CN108864037B

CN108864037B - TRPML1 specific agonist, application of TRPML1 specific agonist as autophagy inhibitor and preparation of tumor treatment drug, and pharmaceutical composition

Info

Publication number: CN108864037B
Application number: CN201810528644.6A
Authority: CN
Inventors: 王午阳
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2018-05-28
Filing date: 2018-05-28
Publication date: 2020-09-29
Anticipated expiration: 2038-05-28
Also published as: CN108864037A

Abstract

The application provides a specific small molecule agonist MK6-83 of a TRPML1 ion channel, and provides application of MK6-83 as an autophagy inhibitor and application of the function in preparation of a tumor treatment drug. The TRPML1 specific small molecule agonist MK6-83 provided by the application has strong lethal capability on various tumor cell lines including pancreatic cancer, breast cancer and gastric cancer under the action of no obvious toxicity on various normal tissue cells by inhibiting autophagy activity. In addition, MK6-83 has obvious effect of inhibiting the growth of breast cancer mass transplanted on mice, and obviously prolongs the life of tumor-forming mice. Solves the technical problem that the anti-cancer drugs and the cancer treatment mode kill tumor cells and cells of normal tissues simultaneously in the prior art.

Description

TRPML1 specific agonist, application of TRPML1 specific agonist as autophagy inhibitor and preparation of tumor treatment drug, and pharmaceutical composition

Technical Field

The application relates to the field of biomedicine, in particular to a TRPML1 specific agonist, and also relates to application of the specific agonist and a tumor treatment drug.

Background

With the increase in human life, cancer has leaped into the leading cause of death in recent years. The 'national latest cancer report in 2018' issued by the national cancer center shows that 380.4 ten thousands of new cases of all malignant tumors in 2014 have more than 1 million confirmed cancers on average each day and 7 confirmed cancers per minute. The first malignant tumor in the country is lung cancer, and the second is gastric cancer, colorectal cancer, liver cancer and breast cancer. Cancer has become the leading cause of death in our country.

Scientists have conducted a great deal of research regarding the treatment of cancer. New anticancer drugs are continuously discovered. At present, the cure rate of more than 20 kinds of tumors reaches more than 30 percent. The research on the subcellular level and the molecular level of the action mechanism of the medicine greatly develops the research on the application aspect of the anti-cancer medicine. The rapid development of the studies in the aspects of cell kinetics, pharmacokinetics and immunology also makes the screening of the drugs, the adjustment of the dosage and the determination of the administration route perfect. The existing combination medicine, large-dose intermittent medicine, auxiliary chemotherapy and the treatment of traditional Chinese medicines are matched, so that the malignant tumor treatment has good curative effect. At present, the treatment of malignant tumor is mainly performed by means of operation, radiotherapy, chemical medicine treatment, Chinese medicine treatment, immunotherapy and the like. The anti-cancer drug has the effects of choice, toxicity and drug resistance, because the anti-cancer drug kills tumor cells and cells of normal tissues, especially bone marrow hematopoietic cells and gastrointestinal tract cells with vigorous proliferation, thus limiting the dose of the anti-cancer drug, reducing the immune function of patients, even making the patients have difficulty in enduring gastrointestinal reactions, and being forced to interrupt the treatment, thus failing the treatment. Anticancer drugs can kill cancer cells, but due to their cytotoxicity, it has been a struggle target of scientists to find a drug which can treat cancer and does not cause harm or harm less to human body.

TRPML1 is a cation releasing channel on the lysosomal membrane. Recent studies have found that TRPML1 functions as a lysosome by participating in various cellular activities and signaling including endocytosis, exocytosis, and cell membrane repair. ML-SA1, ML-SA3, etc. are specific small molecule agonists of the TRPML1 channel. The agonist belonging to the small molecular compound can permeate cell membranes to enter cells, specifically activate a TRPML1 channel on a lysosome, and release cations such as calcium ions, iron ions, zinc ions and the like in the lysosome to enter cytoplasm after the TRPML1 channel is opened. Experiments show that the TRPML1 channel can be specifically activated by using certain small molecular agonists to inhibit the autophagy activity. In order to provide over-increased energy demand for tumor cells, autophagy signals are activated so as to continuously degrade self proteins to meet the malignant proliferation demand of the tumor cells, so that the basic autophagy level of many kinds of tumor cells is remarkably higher than that of normal cells.

