration: 2039-01-31
Also published as: CN109550051A

Abstract

The invention provides application of a histone demethylase KDM6A inhibitor in preparation of a medicament for treating obesity. The histone demethylase KDM6A inhibitor provided by the invention can inhibit the activity of histone demethylase KDM6A, further inhibit the expression of endoplasmic reticulum stress related genes, and reduce leptin resistance, so that obesity can be treated.

Description

Application of histone demethylase KDM6A inhibitor in preparation of obesity treatment drug

Technical Field

The invention relates to the technical field of biology, in particular to application of a histone demethylase KDM6A inhibitor in preparation of a drug for treating obesity.

Background

The prevalence of obesity increases worldwide year by year, and studies report that 1/3 people in the united states are obese. Obesity is an important risk factor for numerous chronic non-infectious diseases such as type 2 diabetes, cardiovascular disease and certain tumors, seriously compromising human health. The recent FDA has aimed at reducing energy intake by suppressing appetite through two new weight loss drugs. On the other hand, increasing energy consumption to intervene obesity is more and more emphasized by clinicians and basic researchers, and finding energy consumption related factors has become a new focus of obesity intervention research at present.

The lysine demethylase 6 (KDM 6) family of histone demethylases contains JMJC domains and is modified by removal of dimethyl and trimethyl groups from histone 3lysine residue 27(histone 3lysine 27, H3K27) to oppose EZH2 enzymatic activity in the PRC2 complex. The KDM6 family includes KDM6A (also known as UTX) and KDM6B (also known as JMJD3) and has been shown to play important roles in a variety of cellular processes, including differentiation, senescence, somatic and germ cell reprogramming, inflammatory responses, and tumors. KDM6A is involved in growth and development and is also considered to be a particularly important target for progression of Acute Lymphoblastic Leukemia (ALL) disease.

However, the physiological role of KDM6A in other human pathological conditions such as obesity has not been reported.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a use of a histone demethylase KDM6A inhibitor for the preparation of a medicament for the treatment of obesity.

To achieve the above and other related objects, the present invention provides, in a first aspect, the use of an inhibitor of histone demethylase KDM6A in the manufacture of a medicament for the treatment of obesity.

In one embodiment, the inhibitor of histone demethylase KDM6A refers to a molecule having an inhibitory effect on histone demethylase KDM 6A.

In one embodiment, the inhibitor of histone demethylase KDM6A inhibits histone demethylase KDM6A activity, or inhibits histone demethylase KDM6A gene transcription or expression.

In one embodiment, the histone demethylase KDM6A inhibitor comprises a KDM6A small molecule compound inhibitor, an anti-KDM 6A antibody, an anti-KDM 6A antisense nucleic acid, and an siRNA, miRNA, or ribozyme specific for KDM 6A.

In one embodiment, the histone demethylase KDM6A protein has the NCBI accession number NP-066963.

The NCBI accession number of the histone demethylase KDM6A gene is NM-021140.

The obesity treatment drug necessarily comprises a histone demethylase KDM6A inhibitor, and the histone demethylase KDM6A inhibitor is used as an effective component of the functions.

The form of the obesity treatment drug is not particularly limited, and the obesity treatment drug can be in the form of various substances such as solid, liquid, gel, semifluid, aerosol and the like.

The subject to which the obesity therapeutic agent is directed is a mammal such as a rodent, a primate, a human, or the like.

In a second aspect, the present invention provides a medicament for the treatment of obesity, comprising an effective amount of an inhibitor of histone demethylase KDM 6A.

In one embodiment, the active ingredient of the therapeutic agent comprises one or more of the following: KDM6A small molecule compound inhibitor, anti-KDM 6A antibody, anti-KDM 6A antisense nucleic acid, siRNA, miRNA and ribozyme specific for KDM 6A.

In one embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

The third aspect of the invention provides a new application of a histone demethylase KDM6A inhibitor, and the application of the histone demethylase KDM6A inhibitor in preparing a medicine for inhibiting the expression of a promoter region H3K27Me3 of an endoplasmic reticulum stress gene and inhibiting the endoplasmic reticulum stress level.

