CN108864037A

CN108864037A - TRPML1 specific agonist, its application as autophagy inhibitor and preparation tumor, pharmaceutical composition

Info

Publication number: CN108864037A
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: 2018-11-23
Anticipated expiration: 2038-05-28
Also published as: CN108864037B

Abstract

This application provides a kind of specific small molecule agonist MK6-83 of TRPML1 ion channel, and provide application of the MK6-83 as the application of autophagy inhibitor and its using the function in preparation tumor therapeutic agent.TRPML1 specific small molecule agonist MK6-83 provided by the present application is by inhibiting autophagy activities, under the action of to various normal tissue cells without overt toxicity, has strong lethal ability in kinds of tumor cells system to including cancer of pancreas, breast cancer and gastric cancer.And MK6-83 has apparent inhibiting effect to the breast cancer bump growth transplanted on mouse, and significantly extends the service life of tumor formation mouse.It solves in traditional technology because anticancer drug and Cancer Treatment Regimens are while killing tumor cell, also the cell of normal tissue has the technical issues of killing.

Description

TRPML1 specific agonist, its as autophagy inhibitor and preparation treatment tumour medicine The application of object, pharmaceutical composition

Technical field

This application involves biomedicine fields to further relate to the spy in particular to a kind of TRPML1 specific agonist The purposes and tumor therapeutic agent of specific agonist.

Background technique

With the extension of human longevity, cancer is risen to as major causes of death in recent years.National Cancer Center publication 《The newest cancer report in the whole nation in 2018》It has been shown that, whole malignant tumour neopathies number of cases 380.4 ten thousand in 2014 are average super daily It crosses 10,000 people and is diagnosed as cancer, there are 7 people to be diagnosed as cancer per minute.Primary national malignant tumor morbidity is lung cancer, It secondly is gastric cancer, colorectal cancer, liver cancer and breast cancer.Cancer has become the first cause of China human mortality death.

About the treatment of cancer, scientists expand a large amount of research work.New anticancer drugs constantly discover.At present The cure rate of existing more than 20 tumours reaches 30% or more.And the subcellsular level of drug mechanism and grinding for molecular level Study carefully, has opened up anticancer drug significantly in the research of application aspect.Cyto-dynamics, drug effect dynamics and immunology aspect are ground The fast development studied carefully, but also the screening of drug, the adjustment of dosage, the determination of administration route are gradually improved.Drug combination, Medication of heavy dose of interval, adjuvant chemotherapy and the treatment for cooperating Chinese medicine, make treating malignant tumor obtain good curative effect.Now, To treating malignant tumor to perform the operation, radiotherapy, chemotherapy, the means such as the treatment of traditional Chinese medicine and immunization therapy be It is main.Playing on the selection of anticancer drug, toxic effect and drug resistance etc. influences its curative effect, because anticancer drug is swollen in killing While oncocyte, also the cell of normal tissue has the myeloid element of killing, especially molecular marker for increased proliferation and gastrointestinal tract thin Born of the same parents, so limit the dosage of anticarcinogen, and make that patient immune function reduces or even patient is difficult to endure gastrointestinal reaction, And be forced to interrupt treatment, make treatment failure.Anticancer drug can kill cancer cell, but due to its cytotoxicity, so to find one Kind can treating cancer simultaneously again do not cause harm to the human body or injure lesser drug be always scientists struggle mesh Mark.

TRPML1 is the cation release channel on a kind of lysosome membrane.In recent years the study found that TRPML1 passes through participation Various cellular activities and signal transduction include cell endocytic, spit outside and the function of lysosome is exercised in cell membrane reparation.ML- SA1, ML-SA3 etc. are the specific small molecule agonists in the channel TRPML1.The agonist for belonging to small molecule compound can be penetrating Cell membrane enters into the cell, the channel TRPML1 on specific activation lysosome, after TRPML1 channel opener, can discharge lyase The cation such as intracorporal calcium ion, iron ion and zinc ion enters cytoplasm.It is found through experiments that and utilizes certain small molecule excitements It can inhibit the progress of autophagy activities behind the channel agent specific activation TRPML1.Tumour cell was in order to provide growth to itself Energy requirement, autophagy signal are activated constantly to degrade oneself protein to meet the malignant proliferation demand of tumour cell, so The basic autophagy level of number of types of tumour cell is more significant than normal cell to be increased, and experiment discovery discovery utilizes the excitomotor After blocking autophagy, source of people tumour cell can be killed, but significant lethal effect is had no for human normal cell.