Through a large number of screens, the specific small molecule agonist of the TRPML1 ion channel can be used as an autophagy inhibitor, particularly has an obvious inhibiting effect on autophagy activity of tumor cells, and is a potential anti-tumor drug.

Disclosure of Invention

In order to solve the problems, the invention obtains an agonist capable of inhibiting autophagy activity of cells by repeatedly screening multiple specific small molecule agonists of TRPML1 ion channels numerous times, and verifies that the agonist has the function of limiting the growth of tumor cells by inhibiting the autophagy activity of the tumor cells.

To achieve the above object, according to a first aspect of the present application, there is provided a specific agonist MK6-83 of TRPML1 ion channel.

MK6-83 is a small molecule compound, and the chemical structural formula is shown as follows:

in order to achieve the above object, according to a second aspect of the present application, there is provided a use of MK6-83 as an autophagy inhibitor or a use thereof in the preparation of an autophagy inhibitor.

In order to achieve the above object, according to a third aspect of the present application, there is provided a use of MK6-83 in the preparation of a medicament for the treatment of tumors.

Further, the tumors include pancreatic cancer, breast cancer and gastric cancer.

In order to achieve the above object, according to a fourth aspect of the present application, there is provided a pharmaceutical composition for treating a tumor. The active ingredient of the pharmaceutical composition is MK 6-83.

Furthermore, the composition can be prepared into oral liquid, granules, tablets, hard capsules, soft capsules, dripping pills, injections, nano preparations, targeting preparations and other medically acceptable formulations.

The TRPML1 specific small molecule agonist MK6-83 provided by the application has strong lethal capability on various tumor cell lines including pancreatic cancer, breast cancer and gastric cancer under the action of no obvious toxicity on various normal tissue cells by inhibiting autophagy activity. In addition, MK6-83 has obvious effect of inhibiting the growth of breast cancer mass transplanted on mice, and obviously prolongs the life of tumor-forming mice. Solves the technical problem that the anti-cancer drugs and the cancer treatment mode kill tumor cells and cells of normal tissues simultaneously in the prior art.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:

FIG. 1 shows the results of the transformation of two forms of the autophagy marker protein LC3 in Hela cells 1 hour after the addition of MK6-83, Baf-A1 and both, detected by Western blotting, wherein the relevant contents in FIG. 1 are translated or explained as follows: LC3 represents Tubulin-related protein 1 light chain 3, Normalized LC3II represents Normalized LC3II value, Tubulin represents Tubulin, CTL represents blank control group, starvation represents starvation treatment group, Baf-A1 represents bafilomycin A1;

FIG. 2 shows the results of transformation of two forms of the autophagy marker protein LC3 in Hela cells 12 hours after the administration of MK6-83, Baf-A1 and both, detected by Western blotting, wherein the relevant elements in FIG. 2 are translated or explained as follows: LC3 represents Tubulin-related protein 1 light chain 3, Normalized LC3II represents Normalized LC3II values, Tubulin represents Tubulin, CTL represents blank control group, Baf-A1 represents bafilomycin A1;

FIG. 3 shows the results of transformation of two forms of the autophagy marker protein LC3 and the results of the level of P62 protein 12 hours after the administration of the CQ with MK6-83, CQ, Rap and MK6-83 in Hela cells detected by Western blotting, wherein the following are translated or explained in FIG. 3: LC3 represents microtubule-associated protein 1 light chain 3, Normalized LC3II represents Normalized LC3II values, Tubulin represents Tubulin, GAPDH represents GAPDH internal reference protein, P62 represents autophagy-selective substrate P62 protein, P62/GAPDH represents Normalized values of GAPDH expression levels, CTL represents blank control, starvation represents starvation treatment, CQ represents chloroquine, Rap represents rapamycin;

FIG. 4 shows the results of MK6-83(5 μ M) treated Trypan blue staining of Patu8988t cells, where relevant information in FIG. 4 is translated or interpreted as follows: control represents control group;

