CN106727548A - Alkyl pyridine class compound is preparing the application of Induces Autophagy medicine and method - Google Patents
Alkyl pyridine class compound is preparing the application of Induces Autophagy medicine and method Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4425—Pyridinium derivatives, e.g. pralidoxime, pyridostigmine
Abstract
A kind of method that the present invention discloses application and Induces Autophagy of alkyl pyridine class compound on the medicine for preparing cell autophagy derivant or preparing the disease that treatment can benefit from autophagy induction, belongs to biomedicine field.The compound is mainly by suppressing the activity of mTORC1 come Induces Autophagy.When compound of the present invention is used as the active ingredient of the medicine that novel autophagy derivant or treatment can benefit from the disease that autophagy is induced, its working concentration is low, and specificity is good, is with a wide range of applications.
Description
Technical field
The present invention relates to biomedicine field, and in particular to alkyl pyridine class compound prepare cell autophagy derivant or
The method for preparing the application and Induces Autophagy treated on the medicine that can benefit from the disease that autophagy is induced.
Background technology
Autophagy refers to that some need the intracellular matters such as the protein and other and organelle of degraded to be wrapped, and
It is final to be transported to the process that lysosome (lysosome) is degraded, amino acid that autophagy degraded is produced and some other small point
Sub- material can be reused or produce energy.This be a warp strictly regulate and control, highly conserved process in evolution, it gives birth in cell
Played an important role in long, development and stable state, help to remain flat between the synthesis of cellular products, degraded and follow-up recycling
Weighing apparatus.So far therefore, at least triformed autophagy is reported：Huge autophagy (macroautophagy), micro- autophagy
(microautophagy) and molecular chaperones mediation autophagy (chaperone-mediated autophagy).Huge autophagy is with shape
It is characterized into unique double membrane structure organelle (i.e. autophagosome, autophagosome)；Micro- autophagy refers to by lysosome membrane
Cave in and directly to wrap up intracellular matter into katabolism in lysosome；The autophagy of molecular chaperones mediation refers to by molecular chaperones
Hsc70, other together participation effect molecular chaperones and lysosomal associated membrane albumen 2A (lysosomal-associated
Membrane protein type 2A, LAMP-2A) mediation a class for the protein containing KFERQ sequences autophagy.
In three kinds of autophagy of form, huge autophagy is considered as a kind of topmost form, and is studied most deep.
Under normal circumstances, intracellular autophagy maintains foundation level, but is induced rapidly under stressed condition, and it is thin
Intracellular plays various functions.Maintained except the organelle by degrade itself aging or damage and other macromolecular substances
Outside the steady state function of physiological status lower body, increasing research shows that autophagy imbalance may be with tumour, infection, aging, generation
Thanking property disease, autoimmune disease are related to nerve degenerative diseases etc..
For example, clinical research find many nerve degenerative diseases (including Alzheimer disease, Parkinson's,
Huntington's chorea, amyotrophic lateral sclerosis etc.) in, lesion region often has substantial amounts of ubiquitin protein matter aggregation or bag
Contain body, these are exactly caused because mutation causes protein conformation to change by the abnormal protein aggregation or inclusion body of ubiquitination more
One of major reason of nerve degenerative diseases.The normal generation of autophagy is to removing these abnormal protein aggregations or inclusion body
Play critical effect.Autophagygene Atg5 or Atg7 mouse in specific knockdown neuron nerve cell (its from
Function is bitten to be blocked) in, the accumulation of a large amount of ubiquitin protein matter polymer is found, and finally trigger nerve degenerative diseases.
In nerve degenerative diseases animal model, there is the removing that can promote protein polymer in induction autophagy, can alleviate neurological
Property disease symptom, point out induction autophagy be likely to become a kind for the treatment of method for treating nerve degenerative diseases.Cell autophagy pair
Infect and immune also with various effects, including：Activation congenital immunity and acquired immunity, maintain the stable state of immunocyte,
Degraded pathogenic microorganism etc..Therefore, for infectious diseases, cell autophagy is also a very attractive therapeutic targets.
In sum, using medicine effect enhancing autophagy, be conducive to tumour, infection, aging, metabolic disease, itself exempt from
The treatment of epidemic disease disease and nerve degenerative diseases etc., however, the working concentration of existing many novel autophagy derivants it is very high and
It is high non-specific.The cell autophagy derivant rapamycin (thunders being such as widely used in Cell Biology Experiment
Handkerchief mycin), its immunosuppressive action immunodepressant cyclosporin more conventional than clinically is also eager to excel upper 10 times, is easily produced big
Side effect, so as to limit its application as novel autophagy derivant clinically.
The content of the invention
In view of this, the application provides a kind of alkyl pyridine class compound and is preparing cell autophagy derivant or preparing treatment
The method of application and Induces Autophagy on the medicine of the disease that can benefit from autophagy induction.
To solve above technical problem, the technical scheme that the present invention is provided is a kind of application of alkyl pyridine class compound,
The structural formula of the alkyl pyridine class compound such as formula I：
In formula, the arbitrary integer in n=0-4, the arbitrary integer in m=12-16, substituent R1Positioned at the ortho position of N atoms,
At least one in meta or para position, substituent R1Selected from hydrogen, hydroxyl, methyl, ethyl, methoxyl group, amino, nitro, sulfonic group,
At least one in halogen, X-Selected from any one in halide ion；
The alkyl pyridine class compound can benefit from autophagy and lure for preparing cell autophagy derivant or preparation treatment
The medicine of the disease led.
Preferably, the alkyl pyridine class compound is by suppressing the activity of mTORC1 come Induces Autophagy.
