CN105534977A - Application of T-type calcium ion channel inhibitor NNC55-0396 to preparation of drug resisting neurodegenerative disease - Google Patents
Application of T-type calcium ion channel inhibitor NNC55-0396 to preparation of drug resisting neurodegenerative disease Download PDFInfo
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- CN105534977A CN105534977A CN201610024789.3A CN201610024789A CN105534977A CN 105534977 A CN105534977 A CN 105534977A CN 201610024789 A CN201610024789 A CN 201610024789A CN 105534977 A CN105534977 A CN 105534977A
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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Abstract
The invention discloses application of a T-type calcium ion channel inhibitor NNC55-0396 to preparation of a drug resisting a neurodegenerative disease. Experiments prove that the compound NNC55-0396 has an obvious effect on enhancing autophagy to relieve cognition impairment, and it is determined that the concentration of NNC55-0396 for effective induction and initiation of autophagy is 5 micron M. It is indicated that the compound is expected to become a potential drug for selective induction of autophagy and effective treatment of the Alzheimer's disease, and the application provides a theoretical foundation for research and control of early intervention of autophagy in the neurodegenerative disease.
Description
Technical field
The present invention relates to the application of a kind of T-shaped calcium channel inhibitor NNC55-0396 (CAS:357400-13-6) in the anti-neurodegenerative diseases medicine of preparation; Belong to biomedical sector.
Background technology
Cell autophagy is a kind of important channel for removing harmful macromolecular substances such as parafunctional organelle, the protein of false folding, oxidized lipid in organism.Its mechanism is from unicellular lower eukaryote yeast to high mammal all high conservatives, most important to the normal vital movement of maintenance.Cell autophagy comprises three kinds of modes, i.e. the autophagy (chaperone-mediatedautophagy) of huge autophagy (macroautophagy), micro-autophagy (microautophagy) and chaperone mediation.Due to the main path that large autophagy is autophagy, thus described herein and autophagy all only refer to large autophagy.
If the protein of false folding can not be removed efficiently, will cause and gather, cause neurocyte afunction and even death, this is the main cause that neurodegenerative diseases comprises Alzheimer's disease (Alzheimer'sdisease, AD).The principal character of Alzheimer's disease (AD) is gathering of amyloid-beta (A β).In cell A β generation and remove the morbidity of Alzheimer's disease is seemed very important.In Alzheimer's disease, cell autophagy sustains damage, and core endoplasm lysosomal pathway occurs abnormal.And pharmacology's activation of autophagy can promote the removing of A β, and then alleviate AD symptom, therefore the research regulation and control early intervention of autophagy to Alzheimer's disease significant (Liu Zhixue etc., 2012).
Compound N NC55-0396 (CAS:357400-13-6) is the derivant of medicine Mibefradil, it is a kind of T-shaped calcium channel inhibitor of high selectivity, be widely used in the research such as tumor, diabetes, also studies have found that it is relevant to embryonic development.But NNC55-0396 is for Induces Autophagy, for the preparation of the research of anti-neurodegenerative diseases medicine aspect, through authoritative institution's Access point, there is not been reported both at home and abroad at present.Given this, research compound N NC55-0396 to Induces Autophagy, improve cognitive dysfunction, the application prepared in anti-neurodegenerative diseases medicine is significant.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is to provide the application of a kind of T-shaped calcium channel inhibitor NNC55-0396 (CAS:357400-13-6) in the anti-neurodegenerative diseases medicine of preparation.
The application of T-shaped calcium channel inhibitor NNC55-0396 of the present invention in the anti-neurodegenerative diseases medicine of preparation.
Wherein: the chemical constitution of described T-shaped calcium channel inhibitor NNC55-0396 is as shown in general formula (I), its chemical name is: (1s, 2s)-2-(2-(N-[3-(2-1H-benzimidazolyl) propyl group]-N-methylamino) ethyl)-6-fluoro-1,2,3,4-tetrahydrochysene-1-isopropyl-beta naphthal-ethylene-acetic acid ester; Described neurodegenerative disease is preferably alzheimer disease.
The present invention uses wild type neurogliocyte U87MG cell and LC3 by the neurogliocyte U87MG cell of green fluorescent protein (GFP) transfection (being called for short LC3-GFP-U87 cell), research in the diseases such as the cognitive dysfunction affecting cell autophagy and association thereof has been carried out on T-shaped calcium channel inhibitor NNC55-0396, confirm: described T-shaped calcium channel inhibitor NNC55-0396 improves in cognitive dysfunction have obvious effect at enhancing cell autophagy, and determine that described T-shaped calcium channel inhibitor NNC55-0396 can effectively induce the concentration of trigger cell autophagy to be 5 μMs.
The effect of essence for a better understanding of the present invention and compound of the present invention, below in conjunction with pharmacological evaluation and the result of NNC55-0396, sets forth it further and is strengthening the effect in cell autophagy.