FIG. 5 shows the differences after trypan blue experiments in the pancreatic ductal epithelial cell line HPDE6c7 and the pancreatic cancer cell line Patu8988t activity control and treatment with different concentrations of MK6-83, wherein the relevant contents in FIG. 5 are translated or explained as follows: CTL on the abscissa represents blank control group, Cell Viability on the ordinate represents Cell activity;

FIG. 6 shows the difference in cell activity of the breast cancer cell line MCF-7 under treatment with different concentrations of MK6-83, where relevant information in FIG. 6 is translated or interpreted as follows: CTL on the abscissa represents blank control, while cellviatility on the ordinate represents cell activity;

FIG. 7 shows the difference in cellular activity of the normal mammary duct epithelial cell line MCF 10A under treatment with different concentrations of MK6-83, where relevant elements in FIG. 7 are translated or explained as follows: CTL on the abscissa represents blank control group, Cell Viability on the ordinate represents Cell activity;

FIG. 8 shows the difference in cell activity of the gastric cancer cell line SGC-7901 under treatment with different concentrations of MK6-83, where relevant information in FIG. 8 is translated or explained as follows: CTL on the abscissa represents blank control, while cellviatility on the ordinate represents cell activity;

FIG. 9 shows the difference in cellular activity of the normal gastric mucosal epithelial cell line GES-1 under treatment with different concentrations of MK6-83, where relevant elements in FIG. 9 are translated or explained as follows: CTL on the abscissa represents blank control group, Cell Viability on the ordinate represents Cell activity;

FIG. 10 shows the survival curves of mice in PBS control group and MK6-83(5 μ M) injection group, wherein the relevant contents in FIG. 10 are translated or explained as follows: the abscissa Days after injection represents the number of Days after injection, and the ordinate survival represents the survival rate;

FIG. 11 shows tumor volumes of PBS control group and MK6-83(5 μ M) injected group, where relevant information in FIG. 11 is translated or interpreted as follows: in the abscissa, PBS represents phosphate buffered saline and in the ordinate, Relative Tumor Volume (fold).

Detailed Description

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the term "comprises" and any variations thereof in the description and claims of this application and the above-described drawings are intended to cover non-exclusive inclusions, such as, for example, integers comprising a list of elements which are not necessarily expressly listed but may include other elements not expressly listed or inherent to such integers.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

It should be noted that the specific operations not specifically recited in the examples of the present application are applicable to the routine operations or routine experimentation in the art, and it should be understood that those skilled in the art can reasonably recognize the operations according to the prior art.

Unless otherwise defined, all terms used herein should be interpreted as being open-ended or closed-ended based on the general knowledge in the art, and should be interpreted as being open-ended or closed-ended based on the general knowledge in the art.

Autophagy: autophagy is a process of phagocytosing self cytoplasmic proteins or organelles and encapsulating them into vesicles, fusing with lysosomes to form autophagosomes, degrading the encapsulated contents, thereby fulfilling the metabolic needs of the cell itself and the renewal of certain organelles.

Western-blot detection method: western blotting is an experimental method frequently used in molecular biology, biochemistry and immunogenetics, and is an analytical method capable of performing qualitative and semi-quantitative analysis on proteins, which is to stain a cell or biological tissue sample treated by gel electrophoresis with a specific antibody, and obtain information on the expression of a specific protein in the analyzed cell or tissue by analyzing the location and depth of staining.

An EP pipe: a miniature centrifugal tube is a small centrifugal tube, is matched with a miniature centrifugal machine for use, and is used for separating a micro reagent.

ddH 2O: the abbreviation of double distilled water is water obtained by redistilling water after primary distillation.

BCA reaction working solution: BCA is an abbreviation for bicinchoninic acid protein, and BCA is a widely used protein quantification method in which the BCA reaction working solution used is a mixture of a reagent A (BCA alkaline solution) and a reagent B (copper sulfate solution) at a ratio of 50: 1.

loadingbuffer: the Western-blot assay uses loading buffer to indicate the progress of electrophoresis and to allow the sample to sink into the wells without floating.

Acr-Bis: acrylamide-methylene bisacrylamide solution.

Tris-HCl buffer: tris-hydroxymethyl aminomethane-hydrochloric acid buffer.

SDS buffer: sodium dodecyl sulfate buffer.

TEMED: tetramethyl ethylenediamine, used for preparing SDS-PAGE glue.