Preferably, in formula I, the arbitrary integer in n=0-3, the arbitrary integer in m=14-16, substituent R1It is former positioned at N
The meta or para position of son, substituent R1Selected from least one in hydrogen, hydroxyl, methyl, amino, sulfonic group, X-Selected from chlorion or
Bromide ion.
It is more highly preferred to, in formula I, n=0, m=15, X-It is chlorion, i.e., preferred structural formula is as shown in formula II：
Preferably, described cell is thin embryonic stem cell, induced multi-potent stem cell, adult stem cell, noble cells, blood
Born of the same parents, hematopoietic cell, vascular endothelial cell, epithelial cell, exocrine cell, endocrine cell, phoirocyte, mucous membrane are thin
Born of the same parents, immunocyte, fat cell, osteocyte, smooth muscle cell, striated muscle cell, nerve cell, sensory cell, visual cell, the heart
Myocyte, liver cell, respiratory tract cell, esophageal cells, gastric cells, enterocyte, splenocyte, pneumonocyte, nephrocyte, reproduction cell
In any one cell；Or from cardiac tumor, liver cancer, splenic tumor, nasopharyngeal carcinoma, the cancer of the esophagus, stomach cancer, bone tumour, myomata,
Leukaemia, lymthoma, lung cancer, kidney, colon cancer, the cancer of the brain, melanoma, breast cancer, oophoroma, cervix cancer, prostate cancer,
The tumour cell of any one in cancer of pancreas, epithelioma, nervous system neoplasm.
Preferably, the novel autophagy derivant or treatment can benefit from the medicine of the disease of autophagy induction also including at least one
Plant and be selected from pharmaceutically acceptable carrier, adjuvant, auxiliary agent, solvent, diluent, excipient, synergist.
Preferably, the formulation that the novel autophagy derivant or treatment can benefit from the medicine of the disease of autophagy induction is molten
It is any one in liquid, tablet, capsule, powder, particle, ointment, suppository, injection, inhalant, gel, microsphere, aerosol
Kind.
Preferably, the described disease that can benefit from autophagy induction includes nerve degenerative diseases, alpha1-antitrypsin
Deficiency disease, angiocardiopathy, IBD, Crohn disease, muscle disease, osteoarthritis, infectious diseases, metabolic disease
Disease, cancer, aging.
Be more highly preferred to, the nerve degenerative diseases include Alzheimer disease, Parkinson's, Huntington's chorea,
Amyotrophic lateral sclerosis.
Preferably, the administering mode of the medicine that the novel autophagy derivant or treatment can benefit from the disease that autophagy is induced is
Orally, through oesophagus, through stomach, through small intestine, per rectum, through eye, intranasal, transtracheal, through ear, through it is sublingual, through skin, transmucosal, warp
It is subcutaneous, through muscle, intravascular, through abdominal cavity, Via vagina, through intrathecal, through ossis be administered in any one.
Technical scheme also provides a kind of method of Induces Autophagy, has been cast to animal in need or patient
Compound described in the formula I of effect amount, the effective dose for casting is 0.001-500mg/kg body weight.
Wherein, the effective dose of described compound administration depends on administration purpose, recipient, the treated illness for the treatment of
And its order of severity, administration time, method of administration, treatment cycle, compound efficiency, its clearance rate and whether have other medicines
Thing co-administered etc..Meanwhile, effective dose still treats any toxicity or unfavorable of the beneficial effect more than the autophagy inducing compounds
Effect is (for example：Side effect and adverse reaction etc.) amount.
Preferably, the mode for casting is orally, through oesophagus, through stomach, through small intestine, per rectum, through eye, intranasal, through gas
Pipe, through ear, through it is sublingual, through skin, transmucosal, through it is subcutaneous, through muscle, intravascular, through abdominal cavity, Via vagina, through intrathecal, through marrow
Chamber administration in any one.
In technical scheme, the compound shown in formula I is a kind of alkyl pyridine class compound, in formula, n=0-4
In arbitrary integer, the arbitrary integer in m=12-16, substituent R1In the ortho position of N atoms, meta or para position at least
One kind, substituent R1Selected from least one in hydrogen, hydroxyl, methyl, ethyl, methoxyl group, amino, nitro, sulfonic group, halogen, X-
Selected from any one in halide ion.Compound shown in formula I is important organic synthesis intermediate, is widely used in doctor
Medicine, agricultural chemicals, spices, the synthesis of industrial products, applicants have discovered that it has the effect of Induces Autophagy, can be used to prepare carefully
Born of the same parents' novel autophagy derivant prepares the medicine that treatment can benefit from the disease of autophagy induction.
Its preferred scheme, n=0, m=15, X-It is chlorion, its structural formula is formula II, the compound chemistry described in formula II
Entitled cetylpyridinium chloride (CPC), its solid pure crystal form is stablized at room temperature.It is 77 DEG C, band without aquosity fusing point
One crystallization water is 80-83 DEG C.Insoluble in acetone, acetic acid, ethanol.At present, cetylpyridinium chloride is primarily as one kind
Antiseptic, is some mouth-washs, and toothpaste, lozenge, throat spray, nasal spray breathes the constituent of spray.
