CN109106715B

CN109106715B - Application of 8-hydroxyquinoline medicine or salt thereof in preparing medicine for treating BRD4 related diseases

Info

Publication number: CN109106715B
Application number: CN201710486433.6A
Authority: CN
Inventors: 罗成; 郑明月; 谢轶谦; 邢婧; 张元元; 蒋昊; 王晨; 陈凯先; 蒋华良
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2017-06-23
Filing date: 2017-06-23
Publication date: 2023-01-31
Anticipated expiration: 2037-06-23
Also published as: CN109106715A

Abstract

The invention relates to application of 8-hydroxyquinoline (8-OH-quinoline) compounds or salts thereof in preparing medicaments for treating BRD 4-related diseases, wherein the BRD 4-related diseases comprise NUT-midline carcinoma, acute lympholeukemia, mixed lineage leukemia, multiple myeloma, burkitt's lymphoma, hepatocellular carcinoma, triple negative breast cancer, non-small cell lung cancer, prostatic cancer, pancreatic cancer, neuroblastoma and other tumor types and heart failure, such as cardiac hypertrophy and myocardial hypertrophy diseases.

Description

Application of 8-hydroxyquinoline medicine or salt thereof in preparing medicine for treating BRD4 related diseases

Technical Field

The invention belongs to the technical field of medicines. In particular to application of 8-hydroxyquinoline (8-OH-quinoline) compounds or salts thereof in preparing medicaments for treating BRD4 related diseases, and more particularly to application of 8-hydroxyquinoline (8-OH-quinoline) compounds or salts thereof in preparing medicaments for treating various tumor types including NUT-midline cancer, acute lymphatic leukemia, mixed lineage leukemia, multiple myeloma, burkitt's lymphoma, hepatocellular carcinoma, triple negative breast cancer, non-small cell lung cancer, prostatic cancer, pancreatic cancer, neuroblastoma and the like and heart failure.

Background

In recent years, protein 4 (Bromodomain-stabilizing protein 4, BRD 4) containing a Bromodomain has been found to be closely related to heart failure, and BRD4 inhibitors relieve the symptoms of cardiomyocyte hypertrophy by reducing the expression of excessive hypertrophy genes, which provides a new direction for developing drugs for treating heart failure. In addition, BRD4 has been identified as a target for a variety of cancers (including NUT-midline cancer, multiple myeloma, acute lymphoid leukemia, etc.) by a major mechanism that, in midline cancer, chromosomal translocation leads to the formation of BRD4 protein fused with NUT, causing the cells to develop dysdifferentiation and malignant proliferation, thereby causing the development of the associated cancer. In c-myc dependent tumors, BRD4 recognizes acetylated lysines on histones, initiating transcription of downstream c-myc, leading to related cancers.

Cardiac hypertrophy, which includes primary myocardial hypertrophy and compensatory myocardial hypertrophy to resist high load caused by other factors (such as hypertension, etc.), is a major cause of heart failure. The patient has symptoms of edema, incapability of lying down and the like, and the life quality is seriously influenced. Heart failure often occurs in the middle-aged and elderly people, but in recent years, many young people have symptoms of heart failure due to high pressure on life. The current treatment method is to increase myocardial contractility by using cardiotonics, but such drugs have the problems of narrow therapeutic window or poor selectivity, etc., and neglect the treatment of hypertrophic myocardial cells. The 8-hydroxyquinoline can be used as a new treatment for heart failure by inhibiting BRD4, relieving myocardial hypertrophy and reducing heart load by being matched with other medicines.

NUT-midline carcinoma (NUT-midline carcinoma) is a rare, highly aggressive and high mortality malignancy that occurs well in children, young adults, and usually less than a year from finding to dying. The pathogenesis is that because the chromosome ectopy causes the fusion of testicular nuclear protein (NUT) and BRD4 protein, BRD4 loses the PID structural domain combined with a transcription factor and can not activate transcription, the cell differentiation can not be completed, and then the cancer is evolved. NUT-midline carcinoma currently has no specific effective drug, which poses a serious threat to the patient's life.