Marker: protein labeling, prestained or non-prestained proteins of various molecular weights, used for labeling the size and tracing of the protein in electrophoresis.

PVDF film: polyvinylidene fluoride (pvdf) is a solid support commonly used in western blotting.

TBST: contains Tris-HCl, NaCl and Tween20, and is one kind of buffering liquid commonly used in Western blotting.

HeLa cells: heila cells are artificially cultured cells with unlimited proliferation capacity, and are widely used in tumor research, biological experiments or cell culture in the medical field.

Trypan blue assay: the method is a rapid, simple and convenient method for detecting the cell survival rate by using a method that trypan blue can only stain dead cells into blue and living cells can not be stained.

The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

Example 1: detection of MK6-83 inhibiting autophagy activity

1. The experimental groups are described below

Blank control group: normal medium + 10% fetal bovine serum

Starvation treatment group: normal medium (without fetal bovine serum)

Test group 1: bafilomycin A1 treatment

Test group 2: 1 μ M MK6-83 treatment

Test group 3: 3 μ M MK6-83 treatment

Test group 4: 5 μ M MK6-83 treatment

Test group 5: treatment with 1 μ M MK6-83 in combination with Bafilomycin A1

Test group 6: treatment with 5 μ M MK6-83 in combination with Bafilomycin A1

Test group 7: 10 μ M CQ treatment

Test group 8: treatment with 5 μ M MK6-83 in combination with CQ

Test group 9: 50 μ M Rap treatment

2. Experimental methods

And (2) carrying out subsequent treatment and detection on each group of cells by adopting a Western-blot detection method (a protein immunoblotting method), wherein the specific method comprises the following steps:

A. protein extraction and concentration determination

(1) Cell: placing six-hole plate for culturing cells on ice, sucking off culture medium, adding 100 μ l cell lysate per hole, standing on ice for 3-5min, scraping off cells with cell scraper, collecting cell lysate in 1.5ml centrifuge tube, shaking on a vortex mixer for 20s, and standing on ice for 30 min.

(3) The standing EP tube was placed in a low-temperature centrifuge and centrifuged at 4 ℃ (15000 rpm. times.18 min).

(4) Gently pipette the supernatant into another well-labeled EP tube to obtain a total protein solution.

(5) And (3) detecting the protein concentration: mu.l of the protein solution was diluted 5-fold by adding 20. mu. lddH2O to each sample, and 10. mu.l was put in a 96-well plate and 2 duplicate wells were made for each sample.

(6) Protein standards A-H were added to the first column of the 96-well plate from high to low concentrations.

(7) Preparing a BCA reaction working solution (A: B ═ 50: 1) under the condition of keeping out of the sun, mixing uniformly, adding 100 mu l of the working solution into each hole, mixing uniformly by gentle shaking, and incubating in an incubator at 37 ℃ for 30min by wrapping the working solution with tinfoil in the condition of keeping out of the sun.

(8) And (3) detecting the Optical Density (OD) value of each hole and the concentration of the protein sample under the wavelength of 562nm by using a microplate reader.

(9) Packaging unused protein, and storing at-80 deg.C.

B. Denaturation of proteins

And calculating the volume of the added protein liquid according to the required protein amount and the experimental sample loading times. Taking the example of electrophoresis performed with 30 μ g total protein per well requiring 4 loading times to detect different assay indices, the volume of protein solution required (μ l) was calculated (30 μ g protein/protein concentration) × 5, then ddH2O was supplemented to 15 μ l × 5 to 75 μ l, 3 μ l × 5 to 15 μ l of 6 × loadingbuffer (6 × loadingbuffer: β -mercaptoethanol ═ 9:1) was added, the mouth was sealed, boiled in boiling water for 5min, and stored at 4 ℃.

C. SDS-PAGE gel electrophoresis

(1) Preparation of 10% polyacrylamide separation gel (2 gel portions): taking a special bottle for preparing the glue, adding the following reagents respectively, and carefully and uniformly mixing: ddH2O4 ml; 30% Acr-Bis3.3ml; 1.5M Tris-HCl buffer (pH8.8)2.5 ml; 0.1ml of 10% SDS buffer; 0.1ml of 10% Ammonium Persulfate (AP) and 0.004ml of TEMED0.004ml are mixed evenly, 1ml of separating glue is sucked by a gun and injected into a glass plate interlayer of an electrophoresis tank, about 2/3 small glass plate height is added without air bubbles, about 1ml of isopropanol is slowly added to seal a pressing line, and polymerization is carried out at room temperature for about 30 min.