As shown in figure 1, autophagy is needed by following several stages:Autophagy precursor (phagophore, also referred to as
Isolation membrane, barrier film) formed；Body extension wraps up the substrate of autophagy before autophagy, and autophagosome is formed；Autophagosome with
Lysosome fusion completes degradation of substrates.The nuclear molecular structure of autophagy by one group of Atg gene (Autophagy-related
Gene) the protein control of coding, this group of Atg gene is highly protected in from saccharomycete to the eucaryote of mammal
Keep.The different phase that autophagy occurs is participated according to it, the autophagy proteins of these cores are divided into five classes:Atg1/ULK1 albumen swashs
Combined enzyme agent, Vps34-Atg6/Beclin1 and type III PI3K complexs, Atg9/mAtg9, Atg12-Atg5-Atg16 class are general
Elementization connection system and Atg8/LC3 ubiquitin-like connection systems.
Atg1/ULK1 (Atg1 being named as in yeast, its mammal homologous protein is named as ULK1) compound is certainly
An important positive regulatory factor during body is formed is bitten, this compound is main by ULK1, FIP200 in mammal
(focal adhesion kinase family interacting protein of 200kDa), Atg13 and Atg101 groups
Into.When nutritional sufficiency, mTORC1 (mechanistic Target Of Rapamycin Complex1) and ULK1 compounds
With reference to so as to suppress autophagy.MTORC1 receives intraor extracellular signal extensively, such as amino acid levels, growth factor, energy and lacks
Oxygen condition etc. adjusts cell growth and metabolism.It is made up of 5 subunits, (is interacted with ULK1) including Raptor
With mTOR (mechanistic Target OfRApamycin, with activity of serine/threonine kinases).MTORC1 passes through phosphorus
ULK1 and Atg13 is acidified so as to suppress the starting of autophagy.Under the conditions of starvation is waited, mTORC1 is separated from ULK1 compounds, from
And induce the formation and extension of autophagosome.
The principle of herein described compound induction autophagy is mainly by suppressing what mTORC1 was carried out.MTORC1 is autophagy
Center adjustment point, mainly played a role by combining and suppressing the crucial startup factor ULK1 compounds of autophagy.Therefore,
CPC mitigates its suppression to ULK1 compounds by suppressing the activity of mTORC1, makes ULK1 complex activating, induction autophagy
Start and carry out.It is demonstrated experimentally that being made carefully by (in vivo) in vivo, in vitro (ex vivo) and/or external (in vitro)
Born of the same parents contact with the application compound of effective dose, can increase the autophagy in cell.
In technical scheme, preparing novel autophagy derivant using above-mentioned compound or preparing treatment to benefit from
During the medicine of the disease of autophagy induction, it may include at least one is selected from pharmaceutically acceptable carrier, adjuvant, auxiliary agent, solvent, dilute
Release agent, excipient, synergist.The novel autophagy derivant or medicine being made can for solution, tablet, capsule, powder, particle, ointment,
The formulation of any one in suppository, injection, inhalant, gel, microsphere, aerosol.In the condition of its therapeutically effective amount
Under, can be to embryonic stem cell, induced multi-potent stem cell, adult stem cell, noble cells, haemocyte, hematopoietic cell, blood vessel endothelium
Cell, epithelial cell, exocrine cell, endocrine cell, phoirocyte, mucomembranous cell, immunocyte, fat cell,
Osteocyte, smooth muscle cell, striated muscle cell, nerve cell, sensory cell, visual cell, cardiac muscle cell, liver cell, respiratory tract
Cell, esophageal cells, gastric cells, enterocyte, splenocyte, pneumonocyte, nephrocyte, reproduction cell；Or from cardiac tumor, liver
Cancer, splenic tumor, nasopharyngeal carcinoma, the cancer of the esophagus, stomach cancer, bone tumour, myomata, leukaemia, lymthoma, lung cancer, kidney, colon cancer, the cancer of the brain,
It is any one in melanoma, breast cancer, oophoroma, cervix cancer, prostate cancer, cancer of pancreas, epithelioma, nervous system neoplasm
The tumor cell induction autophagy planted.The cell autophagy derivant or treatment that are made can benefit from the medicine of the disease of autophagy induction
Under conditions of its therapeutically effective amount, with it is oral, through oesophagus, through stomach, through small intestine, per rectum, through eye, intranasal, transtracheal, warp
Ear, through it is sublingual, through skin, transmucosal, through it is subcutaneous, through muscle, intravascular, through abdominal cavity, Via vagina, through intrathecal, through ossis
Administering mode casts animal in need or subject to improve or increase the autophagy level in these animals or subject,
The disease or situation that can benefit from autophagy induction are treated or improved whereby.
Compared with prior art, the beneficial effect of technical scheme is：Chemical combination described in technical scheme
Thing is widely used in the synthesis of medicine, agricultural chemicals, spices, industrial products, but does not have cell autophagy inducing action on it
Report, particularly cetylpyridinium chloride (CPC) is clinically used only as Cidex-7 at present.And present techniques
Compound described in scheme is used as novel autophagy derivant or treats the active ingredient of the medicine of the disease that can benefit from autophagy induction
When, its working concentration is low, and specificity is good.
Brief description of the drawings
Fig. 1：The mechanism of action figure of CPC Induces Autophagies；
Fig. 2：Without cellular control unit (left side), the CPC treatment groups of cetylpyridinium chloride (CPC) under fluorescence microscope
Cell (in) and the cell (right, as positive control) of known novel autophagy derivant Torin1 treatment in autophagosome mark LC3
The distribution situation (fluorescence represents LC3 albumen, and DIC represents that Nomarski differs picture) of albumen；
Fig. 3：Without the cellular control unit (left side) and the sub- micro- knot of CPC treatment groups cell (right side) of CPC under transmission electron microscope
Structure and autophagosome (autophagosome) quantity (black arrow represents autophagosome in cell)；
Fig. 4：CPC treatment is to cell autophagy and mTORC1 pathway associated proteins changes of contents and phosphorylation
Western Blotting results；
Fig. 5：Cellular control unit (left side) under fluorescence microscope, CPB treatment groups cell (in) or CmThe cell (right side) of PC treatment
The distribution situation of middle autophagosome mark LC3 albumen；
Fig. 6：CPB or CmPC treatment is to cell autophagy and mTORC1 pathway associated proteins changes of contents and phosphorylation
Western Blotting results.