In addition, BRD4 has been identified as a target for many cancers associated with c-myc overexpression, including acute lymphoblastic leukemia, mixed lineage leukemia, multiple myeloma, burkitt's lymphoma, hepatocellular carcinoma, triple negative breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, neuroblastoma, etc., and there are currently no specific therapeutic guidelines or existing drugs that are highly toxic and relapse prone. Therefore, development of anticancer drugs having a new parent nucleus structure is required.

For the case of three-dimensional high resolution structures of known disease targets and target proteins, structure-based drug design (SBDD) is widely used due to its advantages of clear mechanism, strong pertinence, high efficiency in discovering active compounds, and the like. Virtual screening (virtual screening) screens out chemical molecules that may have therapeutic effects by computational methods, and can generally effectively reduce the cost of structural-based drug design. Two requirements for successful virtual drug screening are: (1) Chemical space (chemical space) large enough, that is, the chemical database used is diverse in terms of the structural mother nucleus covering the molecule, and software can efficiently produce conformations (conformations) in which ligands and receptors are more likely to exist; (2) Accurate scoring functions, developed primarily from force-field-based, experience-based and knowledge-based principles, employ energy calculations and statistical learning methods for rapid and efficient assessment of whether an unknown ligand binds stably to a given receptor or even predicts binding intensity (binding affinity) or binding free energy (binding free energy). The invention discovers that the new maternal nucleus BRD4 inhibitor can be used for treating related diseases by combining virtual screening and experimental verification.

To date, no report has been made on biological or chemotherapeutic drugs designed to address the BRD4 binding mechanism. Therefore, there is an urgent need in the art to develop drugs that can effectively treat the above-mentioned diseases with little side effects.

Disclosure of Invention

On the basis of research on biological functions of a target BRD4, through virtual screening of a small molecule drug database which is already on the market, in combination with in vitro activity evaluation and the like, the invention discovers new applications of several 8-hydroxyquinoline (8-OH-quinoline) drugs or salts thereof as inhibitors of the target BRD4 in treating diseases such as certain tumors, heart failure and the like.

According to the first aspect of the invention, the active compound targeting the human BRD4 protein with a new structural entity is obtained by screening in a mode of combining virtual screening and experimental verification. The active compounds are 8-hydroxyquinolines listed in table 1, and the binding sites of the active compounds are two bromodomains BD1 and BD2 of BRD 4.

Table 1: 8-hydroxyquinoline compounds

The invention adopts computer virtual screening, and performs virtual screening on a small molecule drug database which is sold on the market through a method of drug molecule design and virtual screening based on a BRD4BD1 protein domain structure (http:// www. Rcsb. Org/pdb/explorer. Unstructured. Id =4 gpj), and performs biological experimental verification on partial compounds selected through scoring ordering and expert experience judgment, and finally 8 active compounds listed in Table 1 are screened.

According to a second aspect of the present invention there is provided the use of a compound of the 8-hydroxyquinoline class or a salt thereof in the manufacture of a medicament for the treatment of a disease associated with BRD4, preferably, the disease associated with BRD4 includes heart failure, NUT-midline cancer, acute lympholeukemia, mixed lineage leukemia, multiple myeloma, burkitt's lymphoma, hepatocellular carcinoma, triple negative breast carcinoma, non-small cell lung carcinoma, prostate carcinoma, pancreatic carcinoma and neuroblastoma.

In another preferred embodiment, said heart failure comprises cardiac hypertrophy, cardiomyocyte hypertrophy.

According to a third aspect of the present invention there is provided a method of inhibiting BRD4 expression and/or activity in vitro, characterised in that acetylated lysine is competitively inhibited from binding to BRD4 by one or more of the compounds listed in table 1.

In another preferred example, the substrate to which BRD4 binds includes histone acetylated lysine, forward transcription elongation factor b (P-TEFb), PDID domain, and the like.

In another preferred embodiment, the compounds used in the method act on domains of BRD4 including BD1 and BD2.

According to the fourth aspect of the present invention, there is provided a method for inducing apoptosis of various tumor cells and inhibiting hypertrophy of cardiac muscle cells, which is characterized in that the relevant tumor cells or cardiac muscle cells are cultured in the presence of one or more of the 8-hydroxyquinoline compounds, so as to induce apoptosis of the relevant tumor cells or reduce hypertrophy of the cardiac muscle cells; or inhibiting BRD4 expression and/or activity; or inhibit transcription of the c-myc gene.