The preparation of neuroglial cytoma: cultivation wild type neurogliocyte U87MG cell and LC3 are by the neurogliocyte U87MG cell of green fluorescent protein (GFP) transfection (being called for short LC3-GFP-U87 cell) in conventional manner, collect growth conditions good and be in the cell of logarithmic (log) phase, for subsequent use.
Celluar and Molecular Biology method is adopted to test as follows, to observe the impact of NNC55-0396 on LC3-GFP-U87 cell and wild type neurogliocyte U87MG autophagy.
1. green fluorescent protein-autophagy process feature Protein G FP-LC3 method detects cell growth status, to judge the impact of NNC55-0396 on cell autophagy
The LC3-GFP-U87 cell being in exponential phase is inoculated in 96 orifice plates, after the NNC55-0396 of 5 μMs act on 24 hours, the change of LC3 albumen is observed with fluorescence inverted microscope, found that experimental group comparatively matched group there is a large amount of fluorescence speckle, illustrate that cell produces autophagosome, LC3 is gathered in autophagosome, forms fluorescence speckle, demonstrates the generation (see Fig. 1) of autophagy.
2. western blotting (WesternBlot) is analyzed three kinds of concentration and is respectively 1 μM, and the NNC55-0396 of 5 μMs and 10 μMs acts on the expressing quantity after U87MG cell 24 hours (rapamycin is as positive control)
Expression according to protein level changes, find that the NNC55-0396 of 5 μMs and 10 μMs two kinds of concentration doses has significantly raised the expression of LC3 II, confirm the generation of autophagy, and determine that NNC55-0396 can effectively induce the concentration of trigger cell autophagy to be 5 μMs (see Fig. 2).
Above-mentioned experimental data represents with mean+/-standard error, and through t inspection, P<0.05, indicates explicitly difference.
By the above-mentioned experimental data through statistical procedures and result thereof, can obtain drawing a conclusion:
Compound N NC55-0396 of the present invention can significantly induce trigger cell autophagy, experimental result confirms that T-shaped calcium channel inhibitor NNC55-0396 is expected in the anti-neurodegenerative disease medicine of preparation, have application, for novel anti-neurodegenerative disease drug development provides basis.Indication NNC55-0396 is expected to the potential medicine becoming selective induction cell autophagy and effectively treat alzheimer disease, has wide scientific research and clinical development prospect.
Accompanying drawing explanation
The NNC55-0396 solution-treated LC3-GFP-U87 cell 24 hours induced cellular generation autophagy photo of Fig. 1: 5 μMs.
Wherein: A figure is the matched group of solvent process, B figure is the experimental group of the NNC55-0396 solution-treated of 5 μMs.
Fig. 2: 1 μM, the NNC55-0396 of 5 μMs and 10 μMs processes U87MG cell 24 hours respectively, detection display compound concentration be 5 μMs and 10 μMs time, the up-regulated of LC3 II in cell, cell generation autophagy.
Detailed description of the invention
Compound N NC55-0396 of the present invention is purchased from SigmaAldrich.U87MG cell and LC3-GFP-U87 cell be biochemical and cell institute cell bank purchased from Chinese Academy of Sciences Shanghai.
Embodiment 1
The quantitative experiment of autophagosome
After autophagy reporter cell LC3-GFP-U87 cell dissociation is counted, get 1mL density be the LC3-GFP-U87 cell suspension inoculation of 50,000/mL to (the straight 0.2cm of slide) in the burnt culture dish of copolymerization at the bottom of diameter 1cm glass, at 37 DEG C containing 5%CO
2condition under cultivate after 24 hours, add 1mL concentration be the NNC55-0396 solution of 5 μMs at 37 DEG C containing 5%CO
2condition under continue cultivate.Process after 24 hours, abandon culture fluid, add 2% paraformaldehyde and cell is fixed.Then use fluorescence inverted microscope, excite with 488nm, 535nm transmission channel, optimum signal pattern is carried out Fluirescence observation to cell and is taken pictures.
Count the bright punctate fluorescence (autophagosome) in cell in random field, the ability of with the average punctate fluorescence number in individual cells NNC55-0396 being brought out autophagy quantizes.Each sample carries out 3 secondary pollutants to be repeated.
Result: after NNC55-0396 acts on 24 hours, the change of LC3 albumen is observed with fluorescence inverted microscope, find that experimental group occurs a large amount of fluorescence speckle (see Fig. 1) compared with the cell of matched group, illustrate that cell produces autophagosome, LC3 is gathered in autophagosome, form fluorescence speckle, demonstrate the generation of autophagy.
Embodiment 2
Immunoblot experiment
Be 1 × 10 by 3mL density
5the U87MG cell of/mL is seeded in the Tissue Culture Dish of diameter 60mm.At 37 DEG C containing 5%CO
2condition under cultivate 24h after, this cell is respectively 1 μM with concentration respectively, NNC55-0396, rapamycin (positive control), the DMSO of 5 μMs and 10 μMs, at 37 DEG C containing 5%CO
2condition under act on 24h.