By largely screening, it has been found that the specific small molecule agonist of TRPML1 ion channel can be used as autophagy Inhibitor uses, and significantly inhibits especially for the autophagy activity of tumour cell, is potential anti-tumor drug.

Summary of the invention

To solve the above problems, the present invention passes through a variety of specific small molecules excitement many times to TRPML1 ion channel Agent carries out repeated screening, and the agonist of cell autophagy activities can be inhibited by obtaining one kind, and demonstrates it by inhibiting tumour The autophagy activity of cell, has the function of limiting growth of tumour cell.

To achieve the goals above, according to the first aspect of the application, a kind of spy of TRPML1 ion channel is provided Specific agonist MK6-83.

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

To achieve the goals above, according to the second aspect of the application, MK6-83 is provided as autophagy inhibitor Using or its applied in preparing autophagy inhibitor.

To achieve the goals above, in terms of according to the third of the application, MK6-83 is provided in preparation cancer therapeutics Application in object.

Further, the tumour includes cancer of pancreas, breast cancer and gastric cancer.

To achieve the goals above, it according to the 4th of the application the aspect, provides a kind of for treating the drug of tumour Composition.The active constituent of the pharmaceutical composition is MK6-83.

Further, the compositions be made oral solution, granule, tablet, hard capsule, soft capsule, pill, Injection, nanometer formulation, targeting preparation and other medically acceptable dosage forms.

TRPML1 specific small molecule agonist MK6-83 provided by the present application by inhibit autophagy activities, to it is various just Under the action of normal histocyte is without overt toxicity, have strongly to including cancer of pancreas, breast cancer and gastric cancer in kinds of tumor cells system Lethal ability.And MK6-83 has apparent inhibiting effect to the breast cancer bump growth transplanted on mouse, and significant Ground extends the service life of tumor formation mouse.It solves in traditional technology because anticancer drug and Cancer Treatment Regimens are in killing tumour While cell, also the cell of normal tissue has the technical issues of killing.

Detailed description of the invention

The attached drawing constituted part of this application is used to provide further understanding of the present application, so that the application's is other Feature, objects and advantages become more apparent upon.The illustrative examples attached drawing and its explanation of the application is for explaining the application, not Constitute the improper restriction to the application.In the accompanying drawings：

Fig. 1 shows in the Hela cell of method detection of protein immunoblot that MK6-83, Baf-A1 and the two combination add 1 hour after medicine, the conversion results of two kinds of forms of autophagy labelled protein LC3, wherein related content translation or solution in attached drawing 1 It releases as follows：LC3 represents 1 light chain 3 of microtubule associated protein, and Normalized LC3II represents normalized LC3II value, Tubulin Tubulin is represented, CTL represents blank control group, and starvation represents Nature enemy group, and Baf-A1 represents bar bifilomycin A1；

Fig. 2 shows in the Hela cell of method detection of protein immunoblot that MK6-83, Baf-A1 and the two combination add 12 hours after medicine, the conversion results of two kinds of forms of autophagy labelled protein LC3, wherein related content translation or solution in attached drawing 2 It releases as follows：LC3 represents 1 light chain 3 of microtubule associated protein, and Normalized LC3II represents normalized LC3II value, Tubulin Tubulin is represented, CTL represents blank control group, and Baf-A1 represents bar bifilomycin A1；

In the Hela cell that the method that Fig. 3 shows protein immunoblot detects, MK6-83, CQ, Rap and MK6-83 and CQ 12 hours after combination dosing, the conversion results of two kinds of forms of autophagy labelled protein LC3 and the horizontal result of P62 albumen, wherein Related content in attached drawing 3 is translated or is explained as follows：LC3 represents 1 light chain 3 of microtubule associated protein, Normalized LC3II generation The normalized LC3II value of table, Tubulin represent tubulin, and GAPDH represents GAPDH internal reference albumen, and P62 represents autophagy selection Property substrate P62 albumen, P62/GAPDH represent the normalized value of GAPDH expression, and CTL represents blank control group, Starvation represents Nature enemy group, and CQ represents chloroquine, and Rap represents rapamycin；

Fig. 4 shows the processed Patu 8988t cell Trypan Blue result of MK6-83 (5 μM), wherein in attached drawing 4 Related content translation or be explained as follows：Control represents control group；