(2) Preparation of 5% polyacrylamide concentrated gum (2 gum portions): taking a special bottle for preparing the glue, adding the following reagents respectively, and carefully and uniformly mixing: ddH2O2.7ml; 30% Acr-Bis0.67ml; 1.0M Tris-HCl buffer (pH6.8)0.5 ml; 0.04ml of 10% SDS buffer; 0.04ml of 10% ammonium persulfate; TEMED0.004ml and mix well.

(3) Pouring off the isopropanol on the top of the gel, sucking off the residual liquid with filter paper, injecting the concentrated gel on the top of the interlayer, inserting into a comb, and polymerizing at room temperature for 30 min.

(4) After the gel is solidified, the gel plate is fixed in an upper buffer chamber of the electrophoresis apparatus, the electrophoresis solution is added, the comb is pulled out, 18 mul of sample solution is sucked by a microsyringe and added into a sample adding hole, Marker is added into the first sample hole, 1 × loadingbuffer is added into the last sample hole, and the sample is placed into the electrophoresis apparatus poured with 1 × Tris-glycine electrophoresis buffer solution.

(5) And (3) switching on a power supply, wherein the voltage of the concentrated gel is 90V, the electrophoresis is carried out for about 30min, the voltage of the separation gel is 120V, and the voltage and the time for separating the gel are adjusted according to the molecular weight of the target protein in principle after the electrophoresis is carried out until the bromophenol blue reaches the bottom of the separation gel.

(6) And (3) turning off the power supply, taking down the gel, and cutting the gel containing the target protein according to a Marker for further film conversion.

D. Rotary film (Whole wet method)

(1) And (3) shearing a PVDF membrane with a proper size according to the size of the sheared target glue, marking a shearing angle on the PVDF membrane, soaking in methanol for 1-3min, and transferring into a1 Xmembrane transfer buffer solution.

(2) And (3) installing a membrane rotating device according to the sequence of negative electrode side-porous filter cotton-thick filter paper-glue-PVDF membrane-thick filter paper-porous filter cotton-positive electrode side, then placing the membrane rotating device into a Bio-RAD (Bio-Rad electrophoresis) electrophoresis apparatus, pouring 1 multiplied by membrane rotating liquid, carrying out ice-bath membrane rotating, and determining the membrane rotating time according to the molecular weight of target protein, wherein the membrane rotating time is generally determined by using a constant current of 350mA, and the membrane rotating time is transferred for 2 hours or 400mA and the membrane rotating time is transferred for 1.5 hours.

(3) After the film is turned, a corner of the PVDF film is cut off to mark the front and back sides of the film, the Marker is arranged on the left side, the increasing sequence is from bottom to top, the corner is arranged on the upper left, and the PVDF film is rinsed for 3 times by 10min by using 1 XTSST solution.

E. Immune response

(1) And (3) putting the rinsed transfer printing film into 5% skimmed milk powder sealing liquid, sealing for 2h at room temperature, and slowly shaking on a shaking table.

(2) The primary antibody was diluted to the appropriate concentration with 5% nonfat dry milk blocking solution, covered with the appropriate amount of antibody on the membrane, and incubated overnight at 4 ℃.

(3) Wash 3 times with 1 × TBST × 15min, shake on a shaker.

(4) Diluting the secondary antibody to a proper concentration by using 5% skimmed milk powder sealing solution, covering a proper amount of antibody on a membrane, and incubating for 1-2h at room temperature in a wet box.

(5) Wash 3 times with 1 × TBST × 15min, shake on a shaker.

(6) The two reagents A and B in ECL were mixed in equal volume, added to the membrane uniformly (protected from light), and photographed with a chemiluminescence gel imaging system.

3. Results of the experiment

The test results are shown in FIGS. 1 to 3.