In another preferred example, the typical cell lines of the tumor cells are midline cancer cell strains 797, per403, leukemia cells MV4.11, thp-1, pancreatic cancer cell strains Capan-1, aspc-1, panc-1, multiple myeloma cells KMS11, KMS12, U266, H929, MM.1S, MM.1R, triple negative breast cancer cells MDA-MB-231, BT-549, liver cancer cell strains HepG2, SMMC7721, hepB3, bel-7402 and the like.

According to a fifth aspect of the present invention, there is provided a method for treating diseases such as tumors and heart failure, which comprises administering a safe and effective amount of one or more of the 8-hydroxyquinolines or salts thereof to a subject in need thereof.

Preferably, the tumor comprises NUT-midline carcinoma, multiple myeloma, hepatocellular carcinoma, triple negative breast cancer, and the heart failure comprises cardiac hypertrophy disease and cardiomyocyte hypertrophy disease.

In another preferred embodiment, the subject in need thereof comprises a human or non-human mammal, preferably a human, mouse or rat.

Drawings

FIG. 1 shows the molecular biological mechanism of NUT-midline carcinoma, i.e., the fusion of BRD4 to NUT protein, resulting in the loss of the PID domain and failure to activate transcription.

FIG. 2 shows the IC of amplified chemiluminescence affinity homogeneous assay (ALPHA) technique for determining molecular level inhibition of BRD4 by 8-hydroxyquinoline compounds of the present invention ₅₀ Curve line.

FIG. 3 shows the binding curve of compound Nitroxoline to BD1 protein as confirmed by protein thermomigration.

FIG. 4 shows the inhibition of NUT-midline cancer cell proliferation, the activation of differentiation-related target genes and the induction of apoptosis by 8-hydroxyquinoline compound Nitroxoline.

FIG. 5 shows the inhibition of c-MYC proto-oncoprotein dependent AML leukemia cell proliferation, the inhibition of c-MYC transcription and expression, and the induction of cycle arrest and apoptosis by 8-hydroxyquinoline compound Nitroxoline described in this invention.

FIG. 6 shows the inhibition of c-MYC proto-oncoprotein dependent pancreatic cancer cell proliferation, the inhibition of c-MYC transcription and expression, and the inhibition of pancreatic cancer tumor growth in animal models by 8-hydroxyquinoline compound Nitroxoline, in accordance with the present invention.

FIG. 7 shows the inhibitory effect of 8-hydroxyquinoline compound Nitroxoline on cardiac myocyte hypertrophy-associated genes.

Detailed Description

The inventor of the invention, after extensive and intensive research, discovers for the first time that several 8-hydroxyquinoline (8-OH-quinoline) drugs for other diseases have good treatment prospects on certain tumors and heart failure by inhibiting the activity of BRD4 protein through a mode of combining virtual screening and experimental verification. Experiments prove that the 8-hydroxyquinoline compound can inhibit BRD4, reduce the transcription of c-myc gene or hypertrophic gene, has the effects of inducing apoptosis and/or inhibiting the proliferation of related tumor cells, and has the effect of preventing hypertrophy on cardiac muscle cells, thereby having the effect of treating the diseases. In addition, cytotoxicity experiments have also demonstrated that 8-hydroxyquinoline drugs at therapeutic doses, known to be marketed as drugs, are also not significantly toxic when used to treat associated tumors and heart failure. On the basis of this, the present invention has been completed.

As used herein, the terms "8-hydroxyquinoline", "active ingredient of the present invention", "such medicament", "compound listed in table 1" all refer to compounds having the chemical structure as listed in table 1.

The approved uses of the 8-hydroxyquinoline are shown in Table 2.

Table 2: approved use of 8-hydroxyquinoline drugs

The present inventors have found that the above 8-hydroxyquinoline drugs have an inhibitory effect on BRD4 activity in addition to the above approved therapeutic effects. In a molecular level experiment, the 8-hydroxyquinoline compound can compete with acetylated lysine to bind to a bromodomain of BRD4 protein. Meanwhile, in-vitro cell level experiments prove that the medicine has an inhibiting effect on various BRD4 related tumor cells and myocardial mast cells. In vivo animal experiments show that the medicine can induce the reduction or disappearance of the volume of the related tumor or improve the symptoms of cardiac hypertrophy.