Fig. 5 shows the Pancreatic ductal cells system HPDE6c7 and pancreatic carcinoma Patu after trypan blue experiment Difference under 8988t active control and various concentration MK6-83 processing, wherein related content translation or explanation in attached drawing 5 are such as Under：CTL represents blank control group in abscissa, and ordinate Cell Viability represents cell activity；

Fig. 6 shows difference of the cell activity of breast cancer cell line MCF-7 under various concentration MK6-83 processing, In, the related content in attached drawing 6 is translated or is explained as follows：CTL represents blank control group, ordinate Cell in abscissa Viability represents cell activity；

Fig. 7 shows the cell activity of normal breast ductal epithelial cell line MCF 10A in various concentration MK6-83 processing Under difference, wherein in attached drawing 7 related content translation or be explained as follows：CTL represents blank control group in abscissa, indulges and sits Mark Cell Viability represents cell activity；

Fig. 8 shows difference of the cell activity of Gastric caicinoma cell line SGC-7901 under various concentration MK6-83 processing, In, the related content in attached drawing 8 is translated or is explained as follows：CTL represents blank control group, ordinate Cell in abscissa Viability represents cell activity；

Fig. 9 shows the cell activity of normal gastric mucosa epithelial cell line GES-1 under various concentration MK6-83 processing Difference, wherein the related content in attached drawing 9 is translated or is explained as follows：CTL represents blank control group, ordinate in abscissa Cell Viability represents cell activity；

Figure 10 shows the mouse survival curve of PBS control group Yu MK6-83 (5 μM) injection group, wherein in attached drawing 10 Related content is translated or is explained as follows：Abscissa Days after injection represents number of days after injection, ordinate Survival represents survival rate；

Figure 11 shows the gross tumor volume of PBS control group Yu MK6-83 (5 μM) injection group, wherein the correlation in attached drawing 11 Content translation is explained as follows：PBS represents phosphate buffered saline solution, ordinate Relative Tumor Volume in abscissa (fold) relative tumour volume (again) is represented.

Specific embodiment

In order to make those skilled in the art more fully understand application scheme, below in conjunction in the embodiment of the present application Attached drawing, the technical scheme in the embodiment of the application is clearly and completely described, it is clear that described embodiment is only The embodiment of the application a part, instead of all the embodiments.Based on the embodiment in the application, ordinary skill people Member's every other embodiment obtained without making creative work, all should belong to the model of the application protection It encloses.

It should be noted that the description and claims of this application and term " includes " in above-mentioned attached drawing and he Any deformation, it is intended that cover it is non-exclusive include, for example, containing a series of entirety of parts, it is not necessary to be limited to clear Part those of is listed on ground, but may include be not clearly listed or for these whole intrinsic other parts.

It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.

It should be noted that the concrete operations that do not list specifically in the embodiment of the present application be suitable for it is in the art normal Rule operation or routine experiment means, it should be understood that those skilled in the art can rationally be known according to the prior art.

In the case where no specified otherwise, the term in the application can be explained below or those skilled in the art It is done according to common knowledge in the art and reasonably expands or limit explanation, all those skilled in the art can be according to the Shen of the application Please the prior art content before day rationally know.

Autophagy：Autophagy is a phagocytosis own cells matter albumen or organelle and its coating is made to enter vesica, and and lyase Body merges to form autophagy lysosome, the process for the content that it is wrapped up of degrading, thereby realize the metabolism of cell itself need and The update of certain organelles.

Western-blot detection method：Detected by Western blot is applied molecular biology, biochemistry and immune genetic A kind of experimental method that can be used often in, and be a kind of analysis method that quantitative and semi-quantitative can be carried out to albumen, it is The processed cell of gel electrophoresis or biological tissue samples are coloured by specific antibody, and pass through analysis coloring Position obtains the information of expression of the specific protein in the cell or tissue analyzed with color depth.

EP pipe：Micro centrifugal pipe, laboratory consumptive material are a kind of small-sized centrifuge tubes, match with micro centrifuge, use In the separation of trace reagent.

ddH2O：The abbreviation of double distilled water, i.e. distilled water, being will be by the water after single flash again The secondary obtained water of distillation.