The fourth aspect of the invention provides the use of histone demethylase KDM6A as an action target in screening drugs for treating obesity.

The fifth aspect of the present invention provides a method for screening a therapeutic agent for obesity, comprising: and (3) verifying whether the medicament to be screened can inhibit the histone demethylase KDM6A, and if so, determining the medicament to be screened as a candidate medicament for treating the obesity.

Further, the method comprises:

and (3) acting the medicament to be screened on the in vitro cell expressing the histone demethylase KDM6A to determine whether the histone demethylase KDM6A in the cell is inhibited.

For example, the screening method may be: culturing in vitro cells expressing KDM6A under conditions suitable for cell growth, setting two groups of comparison experiments, adding the drug to be screened into a culture dish of the in vitro cells, adding the same amount of physiological saline into the other group, incubating under the same conditions, and testing whether the KDM6A of the in vitro cells is inhibited.

The method for determining whether the activity or the expression of the histone demethylase KDM6A in the cell is reduced is as follows: real-time quantitative PCR and/or Western Blot detection, or mass spectrometric detection.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a new application of histone demethylase KDM6A in a medicine for treating obesity, and the histone demethylase KDM6A inhibitor provided by the invention can inhibit the activity of histone demethylase KDM6A, inhibit the expression of a promoter region H3K27Me3 of a stress gene, inhibit the endoplasmic reticulum stress level, reduce leptin resistance and treat obesity.

Drawings

FIG. 1A: PCR analysis to identify Kdm6a^f/fGenotype electrophoretogram of Cre/ERT2+ mice.

FIG. 1B: kdm6a inducible knock-out protocol.

FIG. 1C: the Kdm6a inducible knock-out results were confirmed by PCR assay.

FIG. 1D: OGTT assay results plot for the detection of blood glucose homeostasis in High Fat Diet (HFD) -induced obese mice.

FIG. 1E: ITT assay results plot for checking DIO mice sensitivity to insulin.

FIG. 1F: the OGTT test measures a plot of blood glucose homeostasis results for standard diet mice.

FIG. 1G: graph of the results of the ITT test used to check insulin sensitivity in standard diet mice.

FIG. 2A: wild-type mice and Kdm6a were observed to induce body weight and percent body weight changes in knockout mice over 21 days (panel a, HFD mice, vehicle, n-10; GSK-J4, n-9).

FIG. 2B: observation of weight and percentage change in weight in 21 days for wild-type mice and Kdm6 a-induced knockout mice (normal diet, ND) mice, vehicle, n ═ 10; GSK-J4, n ═ 10)

FIG. 2C: representative of wild-type and Kdm6 a-induced knockout mice (left: wild-type; right Kdm6 a-induced knockout).

FIG. 2D: body weight change plot ([ p ] 0.001; [ p ] 0.005; [ p ] 0.05. n.s. indicates no significant difference).

FIG. 2E: the first week of treatment daily food intake (. p < 0.001;. p < 0.005;. p < 0.05. n.s. indicates no significant difference).

FIG. 2F: DIO mice had no significant difference in serum insulin concentrations 3 weeks after treatment (p < 0.001;. p < 0.005;. p < 0.05. N.S.).

FIG. 2G: after 3 weeks of treatment, the serum leptin concentrations of DIO and ND mice (p < 0.001;. p < 0.005;. p < 0.05. n.s. indicate no significant difference).

FIG. 2H: serum total cholesterol concentrations in DIO and ND mice 3 weeks after treatment (. p < 0.001;. p < 0.005;. p < 0.05. n.s. indicates no significant difference).

Detailed Description

Endoplasmic reticulum stress is closely related to the pathophysiological mechanism of obesity. The increase of endoplasmic reticulum in the brain plays a key role in the development of leptin resistance, which can induce obesity. As described above, the invention discloses a new application of histone demethylase KDM6A in preparation of a drug for treating obesity, and the histone demethylase KDM6A inhibitor provided by the invention can inhibit the activity of histone demethylase KDM6A, inhibit the expression of a promoter region H3K27Me3 of a stress gene, inhibit the endoplasmic reticulum stress level, reduce leptin resistance and treat obesity.