In addition, as no medicine on the market exists for the BRD4 inhibitor at present, and the BRD4 inhibitor entering clinical trials has a complex and single structure type, the 8-hydroxyquinoline parent nucleus provided by the invention has a simple and novel structure and possibly has better curative effect and safety.

Of course, as the marketed drugs, the 8-hydroxyquinoline compounds have very clear toxicological properties, and the safety is proved, which brings great convenience for the drugs as drugs for treating BRD 4-related diseases.

The 8-hydroxyquinoline compound has the following advantages:

1. has definite effect, and is suitable for BRD4 related diseases including NUT-midline carcinoma, multiple myeloma, hepatocellular carcinoma, triple negative breast cancer and heart failure: in vitro and in vivo experiments show that 8-hydroxyquinoline medicaments can competitively inhibit the activity of BRD4, induce apoptosis and/or inhibit proliferation of related tumor cells and reduce or eliminate the related tumors; or inhibiting cardiac muscle cell hypertrophy, and improving heart failure symptoms.

2. The medicine source is reliable and stable, and the safety is high: as known marketed drugs, the toxicology of the 8-hydroxyquinoline compounds has been widely proven, and cytotoxicity experiments also confirm that the drugs are not obviously toxic when used for treating related tumors and heart failure under the treatment dosage.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental methods, software parameters, which do not indicate specific conditions in the following examples, are generally in accordance with conventional conditions, or in accordance with conditions recommended by the manufacturer, default parameters.

Example 1 virtual screening procedure

(1) And (4) preparing a protein model. The crystal structure of the BRD4BD1 and inhibitor complex was downloaded from the Protein Data Bank, and the nonaqueous solvent molecules in the crystal structure were deleted using Pymol software. Then, a series of Protein Preparation tasks including hydrogenation, repairing of missing residues, energy optimization and the like are completed by a Protein Preparation module in Schrodinger software. After the protein is prepared, non-conserved water molecules are removed. And finally, selecting the inhibitor in the complex as a reference ligand (reference ligand), and generating a docking pocket lattice point file by using a Receptor lattice point Generation program (Receptor Grid Generation) in the Glide 5.6 software.

(2) Ligand preparation. Structures of about 1600 compounds were obtained from a marketed small molecule drug database, and then three-dimensional structures, tautomers, etc. of each small molecule were generated using the "ligand preparation" module in schrodinger software.

(3) And (4) carrying out molecule docking. And (4) carrying out butt joint scoring on the prepared small molecule library by using SP precision of Glide software. 10000 conformations before scoring are subjected to de-duplication and structural clustering, and then 200 compounds are visually selected according to expert experience to prepare for testing the biological activity.

(4) And (4) searching for analogues. The first round of biological activity test finds that the 8-hydroxyquinoline compound may have a good inhibition effect on BRD4, so that other marketed drug small molecules containing 8-hydroxyquinoline substructures are inquired from each large drug database, molecular docking and scoring are carried out, and structures with good scoring and reasonable combination modes are selected for purchase and biological activity test.

Table 3: glide scoring of 8-hydroxyquinoline drug docking BRD4BD1

Serial number	Name of drug	Glide scoring
			1	Nitroxoline	-8.222
2	Chloroxine	-7.832
			3	Broxyquinoline	-7.158
4	Cloxyquin	-7.667
			5	Clioquinol	-7.133
6	Procaterol	-6.846
			7	Indacaterol	-6.582
8	Iodoquinol	-6.373
			9	8-hydroxyquinoline	-6.512

Example 2

Screening and experimental verification of compounds targeting BRD4 molecular level.

By constructing a prokaryotic expression system, the BRD4 protein containing the BD1 structural domain is successfully expressed and purified, 5-position, 8-position, 12-position and 16-position acetylation modified H4 polypeptide is used as a substrate, and homogeneous detection is carried out by means of amplified chemiluminescence affinity (A)ALPHA), successfully establishes a screening platform for targeting BRD4BD1 domain. ALPHA can detect the maximum signal value when no compound affects the binding of BRD4_ BD1 to the substrate polypeptide, and the signal value will be weaker when the small molecule inhibits the binding of BRD4_ BD1 to the substrate polypeptide. Therefore, when the concentration of the compound is gradually reduced from high to low, the signal value is gradually increased from low to high, and an S-shaped curve is presented, so that the IC is calculated by fitting ₅₀ The value is obtained.