BCA reacts working solution：BCA is the abbreviation of bicinchoninicacid (two quinoline formic acid albumen), and BCA method is one The widely used albuminimetry of kind, wherein the BCA reaction working solution used is reagent A (BCA alkaline solution) and reagent B (sulphur Sour copper solution) mixed liquor, the ratio of reagent A and reagent B are 50:1.

loadingbuffer：Sample-loading buffer used in Western-blot detection method, for showing the process of electrophoresis And sink to sample in loading wells without floating.

Acr-Bis：Acrylamide:Methylene-bisacrylamide solution.

Tris-HCl buffer：Tris-HCI buffer.

SDS buffer：Sodium lauryl sulfate buffer.

TEMED：Tetramethylethylenediamine, for preparing SDS-PAGE glue.

Marker：Protein labeling, the albumen of pre-dyed or the various molecular weight of non-pre-dyed, for indicating the size of albumen in electrophoresis And tracer.

Pvdf membrane：PVDF membrane (polyvinylidene fluoride) is common in immunoblotting A kind of solid support.

TBST：It is common a kind of buffer in detected by Western blot containing Tris-Hcl, NaCl and Tween20.

Hela cell：HeLa cell is a kind of manually culture, the cell with unlimited multiplication capacity, wide in medical field It is general to be applied to tumor research, Bioexperiment or cell culture.

Trypan blue experimental method：It is that dead cell can only be dyed to method blue and that living cells cannot be caught using trypan blue Detect the fast and convenient method of one kind of cell survival rate.

The application is described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

Embodiment 1：MK6-83 inhibits the active detection of autophagy

1. experimental group is described as follows

Blank control group：+ 10% fetal calf serum of normal incubation medium

Nature enemy group：Normal incubation medium (is free of fetal calf serum)

Test group 1：Bafilomycin A1 processing

Test group 2：1 μM of MK6-83 processing

Test group 3：3 μM of MK6-83 processing

Test group 4：5 μM of MK6-83 processing

Test group 5：Processing is used in combination in 1 μM of MK6-83 and Bafilomycin A1

Test group 6：Processing is used in combination in 5 μM of MK6-83 and Bafilomycin A1

Test group 7：10 μM of CQ processing

Test group 8：Processing is used in combination in 5 μM of MK6-83 and CQ

Test group 9：50 μM of Rap processing

2. experimental method

Using Western-blot detection method (protein immunoblotting method) to group of cells carry out it is subsequent processing and Detection, specific method are：

A, Protein Extraction and concentration mensuration

(1) cell：Six orifice plates of culture cell are placed on ice, sop up culture medium, and 100 μ l cell pyrolysis liquids are added in every hole, 3-5min is stood on ice, under cell scraper, will be collected cell pyrolysis liquid in 1.5ml centrifuge tube with cell scraper, is shaken in DL instrument 20s is swung, stands 30min on ice.

(3) the EP pipe after standing is placed in refrigerated centrifuge, 4 DEG C of centrifugations (15000rpm × 18min).

(4) gentle aspiration supernatant arrives total protein solution in another EP marked is managed.

(5) protein concentration is detected：Each sample takes 5 μ l protein solutions that 5 times of 20 μ lddH2O dilution is added, and takes 10 μ l in 96 holes In plate, each sample does 2 multiple holes.

(6) protein standard substance A-H is added in the most previous column of 96 orifice plates, concentration is from high to low.

(7) BCA is prepared under the conditions of being protected from light reacts working solution (A：B=50：1) it mixes, 100 μ l working solutions are added in every hole, gently Light concussion mixes, and is protected from light in 37 DEG C of incubators with tinfoil package and is incubated for 30min.

(8) each hole optical density (OD) value and the concentration of protein sample under microplate reader detection 562nm wavelength.

(9) it is saved after unused protein packing in -80 DEG C.

B, albuminous degeneration

The protein liquid volume being added is calculated according to required protein content and experiment loading number.It is needed with every 30 μ g total protein of hole For loading 4 times to detect different test index progress electrophoresis, protein solution volume (μ l)=(30 μ g eggs needed for calculating White/protein concentration) × 5, then ddH2O to 15 μ l × 5=75 μ l is mended, add 6 × loadingbuffer of 3 μ l × 5=15 μ l (6×loadingbuffer:Beta -mercaptoethanol=9:1) mouth, is sealed, 5min, 4 DEG C of preservations are boiled in boiling water.