Inhibitors of histone demethylases KDM6A

Refers to a molecule having inhibitory effect on histone demethylase KDM 6A. Having inhibitory effects on histone demethylase KDM6A include, but are not limited to: inhibit the activity of histone demethylase KDM6A, or inhibit the transcription or expression of histone demethylase KDM6A gene. The histone demethylase KDM6A inhibitor comprises but is not limited to siRNA, shRNA, antibody and small molecule compound.

The inhibition of the activity of the histone demethylase KDM6A refers to the reduction of the activity of the histone demethylase KDM 6A. Preferably, the activity of histone demethylase KDM6A is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, most preferably by at least 90% compared to prior to inhibition.

Inhibiting the transcription or expression of the histone demethylase KDM6A gene refers to: the method comprises the steps of not transcribing the gene of the histone demethylase KDM6A, or reducing the transcriptional activity of the gene of the histone demethylase KDM6A, or not expressing the gene of the histone demethylase KDM6A, or reducing the expression activity of the gene of the histone demethylase KDM 6A.

The skilled person can use conventional methods to modulate the gene transcription or expression of the histone demethylase KDM6A, such as gene knock-out, homologous recombination, interfering RNA, etc.

The inhibition of gene transcription or expression of the histone demethylase KDM6A can be verified by PCR and Western Blot detection of expression level.

Preferably, the histone demethylase KDM6A gene transcription or expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, still more preferably by at least 90%, most preferably completely absent compared to the wild type.

Small molecule compound inhibitors

The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.

Preparation of medicine by histone demethylase KDM6A inhibitor

The histone demethylase KDM6A inhibitor is used as the main active component or one of the main active components in preparing medicine. Generally, the medicament may comprise one or more pharmaceutically acceptable carriers or excipients in addition to the active ingredient, according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with, i.e. capable of being blended with, the histone demethylase KDM6A inhibitor without substantially reducing the efficacy of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as glycerol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1 Effect of KDM6A deficiency on diet-induced obesity (DIO) mouse weight

In view of the correlation between endoplasmic reticulum stress and obesity, in order to study the potential physiological effect of KDM6A in vivo, we established an inducible knockout mouse and selected a high fat diet-induced obesity model, the NCBI accession number of mouse histone demethylase Kdm6a is NM-009483.

1.1 Experimental animals

Kdm6a^f/f(1) Two mouse strains (Jackson laboratory deposit No. 024177, USA) and UBC-Cre/ERT2(2) (Jackson laboratory deposit No. 007179, USA) have been previously reported. Both strains of mice were backcrossed with C57 wild-type mice for more than 10 generations. Kdm6a^iKO/YThe mouse is formed by mating and breeding the two strains, and the genotype of the mouse is identified by adopting a PCR reaction, so that the mouse can inducibly knock out the Kdm6a gene in the whole body range after receiving tamoxifen injection. Mice were housed in a pathogen-free environment at 22 ℃ for a 12 hour day-night cycle with free access to food and water.

1.2 grouping

The experimental animals are divided into four groups, one group is induced to knock out Kdm6a ^iKO/Y10 mice (A), (B) and (C)High fat diet), another group was wild type Kdm6a ^WT/Y10 mice (high fat diet); one group is induced to knock out Kdm6a ^iKO/Y10 mice (standard diet), another group of wild type Kdm6a ^WT/Y10 mice (standard diet).

1.3 Experimental methods

High fat diet feeding (60 kcal% lipid; Research Diets for mulas D12492; Research Diets, Inc., New Brunswick, N.J.) or standard diet feeding (11 kcal% lipid; Research Diets for mulas D12329; Research Diets, Inc.) was started at 6 weeks of age for 16 weeks. Weighing was performed weekly, and after 16 weeks, in order to induce the knockdown of Kdm6a, tamoxifen (150. mu.l, solution concentration 10. mu.g/. mu.l, corresponding to a dose of 75mg/kg) was intraperitoneally injected for 4 consecutive days to activate Cre/ERT2 recombinase, thereby knocking down the KDM6A gene. 3 weeks after tamoxifen injection, animals were sacrificed by anesthesia and blood and tissue collected.