Discarded by culture fluid in ware, the PBS good with 4 DEG C of pre-coolings washs three times.Being abandoned by PBS is only placed in ware on ice, adds 4mLPBS.With cell scraper scraping cells be collected in 15mL centrifuge tube gently, centrifuge tube inserts in ice.At 4 DEG C, abandon supernatant with 1500rpm low-speed centrifugal collecting cell subsequently, then add the PBS buffer solution of 4 DEG C of pre-coolings, vortex by cell dispersal, recentrifuge.Repeat above-mentioned steps once.In the cell mass obtained, add 100 μ L lysates (1mLRipa+10 μ Lproteininhibitorcocktail+1 μ L0.3%PMSF), in cell lysis on ice 20 minutes, period flicked centrifuge tube frequently, makes cell obtain sufficient cracking.Subsequently product of cell lysis is moved into 1.5mL centrifuge tube, at 4 DEG C, 13000rpm high speed centrifugation 10 minutes, collects supernatant and cell protein lysate.Application BCA determination of protein concentration method is carried out quantitatively the protein concentration in cell pyrolysis liquid.Configure isopyknic load solution containing 20 μ g total proteins, add after sample-loading buffer boils 5 minutes in boiling water and prepare loading.
Joined by the above-mentioned sample configured in the corresponding loading duct of SDS-PAGE, under the voltage of 60V, electrophoresis enters separation gel to sample in about 30 minutes and changes 120V continuation electrophoresis about 90 minutes.Take out gel to turn in buffer in electricity and balance after 20 minutes, under 90V, electricity turns and is transferred on pvdf membrane to albumen on glue for about 60 minutes.With after close this film hour containing the TBST solution of 5% defatted milk powder on horizontal shaker, add the LC3 albumen primary antibodie (CST) of the TBST solution dilution of ratio containing 5% defatted milk powder in 1:1000, in 4 DEG C of overnight incubation.Film is washed three times with TBST subsequently, each 5 minutes on shaking table.Two anti-(SantaCruz) that the TBST solution adding use 5% defatted milk powder of certain volume dilutes by 1:5000, incubated at room, after 1 hour, washes 3 times with TBST solution, each 5 minutes.Detect the band on film with chemiluminescence ECL method, expose through development in magazine, fixingly obtain the film that final entry has protein band.
The results are shown in Figure 2.Expression according to protein level changes, find that concentration is 1 μM, after the NNC55-0396 of 5 μMs and 10 μMs acts on U87MG cell 24 hours (rapamycin is as positive control), wherein concentration is the expression that the compound N NC55-0396 of 5 μMs and 10 μMs has all significantly raised LC3 II, confirm the generation of autophagy, and determine that NNC55-0396 can effectively induce the concentration of trigger cell autophagy to be 5 μMs.
Claims (4)
- The application of 1.T type calcium channel inhibitor NNC55-0396 in the anti-neurodegenerative diseases medicine of preparation, the chemical name of wherein said NNC55-0396 is: (1s, 2s)-2-(2-(N-[3-(2-1H-benzimidazolyl) propyl group]-N-methylamino) ethyl)-6-fluoro-1,2,3,4-tetrahydrochysene-1-isopropyl-beta naphthal-ethylene-acetic acid ester.
- 2. apply as claimed in claim 1, it is characterized in that: described neurodegenerative disease is alzheimer disease.
- 3. apply as claimed in claim 1 or 2, it is characterized in that: described T-shaped calcium channel inhibitor NNC55-0396 can effectively induce the concentration of trigger cell autophagy to be 5 μMs.
- 4. apply as claimed in claim 3, it is characterized in that: described cell is neurogliocyte U87MG cell.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110770221A (en) * | 2017-04-26 | 2020-02-07 | 卡维昂公司 | Methods for improving memory and cognition and for treating memory and cognitive disorders |
EP3634410A4 (en) * | 2017-06-05 | 2021-03-03 | The Methodist Hospital System | Tau phosphorylation inhibitors and methods for treating or preventing alzheimer's disease |
Citations (2)
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CN101155548A (en) * | 2005-03-04 | 2008-04-02 | 曼提斯库拉Ehf.公司 | A method and a system for assessing neurological conditions |
CN103764166A (en) * | 2011-06-22 | 2014-04-30 | 通用医疗公司 | Treatment of proteinopathies |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN101155548A (en) * | 2005-03-04 | 2008-04-02 | 曼提斯库拉Ehf.公司 | A method and a system for assessing neurological conditions |
CN103764166A (en) * | 2011-06-22 | 2014-04-30 | 通用医疗公司 | Treatment of proteinopathies |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110770221A (en) * | 2017-04-26 | 2020-02-07 | 卡维昂公司 | Methods for improving memory and cognition and for treating memory and cognitive disorders |
CN110770221B (en) * | 2017-04-26 | 2023-09-08 | 卡维昂公司 | Methods for improving memory and cognition and for treating memory and cognition disorders |
EP3634410A4 (en) * | 2017-06-05 | 2021-03-03 | The Methodist Hospital System | Tau phosphorylation inhibitors and methods for treating or preventing alzheimer's disease |
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