Specific embodiment
In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment and
The present invention is described in further detail for accompanying drawing.
Used experiment material and method explanation in following examples：
LC3 albumen, i.e. (the microtubule-associated protein1light of 1 light chain of MAP 3
Chain3, LC3) (mammalian homologs of yeast Atg8) be a kind of soluble protein, it is after composition (proLC3)
Immediately by cysteine proteinase Atg4B treatment, C-terminal glycine residue (LC3-I) is exposed.During autophagy, autophagosome
Phagocyte matter component, including cytoplasmic protein and organelle.Meanwhile, the LC3 (LC3-I) of cytoplasmatic forms is attached to phosphatidyl ethanol
Amine (PE), forms LC3-PE conjugates (LC3-II), and LC3-II is raised to autophagy film (autophagosomal
membranes：Including phagophore and autophagosome) on, therefore the level of LC3-II reflects to a certain extent
The quantity of autophagosome.Finally, under the effect again of Atg4B, LC3 is from LC3-PE conjugates from discharging, and autophagy lyase
LC3-II in internal portion and inner membrance is then hydrolyzed enzyme and is degraded.So, LC3 is widely used as the mark of autophagy film
(autophagosomal marker)。
P62, also referred to as slider -1 (sequestosome 1, SQSTM1) are autophagy selective substrates, are forming cell
The effect of key is played during matter inclusion bodies of protein, is to include Alzheimer disease (Alzheimer ' s disease), handkerchief
The gloomy disease (Parkinson ' s disease) of gold, amyotrophic lateral sclerosis (ALS) etc. are in interior various nerve degenerative diseases
Mark.P62 also has been identified as LC3 interaction proteins (LC3-interacting protein), in selectivity certainly
Played a significant role in biting.As seen in Figure 1, LC3 is attached to p62 and by its (and any ubiquitin protein for being attached to it
Matter or cellular component) it is transported in autophagosome, p62 finally degrades in autophagy lysosome.Because p62 is dropped by autophagy process in itself
Solution, so p62 is also widely used as the mark of autophagy activity.
Cell autophagy detection method of the present invention is according to " Guidelines for the use and
interpretation of assays for monitoring autophagy(3rd edition).Autophagy
2016,12(1):1-222 " (guide of cell autophagy detection method).
Unless stated otherwise, experiment material, reagent, instrument, the apparatus and method that the present invention is used are for the art is normal
Rule experiment material purchased in market, reagent, instrument, equipment and conventional use of method.
Embodiment 1
CPC is detected to cell autophagy by the distribution in fluorescence microscopy Microscopic observation LC3 albumen (autophagosome mark)
Inducing action
1st, major experimental material and reagent：Cetylpyridinium chloride (cetylpyridinium chloride, CPC, purchase
From Sigma-Aldrich), wherein cetylpyridinium chloride (CPC) structural formula such as formula II (in formula I, n=0, m=15, X-
It is chlorion)：
Above-mentioned CPC ultra-pure waters are configured to 10mM concentrates, -20 DEG C of preservations, cell culture fluid when using after packing
Concentration needed for 0.1 μM, 1 μM and 10 μM grade is diluted to respectively.mTOR(mechanistic Target Of RApamycin) suppress
Agent Torin1 (being purchased from Tocris Bioscience) is configured to 1mM concentrates with dimethyl sulfoxide (DMSO) (DMSO), -20 DEG C after packing
Preserve, 250nM or other required concentration are diluted to cell culture fluid when using.Human cervical carcinoma cell strain (HeLa), people's navel
Venous endothelial cell (HUVEC), MEC (MEF) are purchased from Unite States Standard biology product collecting center (ATCC),
Routine preservation.HeLa and MEF cells are with containing 10% hyclone, 100U/ml penicillin, 100 μ g/ml streptomysins
Dulbecco ' s modified Eagle ' s medium (DMEM) complete medium is in 5%CO2, cultivate under the conditions of 37 DEG C.
Condition of culture culture of the HUVEC cells according to explanation and the recommendation of ATCC.Anti- LC3 mouse monoclonal antibodies are purchased from CosmoBio
Co.(clone 1703)。
2nd, experimental technique：By cell dissociation, it is centrifuged and is resuspended in corresponding nutrient solution, is planted in glimmering with suitable density
On the special cover glass of light microscope (coverslips), 37 DEG C, 5%CO are placed in2After cultivating 24h in incubator, former training is removed
Nutrient solution, adds Fresh, and containing 0.1 μM, the 1 μM and 10 μM nutrient solution of CPC or 250nM Torin1 continues to cultivate
2h.Then by with cell cover glass with phosphate buffer (PBS) rinse, then with 4% paraformaldehyde at room temperature consolidate
It is fixed 10 minutes, with 50 μ g/ml digitonin (being purchased from Sigma-Aldrich) saturatingization after PBS flushings
(permeabilization) 5 minutes.Then closed 30 minutes at room temperature with 3% bovine serum albumin(BSA) (BSA), then with 1:
The anti-LC3 antibody incubations 1h of 400 dilutions.PBS is incubated 1h after rinsing with the secondary antibody of fluorescence labeling, is finally seen under fluorescence microscope
Examine the intensity and distribution situation of fluorescence.