1.3.1OGTT glucose tolerance test

After animals were starved for 15 hours, 1g/kg of glucose was orally injected. Blood glucose was measured by a handheld glucometer after tail blood at 15, 30, 60, 90 and 120 minutes before and after oral glucose injection, respectively.

1.3.2ITT glucose tolerance test

Animals were starved for 6 hours from 8 am to 2 pm and were injected intraperitoneally with 1IU/kg of insulin. Blood glucose was measured by a handheld glucometer after tail bleed at 15, 30, 45 and 60 minutes before and after injection, respectively.

1.3.3 serum index detection

Serum was assayed for leptin (R & D), insulin (Millipore), cholestrol (Roche) and trigyceride (Roche) by ELISA.

1.4 results show

Identification of Kdm6a by PCR analysis^f/f,Cre/ERT2⁺Genotype of mice (fig. 1A). Mice with a validated genotype administered tamoxifen i.p. for 4 days daily resulted in functional activation of CRE/ERT2 to knock out the Kdm6a gene (fig. 1B, C). Notably, induction of KDM6A knock-out significantly reduced body weight in DIO mice (fig. 2A).The last day of administration, three weeks later, Kdm6a^iKO/YThe body weight of the mice was approximately 30% lower than the body weight before treatment (fig. 2B, C). Consistent with this, KDM6A knockout significantly reduced perirenal and epididymal adipose tissue compared to the wild-type high fat diet group (fig. 2D). We also investigated the effect of KDM6A gene knock-out on body weight in normal diet mice. The normal diet Kdm6a after being treated with tamoxifen at the same dose as the high fat diet group^iKO/YThe body weight of the mice was comparable to that of wild-type mice.

Since a decrease in food intake is the main cause of weight loss, the present invention measures the average food intake for the second week of the treatment given. Kdm6a^iKO/YThe amount of food consumed by the mice was compared to Kdm6a of the DIO mice^WT/YThe group is much lower (fig. 2D). It has also been found that Kdm6a^iKO/YConcentration ratio of insulin to leptin in mice Kdm6a^WT/YMice (fig. 2E, F) were much lower. KDM6A knockout significantly improved the metabolic state of DIO mice (fig. 2G). Although food intake was significantly reduced in normally fed mice, there was no significant change in insulin, leptin or total cholesterol levels compared to the wild-type group after administration of tamoxifen to knock out Kdm6a 3 for weeks. In the OGTT assay, induced knock-out of KDM6A selectively enhanced glucose homeostasis in obese mice (fig. 1D), but not lean mice (fig. 1F). Knockout of Kdm6a had no significant effect on insulin stimulation in ITT analysis of both obese and lean mice (fig. 1E, G).

In conclusion, the KDM6A knockout effect can obviously reduce the food intake and the body weight of the high-fat-induced obese mice, the KDM6A is a drug target for treating obesity, and the KDM6A inhibitor can be used for treating obesity.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. An application of a histone demethylase KDM6A inhibitor in preparing a medicament for treating obesity.

2. The use according to claim 1, wherein the inhibitor of histone demethylase KDM6A is a molecule having inhibitory effect on histone demethylase KDM 6A.

3. The use of claim 1, wherein the inhibitor of histone demethylase KDM6A inhibits histone demethylase KDM6A activity or inhibits histone demethylase KDM6A gene transcription or expression.

4. The use of claim 1, wherein the histone demethylase KDM6A inhibitor comprises a KDM6A small molecule compound inhibitor, an anti-KDM 6A antibody, an anti-KDM 6A antisense nucleic acid, and an siRNA, miRNA, or ribozyme specific for KDM 6A.

5. The use according to claim 1, wherein the histone demethylase KDM6A has NCBI accession No. NP-066963.