In the experiment, two forms of transformation of autophagy marker protein LC3 before and after adding drugs of MK6-83(1 mu M, 3 mu M and 5 mu M) in Hela cells are detected by a protein immunoblotting method. When the level of conversion of LC3I to LC3II increased, it was suggested that the level of autophagy was regulated. As shown in FIGS. 1 and 2, we see a significant increase in LC3II transformation from one hour after MK6-83 dosing, revealing that MK6-83 has a rapid regulatory effect on autophagy. Furthermore, when MK6-83 was administered in combination with Bafilomycin A1 (Baf-A1; a lysosomal inhibitor), there was no further increase in LC3II transformation, suggesting that MK6-83 inhibits autophagy activity.

As shown in FIG. 3, the use of another lysosomal inhibitor, Chloroquine (CQ), in combination with MK6-83 (test group 8), was found to have no further increase in the transformation effect of LC3II, revealing the inhibitory effect of MK6-83 on autophagy. In addition, rapamycin (rapamycin) treated group (test group 9) and starvation treated group were found to have the effect of reducing the level of P62 protein, and in contrast, MK6-83 treated group increased the level of P62 protein, further verifying that MK6-83 inhibited autophagy activity.

4. Conclusion

The TRPML1 specific small molecule agonist MK6-83 can inhibit the progress of the hela cell autophagy activity and can be used as an autophagy inhibitor.

Example 2: testing of MK6-83 for lethality in human pancreatic cancer cells Patu8988t

1. Test method

Detecting cells by trypan blue experiment method, specifically, digesting control group (control) cells and drug-added group cells with pancreatin, and mixing with trypan blue reagent with fixed volume. Then, a pipette is used to suck 10. mu.L of the mixed solution, the mixed solution is added into a leukocyte counter, and the leukocyte counter is placed under a microscope for counting. Cells stained with trypan blue were counted as dead cells, whereas viable cells. The final ratio of viable cells to total cells per group was counted as cell viability.

2. Test results

FIG. 4 is a graph showing the results of Trypan blue staining of MK6-83(5 μ M) treated Patu8988t cells (three graphs are a control group image, an image after 24h of drug addition, and an image after 72h of drug addition); FIG. 4 shows that both mortality of Patu8988t cells treated with 5. mu.M MK6-83 was significantly increased compared to the control group, indicating that MK6-83 has a lethal effect on Patu8988 t. Arrows indicate dead cells stained with trypan blue.

FIG. 5 is a graph showing the difference between the cell activity control of the pancreatic ductal epithelial cell line HPDE6c7 and the pancreatic cancer cell line Patu8988t and the treatment with different concentrations of MK6-83 after trypan blue experiment; the histogram shows that in the normal pancreatic ductal epithelial cell line HPDE6c7 cells, the cell activities of the drug-added group and the control group are not significantly different (P > 0.05); in contrast, in pancreatic cancer Patu8988t cells, MK6-83 significantly decreased cell activity and was in a time and dose dependent trend.

3. Conclusion

The TRPML1 specific small molecule agonist MK6-83 has lethal capability to pancreatic cancer cells Patu8988t, but has no killing capability to normal pancreatic duct epithelial cells HPDE6c 7.

Example 3: testing of MK6-83 for killing human breast cancer cells MCF-7

1. Test method

2. Test results

FIG. 6 is a graph showing the results of the cell activities of the breast cancer cell line MCF-7 treated with different concentrations of MK6-83 (three data bars correspond to a blank control group CTL, 1. mu.M MK6-83 drug-added group and 5. mu.M MK6-83 drug-added group, respectively); FIG. 7 is a graph showing the results of the cell activities of a normal mammary duct epithelial cell line MCF 10A treated with different concentrations of MK6-83 (three data bars correspond to a blank control group CTL, a 1. mu.M MK6-83 drug addition group and a 5. mu.M MK6-83 drug addition group, respectively);

FIG. 7 shows that the cell activities of the drug-added group and the control group are not significantly different in the normal mammary duct epithelial cell line MCF 10A cells (P > 0.05); FIG. 6 shows that MK6-83 treatment at 48 hours significantly reduced cell activity in breast cancer MCF-7 cells and was dose dependent.

3. Conclusion

The TRPML1 specific small molecule agonist MK6-83 has lethal ability to the breast cancer cell MCF-7, but has no lethality to the normal mammary duct epithelial cell MCF 10A.