C, PAGE gel electrophoresis

(1) resolving polyacrylamide gel (2 pieces of glue amounts) of preparation 10%：It takes with glue special-purpose bottle, is separately added into following reagent, Pay attention to mixing：ddH2O4ml；30%Acr-Bis3.3ml；1.5MTris-HCl buffer (pH8.8) 2.5ml；10%SDS buffering Liquid 0.1ml；10% Ammonium Persulfate 98.5 (AP) 0.1ml, TEMED0.004ml is mixed, and draws separation gel injection electrophoresis tank with 1ml rifle It in glass plate interlayer, is careful not to form bubble, the small glass plate height of glue amount about 2/3 is added, then be slowly added into about 1ml Isopropanol fluid-tight crimping, polymerized at room temperature 30min or so.

(2) preparation 5% polyacrylamide concentration glue (2 pieces of glue amounts)：It takes with glue special-purpose bottle, is separately added into following reagent, infuse Meaning mixes：ddH2O2.7ml；30%Acr-Bis0.67ml；1.0MTris-HCl buffer (pH6.8) 0.5ml；10%SDS is slow Fliud flushing 0.04ml；10% Ammonium Persulfate 98.5 0.04ml；TEMED0.004ml is mixed.

(3) isopropanol at the top of the separation gel coagulated is gently gone, blots remaining liquid with filter paper, by concentration glue injection At the top of interlayer, it is inserted into comb, polymerized at room temperature 30min.

(4) after gelling is solid, gel slab is fixed on to the upper buffer chamber of electrophoretic apparatus, electrophoresis liquid is added, extracts comb, is used Microsyringe draws 18 μ l sample solutions and adds to well, Marker is added in first sample hole, the last one sample hole is added 1 × loadingbuffer is put into the electrophoretic apparatus of 1 × Tris- glycine running buffer.

(5) power on, concentrate glue voltage 90V, electrophoresis about 30min, separation gel voltage 120V, electrophoresis is until bromine phenol Orchid arrive at separation gel bottom, according to the size of destination protein molecular weight, should adjust in principle voltage size and separation gel when Between.

(6) power supply is closed, gel is removed, the glue comprising destination protein is cut according to Marker, for the transferring film of next step.

D, transferring film (Whote-wet method)

(1) pvdf membrane appropriately sized according to the size clip of the purpose glue of cutting, and shear angle makes marks on film, is placed in 1-3min is soaked in methanol, is then transferred in 1 × transferring film buffer.

(2) by the porous filter pulp of negative side-- thickness filter paper-glue-pvdf membrane-thickness filter paper-porous filter pulp-side of the positive electrode sequence dress Then improvement film device is placed in Bio-RAD electrophoresis apparatus, pours into 1 × transferring film liquid, ice bath transferring film, according to the molecule of destination protein It measures and determines the transferring film time, generally multi-purpose constant current 350mA shifts 2h or 400mA, shifts 1.5h.

(3) after transferring film, pvdf membrane is cut off into one jiao of front and back sides with label film, makes Marker in left side and under To it is upper be increase sequence, shear angle rinses 3 times × 10min in upper left, with 1 × TBST solution.

E, it is immunoreacted

(1) transfer film after rinsing is put into room temperature in 5% skimmed milk power confining liquid and closes 2h, and slowly shaken on shaking table It is dynamic.

(2) primary antibody is diluted to debita spissitudo with 5% skimmed milk power confining liquid, suitable antibody is covered on film, 4 DEG C incubate It educates overnight.

(3) 3 times × 15min is washed with 1 × TBST, is shaken on shaking table.

(4) 5% skimmed milk power confining liquids dilute secondary antibody to debita spissitudo, and suitable antibody, room temperature wet box are covered on film It is incubated for 1-2h.

(5) 3 times × 15min is washed with 1 × TBST, is shaken on shaking table.

(6) two kinds of reagents of A and B in ECL are mixed in equal volume, is uniformly added on film and (is protected from light), with chemiluminescence gel Picture system is taken pictures.

3. experimental result

Test result is referring to figures 1-3.