3rd, experimental result：As shown in Fig. 2 in the control group MEF cells without addition CPC, LC3 fluorescence is uniform, disperse
Be distributed in cytoplasm；And in 1 μM of CPC treatment group cell, the LC3 fluorescent grains (LC3puncta) of state of aggregation have substantially
Increase trend, 1 μm or so of diameter, fluorescence intensity also substantially increases, and the autophagosome number for pointing out LC3-II to represent substantially increases,
That is CPC being capable of effective Induces Autophagy.It is (right as the positive herein in known novel autophagy derivant Torin1 treatment groups cell
According to) in, also obtain the result similar with CPC treatment groups.
Embodiment 2
Inductions of the CPC to cell autophagy is detected by the observation of cell ultra microstructure (including autophagosome) under transmission electron microscope
Effect
1st, experimental technique：By cell dissociation, it is centrifuged and is resuspended in corresponding nutrient solution, is planted in electricity with suitable density
In the treated plastic coverslip (collagen-coated plastic coverslips) of the special collagen of mirror, it is placed in
37 DEG C, 5%CO2After cultivating 24h in incubator, original fluid is removed, add Fresh, contain 0.1 μM, 1 μM and 10 μM
The nutrient solution of CPC or 250nM Torin1 continues to cultivate 2h.Then by the cover glass with cell with 0.1M PBS (pH7.4)
After rinsing 2 times, 2.5% glutaraldehyde is added to fix 2h.Glutaraldehyde is blotted, after rinsing 2 times using 0.1M PBS, with 1.5% 4 oxygen
2h is fixed after changing osmium.Subsequent dewatering, Epon812 epoxy resin embeddings.Finally repair block and ultra-thin section.Gained 4% acetic acid of section
Uranium is dyeed 20 minutes, and lead citrate is dyeed 5 minutes.Hitachi H-7100 transmission electron microscope observings.
2nd, experimental result：As shown in figure 3, by transmission electron microscope observing MEF cell ultrastructures, finding through 1 μM of CPC treatment
The autophagosome (autophagosome, at black arrow meaning) of a large amount of double membrane structures of intracellular appearance afterwards, diameter about 200-
800nm, autophagosome includes the organelles such as mitochondrial, endoplasmic reticulum and other cellular contents, points out autophagosome after CPC treatment
Number substantially increases, i.e. CPC being capable of effective Induces Autophagy.
Embodiment 3
CPC is monitored by Western Blotting to process to cell autophagy and mTORC1 pathway associated protein changes of contents
Inducing actions of the CPC to cell autophagy is detected with phosphorylation
1st, major experimental material and reagent：Anti- LC3 rabbit polyclonal antibodies are purchased from MBL (catalog number:PM036)；
Anti- p62 rabbit polyclonal antibodies are purchased from MBL (catalog number:PM045)；Anti- phospho-p70S6Kinase (Thr389)
Rabbit polyclonal antibody is purchased from Cell Signaling Technology (catalog number:9234)；Anti- p70S6Kinase
Rabbit polyclonal antibody is purchased from Cell Signaling Technology (catalog number:9202)；Many grams of anti-4E-BP1 rabbits
Grand antibody is purchased from Cell Signaling Technology (catalog number:9452)；Anti- β-actin mouse monoclonals
Antibody is purchased from Sigma-Aldrich (catalog number:A2228).
2nd, experimental technique：By cell dissociation, it is centrifuged and is resuspended in corresponding nutrient solution, is planted in thin with suitable density
In born of the same parents' culture dish, 37 DEG C, 5%CO are placed in2After cultivating 24h in incubator, original fluid is removed, add Fresh, contained
0.1 μM, 1 μM and 10 μM CPC or removal amino acid nutrient solution (amino acid-free starvation medium,
Classical novel autophagy derivant, as positive control) continue to cultivate 2h.Cell is then collected, total protein of cell, Western is extracted
Blotting methods detect cell autophagy (LC3 and p62) and mTORC1 pathway associated proteins (phospho-p70S6Kinase,
P70S6Kinase and 4E-BP1) changes of contents and phosphorylation.
3rd, experimental result：As shown in figure 4, compare with control group HeLa cells (first row) without addition CPC, at CPC
P62 (selective substrate of autophagy, itself is degraded by autophagy) content is substantially reduced in reason group cell (secondary series), cytoplasmatic forms
LC3 (LC3-I) content reduction, LC3-PE conjugates (LC3-II, it is recruited to autophagy film) content raise, point out CPC
Treatment being capable of effective Induces Autophagy.MTORC1 is mainly opened as the central regulator point of autophagy by suppressing the key of autophagy
The sub- ULK1 compounds of reason are active (Fig. 1) to adjust autophagy.P70S6Kinase and 4E-BP1 (eukaryotic
Initiation factor 4E-binding protein1) as two classical substrates of mTORC1, respectively by mTORC1 phosphorus
Acidifying regulation and control (Fig. 1).Therefore, p70S6Kinase Thr389 sites and 4E-BP1 phosphorylation states are usually utilized to reflection
The activity of mTORC1.As shown in figure 4, compare with the cellular control unit (first row) without addition CPC, CPC treatment groups cell (the
Two row) in p70S6Kinase Thr389 sites and 4E-BP1 phosphorylations be substantially suppressed, point out the CPC treatment can be obvious
Suppress mTORC1 activity.In amino acid starvation treatment group cell, (the 3rd row, classical novel autophagy derivant is right as the positive herein
According to) in, also obtain the result similar with CPC treatment groups.