Meanwhile, the binding of the compound Nitroxoline and the BD1 protein is verified by adopting a biochemical experiment of protein heat migration. Protein thermomigration experiments are a temperature-based biochemical method for detecting protein unfolding by binding of fluorescent dyes to hydrophobic amino acids. With the continuous rise of the temperature, the hydrophobic amino acid of the protein is gradually exposed, and the fluorescence signal combined with the hydrophobic amino acid becomes stronger. The dissolution temperature can thus be fitted by means of the boltzmann equation. When a small molecule is added to a protein, if the small molecule can be combined with the protein to increase the stability of the protein, the unfolding process of the protein is slowed down along with the increase of the temperature, and the half melting temperature is increased. In the experiment we observed that BRD4_ BD1 exhibited a concentration-dependent increase in half melting temperature with increasing compound concentration, indicating that the compound bound to and stabilized BRD4_ BD1, as shown in figure 3.

Table 4: molecular level inhibitory activity of 8-hydroxyquinoline drugs on BRD4

Serial number	Name (R)	CAS	IC ₅₀ (μM)
				1	Nitroxoline	4008-48-4	0.27
2	Chloroxine	773-76-2	12.2
				3	Broxyquinoline	521-74-4	2.74
4	Cloxyquin	130-16-5	1.62
				5	Clioquinol	130-26-7	10.0
6	Procaterol	72332-33-3	119
				7	Indacaterol	312753-06-3	71.9
8	Iodoquinol	83-73-8	4.93
				9	8-hydroxyquinoline	148-24-3	2.81

IC of the 8-hydroxyquinoline compound for inhibiting BRD4 at molecular level ₅₀ See fig. 2 for a graph.

Example 3

The Nitroxoline has the effects of inhibiting NUT-midline cancer cell proliferation, activating differentiation related target genes and inducing apoptosis.

First, two NUT-midline carcinoma cell lines 797 and per433 were used as cell models to examine the IC of the compound Nitroxoline in inhibiting the proliferation of the two cells ₅₀ . Adding 10% fetal calf serum into DMEM/F12 culture medium, adding double antibody for culture, and performing trypsinization passage. After about 70% of cells are fused, the cells are digested by trypsin, made into cell suspension, and then counted under a microscope, and then inoculated into a 96-well plate, 6 plates per well, with each cell. After cell attachment, nitroxoline treatment was given and the concentration gradient was 100 μ M as the starting concentration, diluted in a two-fold gradient. Changes in cell proliferation were detected by the Alrma blue method 72 hours after administration. The cell viability was plotted on the ordinate versus the drug concentration on the ordinate, and the IC of proliferation inhibition of two cell lines by Nitroxoline was calculated ₅₀ The specific inhibition rate is shown in FIG. 4A. The cell survival rate (%) was calculated as: survival (%) = (administration well OD-blank well OD)/(control well OD-blank well OD) × 100%.

Next, the effect of compound Nitroxoline on transcription of genes targeted to genes associated with differentiation of linear carcinomas was examined. 797 the cells are digested, plated on a 6-well plate, dosed after adherence (including a control group and a compound group), treated with the drug for 48 hours, rinsed with PBS, sampled with trizol, and subjected to RNA extraction and reverse transcription. And (3) taking the cDNA obtained by reverse transcription as a template, and carrying out realtimePCR on the cDNA sample obtained by reverse transcription by using primers of the internal reference b2MG gene, the differentiation related genes KRT14 and KRT10 to obtain an amplified Ct value.

The gene expression abundance of the control DMSO group was normalized to 1 using the Δ Δ Ct method:

ΔΔCt＝ΔCt(GENE-b2MG) _DMSO -ΔCt(GENE-b2MG) _{DMSO group} ＝0。

Abundance of gene expression =1/2 in control group ^ΔΔCt ＝1。

Similarly, the Δ Δ Ct method was used to calculate the expression of genes in the experimental group versus the control DMSO group:

ΔΔCt＝ΔCt(GENE-b2MG) _DMSO -ΔCt(GENE-b2MG) _{experimental group} . +1 represents up-regulation by a factor of two, -1 represents down-regulation to 1/2.