This experiment is had detected in Hela cell using the method for protein immunoblot, MK6-83 (1 μM, 3 μM and 5 μM) dosing The conversion of two kinds of forms of front and back autophagy labelled protein LC3.When LC3I is converted to the horizontal increase of LC3II, illustrate autophagy water It puts down.As depicted in figs. 1 and 2, it is seen that since a hour, the conversion of LC3II is obvious after MK6-83 dosing Increase, disclosing MK6-83 has rapid regulating and controlling effect to autophagy.And as joint MK6-83 and Bafilomycin A1 (Baf- A1；A kind of lysosomal inhibitor) after medication, the conversion of LC3II does not further increase, and MK6-83 is prompted to inhibit autophagy living It is dynamic.

As shown in figure 3, combining MK6-83 using another lysosomal inhibitor chloroquine (CQ) uses (test group 8), discovery For LC3II transformation either with or without further increasing, disclose MK6-83 for the inhibiting effect of autophagy.And it was found that thunder Pa mycin (rapamycin) processing group (test group 9) and Nature enemy group have the function of reduction P62 protein level, on the contrary, MK6-83 processing group increases the level of P62 albumen, further demonstrates MK6-83 and inhibits autophagy activities.

4. conclusion

TRPML1 specific small molecule agonist MK6-83 can inhibit the progress of hela cell autophagy activities, can be used as Autophagy inhibitor uses.

Embodiment 2：Test of the MK6-83 to the lethal ability of human pancreatic cancer cell Patu 8988t

1. test method

Cell is detected using trypan blue experimental method, it is specially that control group (control) cell and dosing group is thin It is mixed after born of the same parents are digested with pancreatin with the trypan blue reagent of fixed volume.It is added again with the mixing liquid that pipettor draws 10 μ L white thin Born of the same parents' counter is placed under microscope and is counted.The cell for being infected with trypan blue is calculated as dead cell, otherwise is living cells.Finally The ratio of every group of living cells and total cell number is calculated as cell activity.

2. test result

Fig. 4 is that (three width figures are respectively pair to MK6-83 (5 μM) processed Patu 8988t cell Trypan Blue result figure According to group, dosing group for 24 hours after image and dosing group 72h after image)；Fig. 4 shows through 5 μM of processed Patu of MK6-83 The death rate of 8988t cell all obviously increases compared with the control group, illustrates that MK6-83 has lethal effect to Patu 8988t.Arrow What is indicated is by the dead cell of Trypan Blue.

Fig. 5 is the Pancreatic ductal cells system HPDE6c7 and pancreatic carcinoma Patu 8988t after trypan blue experiment Disparity map under cell activity control and various concentration MK6-83 processing；Histogram is shown in Normal Pancreas ductal epithelial cell system In HPDE6c7 cell, the cell activity of dosing group and control group there were significant differences (P>0.05)；In contrast to this, in pancreas In cancer Patu 8988t cell, MK6-83 significantly reduces cell activity, and in the time and measures interdependent trend.

3. conclusion

TRPML1 specific small molecule agonist MK6-83 has a lethal ability to pancreatic cancer cell Patu 8988t, but for Normal Pancreas ductal epithelial cell HPDE6c7 is without lethality.

Embodiment 3：Test of the MK6-83 to the lethal ability of human breast cancer cell line Bcap-37

1. test method

2. test result

Result figure (three numbers of the cell activity that Fig. 6 is breast cancer cell line MCF-7 under various concentration MK6-83 processing Blank control group CTL, 1 μM of MK6-83 dosing group and 5 μM of MK6-83 dosing groups are respectively corresponded according to column)；Fig. 7 leads for normal breast (three data columns are right respectively for result figure of the cell activity of pipe epithelial cell line MCF 10A under various concentration MK6-83 processing Answer blank control group CTL, 1 μM of MK6-83 dosing group and 5 μM of MK6-83 dosing groups)；

Fig. 7 shows in normal breast ductal epithelial cell line MCF 10A cell, the cell activity of dosing group and control group There were significant differences (P>0.05)；Fig. 6 shows that in MCF-7 Breast Cancer Cell, 48 hours MK6-83 are handled and significantly reduced Cell activity, and in measuring interdependent trend.

3. conclusion

TRPML1 specific small molecule agonist MK6-83 has lethal ability to breast cancer cell MCF-7, but for normal Breast ductal epithelial cells MCF 10A is without lethality.