Example 4: testing of MK6-83 ability to kill human gastric adenocarcinoma cells SGC-7901

1. Test method

2. Test results

FIG. 8 is a graph showing the results of the cell activities of the gastric cancer cell line SGC-7901 treated with different concentrations of MK6-83 (three data bars correspond to a blank control group CTL, 1. mu.M MK6-83 drug-added group and 5. mu.M MK6-83 drug-added group, respectively); FIG. 9 is a graph showing the results of the cell activities of the normal gastric mucosal epithelial cell line GES-1 treated with MK6-83 at different concentrations (three data bars correspond to a blank control group CTL, 1. mu.M MK6-83 drug addition group and 5. mu.M MK6-83 drug addition group, respectively);

FIG. 9 shows that in the normal gastric mucosal epithelial cell line GES-1 cells, the cell activities of the drug-added group and the control group are not significantly different (P > 0.05); FIG. 8 shows that 48 hours of MK6-83 treatment significantly reduced cell activity in gastric carcinoma SGC-7901 cells, with a dose dependent trend.

3. Conclusion

The TRPML1 specific small molecule agonist MK6-83 has lethal capability on gastric adenocarcinoma cells SGC-7901, but has no lethality on normal gastric mucosal epithelial cells GES-1.

Example 5: experiment for MK6-83 inhibiting growth of mouse pancreatic cancer tumor and prolonging survival life of mouse

1. Experimental methods

A nude mouse subcutaneous tumor formation experiment:

1. when the Patu8988t cells reach about 80-90% density, the fresh culture medium is replaced the night before the cells are collected.

2. Cells were trypsinized and washed twice with pre-cooled PBS in order to remove serum from the cells.

3. The cell pellet is blown with PBS or serum-free medium to a suitable concentration, and the amount of the cells inoculated to the subcutaneous tumor is (1-5) × 10⁶The inoculation volume of each cell per branch was 0.1ml, so that the concentration of the cell suspension was 1-5 × 10⁷Individual cells/ml.

4. After the cells are digested, the cells should be inoculated under the skin of the nude mice as soon as possible, and the inoculation is generally completed within half an hour as possible, and the cell suspension is placed on ice in the process to reduce the metabolism of the cells.

5. The selected nude mice are 5-8 weeks old, and have a body weight of about 18-20g, and the planting parts are selected from areas rich in blood supply, such as the middle and rear parts of armpits and the middle and upper parts of groin.

6. Before inoculation, the cell suspension is blown off fully by a gun, so that cell agglomeration is prevented, and the cell survival rate is reduced.

7. During inoculation, the needle head is inserted a little bit deep into the subcutaneous needle, about 1cm deep, and the overflow of the cell suspension from the needle eye after injection is reduced.

Method for injecting tumor-in-tumor drug and measuring tumor volume of B mouse

1. After the tumor of the mice exceeded one centimeter, the tumor was injected with PBS or MK6-83 at the corresponding concentration every day at approximately 100l, and the tumor volume was measured and calculated daily with a vernier caliper. Tumor volume ═ major diameter ═ minor diameter/2

2. Mice were waited for natural death and survival time was recorded.

3. After death, the tumor mass was removed and the final tumor volume was measured and recorded.

2. Test results

FIG. 10 shows the survival curves of mice in PBS control group and MK6-83(5 μ M) injected group; survival curves of mice from the PBS (lower plot) control group and MK6-83 (upper plot) injected group are plotted. After MK6-83 (5. mu.M) was injected, the survival time of the mice was significantly increased compared to the control group.

FIG. 11 shows tumor volumes for PBS control and MK6-83(5 μ M) injected groups. The final tumor volume of 12 PBS control groups and 11 MK6-83 injected groups of mice are plotted. The tumor volume of MK6-83 injected mice in group 11 was significantly reduced overall compared to PBS mouse tumors in group 12. (. P <0.001)

3. Conclusion

The TRPML1 specific small molecule agonist MK6-83 can inhibit the growth of mouse pancreatic cancer tumor and prolong the survival life of mouse.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of the TRPML1 specific small molecule agonist in preparing tumor therapeutic drugs is characterized in that the tumors are pancreatic cancer and breast cancer;

the chemical structural formula of the TRPML1 specific small molecule agonist is shown as