Relative expression of GENE＝＝1/2 ^-ΔΔCt

The results showed that nitroxoline significantly promoted the expression of genes associated with the differentiation of midline cancer cells as shown in fig. 4 b.

Again, the effect of the compound Nitroxoline on apoptosis of mid-line carcinomas was examined. 797 cells were plated on 6-well plates after digestion, dosed after attachment (including control and compound), and drug treated for 72 hours. The cells in the supernatant are combined with the adherent cells digested by pancreatin, and collected by centrifugation. The collected cells were washed with cold PBS and collected by centrifugation. The supernatant was discarded, 1 × binding buffer was added, and the cell concentration was adjusted. Annexin V and PI, after incubation and staining for 15 minutes at room temperature in the dark, the samples were diluted with binding buffer, stored on ice, and the proportion of normal cells, early apoptotic cells and late apoptotic cells in the samples was determined and calculated by flow cytometry, and the results are shown in fig. 4C. Experimental results show that the compound Nitroxoline can effectively induce the apoptosis of midline cancer cells.

Example 4

The inhibition of c-MYC proto-oncoprotein dependent AML leukemia cell proliferation, the inhibition of c-MYC transcription and expression, and the induction of cycle arrest and apoptosis by Nitroxoline.

First, two AML leukemia cell strains MV4.11 and THP1 were used as cell models, and the IC of the compound Nitroxoline for inhibiting the proliferation of two cells was examined ₅₀ . By 1640 cultureThe nutrient medium is added with 10% fetal calf serum. After cell counting, 10 cells per well ⁴ One/100 ul was inoculated in 96 well plates with Nitroxoline treatment, with a concentration gradient of 100 μ M as the starting concentration, and a two-fold gradient dilution. Changes in cell proliferation were detected by the alrma blue method 72 hours after administration. The cell viability was plotted on the ordinate versus the drug concentration on the ordinate, and the IC of proliferation inhibition of two cell lines by Nitroxoline was calculated ₅₀ . The cell survival rate (%) calculation method was: survival (%) = (administration well OD-blank well OD)/(control well OD-blank well OD) × 100. The results show that the compound Nitroxoline can effectively inhibit the proliferation of leukemia cells, and has specific inhibition rate and IC ₅₀ See figure 5A for values.

Secondly, it was examined whether the inhibitory effect of the compound Nitroxoline on cell proliferation was caused by affecting the cell cycle and apoptosis of leukemia. MV4.11 cells were plated at a density of 2.5X 105/ml in six well plates, 2ml per well, and the plate was dosed the day. The effect of the compounds on cell cycle and apoptosis was examined at 48 and 72 hours, respectively. In cell cycle detection, cells were collected by centrifugation, washed with PBS, and fixed overnight with-20 ℃ pre-chilled ethanol. Fixed cells are collected by centrifugation, washed by PBS and stained by PI, the stained cells are detected by a flow cytometer, and the proportion of cells in different cell cycle images (G1, G2/M, S phase) is analyzed. In the detection of apoptosis, cells are collected by centrifugation, washed by PBS, stained with Annexin V and PI at room temperature in a dark place, and the proportion of normal cells, early apoptosis and late apoptosis cells in a sample is determined and calculated by a flow cytometer. The experimental results show that the compound Nitroxoline promotes the G1 phase arrest (FIG. 5B) and effectively induces apoptosis (FIG. 5C) of MV4.11 leukemia cells, and has a concentration-dependent gradient for the cycle arrest and apoptosis induction.

Finally, the effect of the compounds on the transcription and expression of the c-myc proto-oncogene was examined. MV4.11 cells were seeded at a density of 1X 106/ml in six well plates, 2ml per well. And after the drug treatment is carried out for 6 hours and 24 hours, the influence of the compound on the transcription of the c-myc gene and the protein expression quantity of the c-myc gene is respectively detected. The results show that treatment with the compound Nitroxoline inhibited BRD4 from regulating transcription (FIG. 5E) and protein expression (FIG. 5D) of the target gene, c-myc.

Example 5

The inhibition effect of Nitroxoline on c-MYC proto-oncoprotein dependent pancreatic cancer cell proliferation, c-MYC transcription and expression, and the inhibition of pancreatic cancer tumor growth on an animal model.