Embodiment 4：Test of the MK6-83 to the lethal ability of human gastric adenocarcinoma SGC-7901

1. test method

2. test result

Fig. 8 is result figure (three of the cell activity of Gastric caicinoma cell line SGC-7901 under various concentration MK6-83 processing Data column respectively corresponds blank control group CTL, 1 μM of MK6-83 dosing group and 5 μM of MK6-83 dosing groups)；Fig. 9 is glutinous for normal gastric (three data columns respectively correspond result figure of the cell activity of film epithelial cell line GES-1 under various concentration MK6-83 processing Blank control group CTL, 1 μM of MK6-83 dosing group and 5 μM of MK6-83 dosing groups)；

Fig. 9 shows in normal gastric mucosa epithelial cell line GES-1 cell that the cell activity of dosing group and control group is not There were significant differences (P>0.05)；Fig. 8 shows that in SGC-7901 cell, MK6-83 processing in 48 hours significantly reduces Cell activity, and in the interdependent trend of metering.

3. conclusion

TRPML1 specific small molecule agonist MK6-83 has lethal ability to gastric adenocarcinoma cells SGC-7901, but for just Normal gastric epithelial cells GES-1 is without lethality.

Embodiment 5：MK6-83 inhibits the growth of mice pancreatic tumor and extends the experiment in mouse survival service life

1. experimental method

A nude mice by subcutaneous tumor formation experiment：

1, when Patu 8988t cell reaches 80-90% or so density, day before cell replacement fresh cultured at night is collected Base.

2, it is washed twice after trypsin digestion cell with the PBS of pre-cooling, purpose is the serum removed in cell.

3, cell precipitation is blown and beaten to suitable concentration, the cell of general subcutaneous tumors inoculation with PBS or serum free medium Amount is (1-5) × 10⁶A cell/, inoculation volume is 0.1ml, therefore the concentration of cell suspension is 1-5 × 10⁷A cell/ml.

4, nude mice by subcutaneous should be inoculated into after cell dissociation as early as possible, is generally completed within half an hour as far as possible, on the way hangs cell Liquid is placed on the metabolism for reducing cell on ice.

5, the nude mice selected plants position selection blood supply and enriches region, such as armpit in 5-8 week old, weight 18-20g or so Middle and back, groin middle and upper part.

6, cell suspension is adequately dispelled with rifle before inoculation, prevents cell agglomerating and reduces cell survival rate.

7, when being inoculated with, syringe needle is a little deep in subcutaneous inserting needle, and about 1cm is deep, and cell suspension is overflowed from pinprick after reducing injection.

B mouse is in tumor medicine injection and tumor volume measurement method

1, after mouse tumor is more than one centimeter, the MK6-83 about 100l of PBS or respective concentration is injected in tumor daily, often It is with vernier caliper measurement gross tumor volume and calculates.Gross tumor volume=minor axis/2 major diameter * minor axis *

2, mouse natural death is waited, and records life span.

3, lump is taken out after dead mouse, measures final gross tumor volume and record.

2. test result

Figure 10 shows the mouse survival curve of PBS control group Yu MK6-83 (5 μM) injection group；Depict PBS (lower section figure Line) control group and MK6-83 (upper figure) injection group mouse survivorship curve.After injecting MK6-83 (5 μM), the life of mouse Deposit the time obviously increases than control group.

Figure 11 shows the gross tumor volume of PBS control group Yu MK6-83 (5 μM) injection group.Depict 12 groups of PBS control groups Final gross tumor volume compares with the mouse of 11 groups of MK6-83 injection group.Compared with 12 groups of PBS mouse tumor, 11 groups The gross tumor volume of MK6-83 injection mouse integrally significantly reduces.(***P<0.001)

3. conclusion

TRPML1 specific small molecule agonist MK6-83 is able to suppress the growth of mice pancreatic tumor and extends mouse It survives the service life.

The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

Claims

1. a kind of TRPML1 specific small molecule agonist, which is characterized in that its chemical structural formula is

2. application of the TRPML1 specific small molecule agonist described in claim 1 as autophagy inhibitor.

3. application of the TRPML1 specific small molecule agonist described in claim 1 in preparation tumor therapeutic agent.

4. application according to claim 3, which is characterized in that the tumour includes cancer of pancreas, breast cancer and gastric cancer.

5. a kind of for treating the pharmaceutical composition of tumour, which is characterized in that active constituent is TRPML1 described in claim 1 Specific small molecule agonist.

6. pharmaceutical composition according to claim 5, which is characterized in that oral solution, granule, piece is made in pharmaceutical composition Agent, hard capsule, soft capsule, pill, injection and nanometer formulation, targeting preparation.