In sum, CPC mainly induces autophagy by suppressing mTORC1 activity.
Experimental result can draw in integrated embodiment 1-3, and CPC can be thin effectively to induce by suppressing mTORC1 activity
Born of the same parents' autophagy, based on this, can be by CPC or CPC combinations pharmaceutically acceptable carrier, adjuvant, auxiliary agent, solvent, diluent, excipient
Or synergist is made pharmaceutically acceptable solution, tablet, capsule, powder, particle, ointment, suppository, injection, inhalant, solidifying
Glue, microsphere or aerosol, by oral administration, through oesophagus, through stomach, through small intestine, per rectum, through eye, intranasal, transtracheal, through ear, through tongue
Under, through skin, transmucosal, through it is subcutaneous, be administered to through muscle, intravascular, through abdominal cavity, Via vagina, through intrathecal or through ossis
The animal of needs or patient, can reach the effect of Induces Autophagy.Based on this, treatment can benefit from the disease of autophagy induction
Disease, such as nerve degenerative diseases, α1-antitrypsin deficiency, angiocardiopathy, IBD, Crohn disease, muscle disease
Disease, osteoarthritis, infectious diseases, metabolic disease, cancer, aging.When the compound is as cell autophagy derivant
Cell can be embryonic stem cell, induced multi-potent stem cell, adult stem cell, noble cells, haemocyte, hematopoietic cell, blood vessel
Endothelial cell, epithelial cell, exocrine cell, endocrine cell, phoirocyte, mucomembranous cell, immunocyte, fat are thin
Born of the same parents, osteocyte, smooth muscle cell, striated muscle cell, nerve cell, sensory cell, visual cell, cardiac muscle cell, liver cell, breathing
Any one cell in road cell, esophageal cells, gastric cells, enterocyte, splenocyte, pneumonocyte, nephrocyte, reproduction cell；
Or from cardiac tumor, liver cancer, splenic tumor, nasopharyngeal carcinoma, the cancer of the esophagus, stomach cancer, bone tumour, myomata, leukaemia, lymthoma, lung
Cancer, kidney, colon cancer, the cancer of the brain, melanoma, breast cancer, oophoroma, cervix cancer, prostate cancer, cancer of pancreas, epithelioma, god
Through the tumour cell of any one in system tumor.
Embodiment 4
Hexadecyl pyrrole is detected by the distribution in fluorescence microscopy Microscopic observation LC3 albumen (autophagosome mark)
Pyridine (CPB) and chlorination 1- cetyl -4- picolines (CmPC) to the inducing action of cell autophagy
1st, major experimental material and reagent：Brocide (cetylpyridinium bromide, CPB, purchase
From Sigma-Aldrich) and chlorination 1- cetyl -4- picolines (1-hexadecyl-4-methylpyridinium
chloride,CmPC, purchased from Santa Cruz Biotechnology), the wherein structural formula of brocide (CPB)
For formula III (in formula I, n=0, m=15, X-It is bromide ion), chlorination 1- cetyl -4- picolines (CmPC structural formula) is
Formula IV (in formula I, n=1, m=15, substituent R1Positioned at the contraposition of N atoms, substituent R1It is methyl, X-It is chlorion)：
By above-mentioned CPB and CmPC is configured to 10mM concentrates with ultra-pure water respectively, and -20 DEG C of preservations, use when using after packing
Cell culture fluid is diluted to concentration needed for 0.1 μM, 1 μM and 10 μM grade respectively.Human cervical carcinoma cell strain (HeLa), human umbilical vein
Endothelial cell (HUVEC), MEC (MEF) are purchased from Unite States Standard biology product collecting center (ATCC), conventional
Preserve.HeLa and MEF cells are with containing 10% hyclone, 100U/ml penicillin, the Dulbecco ' s of 100 μ g/ml streptomysins
Modified Eagle ' s medium (DMEM) complete medium is in 5%CO2, cultivate under the conditions of 37 DEG C.HUVEC cells according to
The condition of culture culture of explanation and the recommendation of ATCC.Anti- LC3 mouse monoclonal antibodies are purchased from Cosmo Bio Co.
(clone1703)。
2nd, experimental technique：By cell dissociation, it is centrifuged and is resuspended in corresponding nutrient solution, is planted in glimmering with suitable density
On the special cover glass of light microscope (coverslips), 37 DEG C, 5%CO are placed in2After cultivating 24h in incubator, former training is removed
Nutrient solution, adds Fresh, contains 0.1 μM, 1 μM and 10 μM CPB or CmThe nutrient solution of PC continues to cultivate 2h.Then will
Cover glass with cell is rinsed with phosphate buffer (PBS), then fixes 10 minutes at room temperature with 4% paraformaldehyde,
PBS rinse after with 50 μ g/ml digitonin (purchased from Sigma-Aldrich) saturatingization (permeabilization) 5 minutes.With
Closed 30 minutes at room temperature with 3% bovine serum albumin(BSA) (BSA) afterwards, then with 1:The anti-LC3 antibody incubations 1h of 400 dilutions.
PBS is incubated 1h after rinsing with the secondary antibody of fluorescence labeling, finally in the intensity and distribution situation of fluorescence microscopy Microscopic observation fluorescence.
3rd, experimental result：As shown in figure 5, in control group MEF cells (Fig. 5 is left), LC3 fluorescence is uniform, the distribution of disperse ground
In cytoplasm；And in 1 μM of CPB (in Fig. 5) and CmIn PC treatment groups cell (Fig. 5 is right), the LC3 fluorescent grains of state of aggregation
(LC3puncta) have and significantly increase trend, 1 μm or so of diameter, fluorescence intensity also substantially increases, and points out LC3-II representatives oneself
Biting body number substantially increases, i.e. CPB and CmPC being capable of effective Induces Autophagy.