Firstly, three pancreatic cancer cell strains, aspc-1, capan-1 and Panc-1, were taken as cell models, and the IC of the compound Nitroxoline for inhibiting pancreatic cancer cell proliferation was examined ₅₀ . Culturing in DMEM medium (containing 10% fetal calf serum), and performing trypsinization passage. Cells were trypsinized and plated at 3000-6000 cells/well on 96-well 3D plates (low adsorption plates, non-adherent cells) and were treated with Nitroxoline. The Alrma blue method measures changes in cell proliferation 72 hours after administration. IC for cell proliferation inhibition by Nitroxoline ₅₀ The calculation method is consistent with the previously described method. The results show that the compound Nitroxoline effectively inhibits pancreatic cancer cell proliferation (FIG. 6A)

Next, the effect of the compounds on the transcription and expression of the c-myc proto-oncogene was examined. Capan-1 cells were seeded into six-well plates, 2ml per well. After the drug treatment is carried out for 24 hours and 48 hours, the influence of the compound on the transcription of the c-myc gene and the protein expression quantity of the c-myc gene is respectively detected. The results show that treatment with the compound Nitroxoline inhibited transcription of the BRD4 target gene C-myc (fig. 6B) and protein expression (fig. 6C) in pancreatic cancer cells.

Finally, the inhibition of pancreatic cancer tumor body growth by the compounds was examined on a capan-1 subcutaneous transplantation tumor model. Capan-1 cells at 2X 10 ⁶ Cells/200 ul were inoculated to the axilla of nude mice of 4 weeks of age, tumors were formed after one week, and divided evenly into groups (8/group) in tumor size, and solvent, nitroxoline (50 mg/kg) and Gemcitabine (Gemcitabine) (50 mg/kg), a first-line drug for pancreatic cancer, were administered separately every other day, and tumor size and mouse body weight were measured. After 3 weeks of continuous administration, mice were sacrificed, tumor bodies were weighed, and color change of organs of the mice was observed. Animal experiment results show that the compound Nitroxoline can effectively inhibit tumor body growth on a capan-1 pancreatic cancer animal model (figure 6D), the inhibition effect of the compound Nitroxoline is similar to the effect of a first-line clinical pancreatic cancer medicament, and the compound has no obvious effect on the weight (figure 6E) and the color and texture of each organ of a mouseHas obvious influence and indicates that the traditional Chinese medicine has small toxic and side effects.

Example 6

The proliferation inhibition effect of the compound Nitroxoline on c-myc dependent other tumor cells (such as multiple myeloma cells, liver cancer cells and triple negative breast cancer cells).

TABLE 5 proliferation inhibition of c-myc dependent other tumor cells by Nitroxline

Example 7

Inhibition of cardiac myocyte hypertrophy-associated genes by compounds

First, a cell model of myocardial hypertrophy was constructed. The primary isolated and cultured neonatal rat myocardial cells neonatal ventrial myocytes (NRVMs) generate myocardial hypertrophy lesion under the induction of Phenylephrine (PE), and are characterized in that the expression of myocardial hypertrophy related protein markers is changed: such as marked up-regulation of markers ANF (intrinsic natural factor), BNP (brain natural peptide) and marked down-regulation of SERCA2a (sarcoendoplastic reticulum ATPase). On this model of induction of myocardial hypertrophy lesions, we examined the regulatory effect of the compound, niroxolin, on the expression of markers associated with myocardial hypertrophy lesions. The results are shown in fig. 7, the pathological markers ANF and BNP are obviously up-regulated under the induction of PE, and the compound Nitroxoline can effectively inhibit the expression of the compound Nitroxoline; PE induces the up-regulation of the marker SERCA2a, whereas the compound significantly inhibits its up-regulation. Therefore, in a myocardial hypertrophy induction model, the compound Niroxoline remarkably reverses the expression regulation of PE on myocardial hypertrophy lesion markers, so that the myocardial hypertrophy can be effectively improved.

Claims

1. Use of an 8-hydroxyquinoline compound or a salt thereof in the manufacture of a medicament for the treatment of a BRD 4-related disease, the 8-hydroxyquinoline compound having the structure:

；

the BRD 4-associated disease is heart failure.

2. The use according to claim 1, said heart failure being selected from cardiac hypertrophy diseases and cardiomyocyte hypertrophy diseases.