Embodiment 5
Brocide (CPB) and chlorination 1- cetyl -4- methyl are monitored by Western Blotting
Pyridine (CmPC) treatment detects it to thin to cell autophagy and mTORC1 pathway associated proteins changes of contents and phosphorylation
The inducing action of born of the same parents' autophagy
1st, major experimental material and reagent：Anti- LC3 rabbit polyclonal antibodies are purchased from MBL (catalog number:PM036)；
Anti- p62 rabbit polyclonal antibodies are purchased from MBL (catalog number:PM045)；Anti- phospho-p70S6Kinase (Thr389)
Rabbit polyclonal antibody is purchased from Cell Signaling Technology (catalog number:9234)；Anti- p70S6Kinase
Rabbit polyclonal antibody is purchased from Cell Signaling Technology (catalog number:9202)；Many grams of anti-4E-BP1 rabbits
Grand antibody is purchased from Cell Signaling Technology (catalog number:9452)；Anti- β-actin mouse monoclonals
Antibody is purchased from Sigma-Aldrich (catalog number:A2228).
2nd, experimental technique：By cell dissociation, it is centrifuged and is resuspended in corresponding nutrient solution, is planted in thin with suitable density
In born of the same parents' culture dish, 37 DEG C, 5%CO are placed in2After cultivating 24h in incubator, original fluid is removed, add Fresh, contained
0.1 μM, 1 μM and 10 μM CPB or CmThe nutrient solution of PC continues to cultivate 2h.Cell is then collected, total protein of cell is extracted,
Western Blotting methods detect cell autophagy (LC3 and p62) and mTORC1 pathway associated proteins (phospho-
P70S6Kinase, p70S6Kinase and 4E-BP1) changes of contents and phosphorylation.
3rd, experimental result：As shown in fig. 6, comparing with control group HeLa cells (first row), CPB treatment groups cell (second
Row) and CmP62 (selective substrate of autophagy, itself is degraded by autophagy) content substantially drops in PC treatment groups cell (the 3rd row)
It is low, LC3 (LC3-I) content reduction of cytoplasmatic forms, LC3-PE conjugates (LC3-II, it is recruited to autophagy film) content liter
Height, points out CPB or CmPC treatment being capable of effective Induces Autophagy.MTORC1 mainly leads to as the central regulator point of autophagy
The crucial startup factor ULK1 compounds of suppression autophagy are crossed to adjust autophagy activity (Fig. 1).P70S6Kinase and 4E-BP1
(eukaryotic initiation factor 4E-binding protein 1) as two of mTORC1 classical substrates,
Respectively by mTORC1 phosphorylations (Fig. 1).Therefore, p70S6Kinase Thr389 sites and 4E-BP1 phosphorylation states are passed through
Often it is used to reflect the activity of mTORC1.As shown in fig. 6, compare with cellular control unit (first row), CPB treatment groups cell (the
Two row) and CmP70S6Kinase Thr389 sites and 4E-BP1 phosphorylations are substantially subject in PC treatment groups cell (the 3rd row)
Suppress, point out CPB or CmPC treatment can substantially suppress mTORC1 activity.
In sum, CPB and CmPC also mainly induces autophagy by suppressing mTORC1 activity.
Experimental result can draw in integrated embodiment 4 and 5, CPB and CmPC can have by suppressing mTORC1 activity
Effect Induces Autophagy, based on this, can be by CPB, CmIt is PC and/or CPC combinations pharmaceutically acceptable carrier, adjuvant, auxiliary agent, molten
Agent, diluent, excipient or synergist be made pharmaceutically acceptable solution, tablet, capsule, powder, particle, ointment, suppository,
Injection, inhalant, gel, microsphere or aerosol, by oral administration, through oesophagus, through stomach, through small intestine, per rectum, through eye, intranasal,
Transtracheal, through ear, through it is sublingual, through skin, transmucosal, through it is subcutaneous, through muscle, intravascular, through abdominal cavity, Via vagina, through intrathecal or
Animal in need or patient are administered to through ossis, the effect of Induces Autophagy can be reached.Based on this, treatment can be received
Beneficial to the disease of autophagy induction, such as nerve degenerative diseases, α1-antitrypsin deficiency, angiocardiopathy, inflammatory bowel
Disease, Crohn disease, muscle disease, osteoarthritis, infectious diseases, metabolic disease, cancer, aging.The compound is used as
Cell during cell autophagy derivant can be embryonic stem cell, induced multi-potent stem cell, adult stem cell, noble cells, blood
Cell, hematopoietic cell, vascular endothelial cell, epithelial cell, exocrine cell, endocrine cell, phoirocyte, mucous membrane are thin
Born of the same parents, immunocyte, fat cell, osteocyte, smooth muscle cell, striated muscle cell, nerve cell, sensory cell, visual cell, the heart
Myocyte, liver cell, respiratory tract cell, esophageal cells, gastric cells, enterocyte, splenocyte, pneumonocyte, nephrocyte, reproduction cell
In any one cell；Or from cardiac tumor, liver cancer, splenic tumor, nasopharyngeal carcinoma, the cancer of the esophagus, stomach cancer, bone tumour, myomata,
Leukaemia, lymthoma, lung cancer, kidney, colon cancer, the cancer of the brain, melanoma, breast cancer, oophoroma, cervix cancer, prostate cancer,
The tumour cell of any one in cancer of pancreas, epithelioma, nervous system neoplasm.
The above is only the preferred embodiment of the present invention, it is noted that it is right that above-mentioned preferred embodiment is not construed as
Limitation of the invention, protection scope of the present invention should be defined by claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these change
Enter and retouch and also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of application of alkyl pyridine class compound, it is characterised in that：The structural formula such as formula of the alkyl pyridine class compound
Ⅰ：
In formula, the arbitrary integer in n=0-4, the arbitrary integer in m=12-16, substituent R1Positioned at the ortho position of N atoms, meta
Or at least one in contraposition, substituent R1Selected from hydrogen, hydroxyl, methyl, ethyl, methoxyl group, amino, nitro, sulfonic group, halogen
In at least one, X-Selected from any one in halide ion；
The alkyl pyridine class compound is used to prepare cell autophagy derivant or prepare treatment that autophagy induction can be benefited from
The medicine of disease.
2. application according to claim 1, it is characterised in that：The alkyl pyridine class compound is by suppressing mTORC1
Activity carry out Induces Autophagy.
3. application according to claim 1, it is characterised in that：In formula I, the arbitrary integer in n=0-3, in m=14-16
Arbitrary integer, substituent R1Positioned at the meta or para position of N atoms, substituent R1Selected from hydrogen, hydroxyl, methyl, amino, sulfonic group
In at least one, X-Selected from chlorion or bromide ion.
4. application according to claim 3, it is characterised in that：In formula I, n=0, m=15, X-It is chlorion.
5. application according to claim 1, it is characterised in that：Described cell is embryonic stem cell, induced multi-potent is dry thin
Born of the same parents, adult stem cell, noble cells, haemocyte, hematopoietic cell, vascular endothelial cell, epithelial cell, exocrine cell, interior point
Secrete cell, phoirocyte, mucomembranous cell, immunocyte, fat cell, osteocyte, smooth muscle cell, striated muscle cell,
Nerve cell, sensory cell, visual cell, cardiac muscle cell, liver cell, respiratory tract cell, esophageal cells, gastric cells, enterocyte, spleen
Any one cell in cell, pneumonocyte, nephrocyte, reproduction cell；Or from cardiac tumor, liver cancer, splenic tumor, nasopharynx
Cancer, the cancer of the esophagus, stomach cancer, bone tumour, myomata, leukaemia, lymthoma, lung cancer, kidney, colon cancer, the cancer of the brain, melanoma, mammary gland
The tumour cell of any one in cancer, oophoroma, cervix cancer, prostate cancer, cancer of pancreas, epithelioma, nervous system neoplasm.
6. application according to claim 1, it is characterised in that:The novel autophagy derivant or treatment can benefit from autophagy and lure
The medicine of the disease led also includes at least one selected from pharmaceutically acceptable carrier, adjuvant, auxiliary agent, solvent, diluent, figuration
Agent, synergist.
7. application according to claim 1, it is characterised in that:The novel autophagy derivant or treatment can benefit from autophagy and lure
The formulation of the medicine of the disease led be solution, tablet, capsule, powder, particle, ointment, suppository, injection, inhalant, gel,
Any one in microsphere, aerosol.
8. application according to claim 1, it is characterised in that:The described disease that can benefit from autophagy induction includes god
Closed through DD, α1-antitrypsin deficiency, angiocardiopathy, IBD, Crohn disease, muscle disease, bone
Section inflammation, infectious diseases, metabolic disease, cancer, aging.
9. application according to claim 1, it is characterised in that：The novel autophagy derivant or treatment can benefit from autophagy and lure
The administering mode of the medicine of the disease led is orally, through oesophagus, through stomach, through small intestine, per rectum, through eye, intranasal, transtracheal, warp
Ear, through it is sublingual, through skin, transmucosal, through it is subcutaneous, give through muscle, intravascular, through abdominal cavity, Via vagina, through intrathecal, through ossis
Any one in medicine.
10. a kind of method of Induces Autophagy, it is characterised in that：The formula I of effective dose is cast to animal in need or patient
The compound, the effective dose for casting is 0.001-500mg/kg body weight.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628451A (en) * | 2019-01-10 | 2019-04-16 | 广西大学 | It is a kind of inhibit rabbit Deptor gene expression shRNA, Lentiviral and its construction method and application |
| CN112166977A (en) * | 2019-11-08 | 2021-01-05 | 山东省农业科学院作物研究所 | Method for creating physiological phenotype of drought and premature senility of wheat seedling by using cell autophagy inhibitor |
| CN113924120A (en) * | 2019-06-20 | 2022-01-11 | 雀巢产品有限公司 | Compositions and methods for enhancing the musculoskeletal effects of one or more anabolic amino acids |
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| WO2016201323A1 (en) * | 2015-06-10 | 2016-12-15 | Board Of Regents, The University Of Texas System | Use of exosomes for the treatment of disease |
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2016
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| WO2016201323A1 (en) * | 2015-06-10 | 2016-12-15 | Board Of Regents, The University Of Texas System | Use of exosomes for the treatment of disease |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628451A (en) * | 2019-01-10 | 2019-04-16 | 广西大学 | It is a kind of inhibit rabbit Deptor gene expression shRNA, Lentiviral and its construction method and application |
| CN113924120A (en) * | 2019-06-20 | 2022-01-11 | 雀巢产品有限公司 | Compositions and methods for enhancing the musculoskeletal effects of one or more anabolic amino acids |
| CN112166977A (en) * | 2019-11-08 | 2021-01-05 | 山东省农业科学院作物研究所 | Method for creating physiological phenotype of drought and premature senility of wheat seedling by using cell autophagy inhibitor |
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