CN102854176B - Screening method of TMEM16A calcium-activated chloride channel inhibitor - Google Patents
Screening method of TMEM16A calcium-activated chloride channel inhibitor Download PDFInfo
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- CN102854176B CN102854176B CN201210245365.1A CN201210245365A CN102854176B CN 102854176 B CN102854176 B CN 102854176B CN 201210245365 A CN201210245365 A CN 201210245365A CN 102854176 B CN102854176 B CN 102854176B
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Abstract
The invention relates to a screening method of a TMEM16A calcium-activated chloride channel inhibitor; with the screening method of the invention, a TMEM16A calcium-activated chloride channel inhibitor can be screened, which can be developed into a medicament for treating hypertension, CF disease, asthma, stomach and intestine dynamic dysfunction diseases, and diseases of some types of tumors.
Description
Technical field
The present invention relates to biology field, particularly a kind of TMEM16A calcium activates the screening technique of chloride channel inhibitor.
Background technology
Calcium activates chloride channel (Calcium-activated Chloride Channels, CaCCs) owing to being activated and gaining the name by cell interior calcium ion, be found in the earliest in Xenopus Oocytes, in succession have subsequently report to show all to exist in epithelial cell, vascular endothelial cell, neuron, smooth muscle and cardiac muscle cell.The signal transduction pathway of calcium-mediated is the important component part of eukaryotic signal transduction, therefore CaCCs carries out several functions, comprises that the fertilization, transepithelial ion/fluid transport, cardiac muscle cell's repolarization of egg cell and action potential occur, sense of smell conduction and the flexible adjusting of smooth muscle etc.
Due to technology, the molecular basis problem of CaCCs fails to solve always, causes Related Drug pharmacological research process very slow.Until have three research groups independently to report that the molecular basis of CaCCs is transmembrane protein 16A(transmembrane16A the end of the year 2008, be called for short TMEM16A) (Schroeder BC etc., Cell, 2008,134:1019-1029; Caputo A etc., Science, 2008,322:590-594; Yang YD etc., Nature, 2008,455:1210-1215).This is found to be the problem of studying the aspect such as physiology and pharmacology of CaCCs in specific cells and tissue new research platform is provided.
Recent findings TMEM16A passage and multiple major disease are closely related.Bibliographical information, TMEM16A ion channel may be the drug target of pulmonary cystic fibrosis (CF) disease, activate TMEM16A passage and can play chloride channel cystic fibrosis transmembrane conductance regulator (cystic fibrosis transmembrane conductance regulator on compensation epithelial tissue, be called for short CFTR) ion/fluid transport imbalance (Rock JR etc. due to gene mutation, J.Biol.Chem., 2009,284:14875-14880).In addition, 2012, Wang etc. (Wang M etc., Circulation, 2012,125:697-707) proved that TMEM16A is the molecular basis of cerebrovascular smooth muscle cells CaCCs, and found that activity and the blood pressure of TMEM16A ion channel are negative correlation.On the other hand, the inhibitor of CaCCs is in the news and can be used for tumour for the treatment of asthma, stomach and intestine dynamics disorder disease and some type etc.As can be seen here, the treatment that screening TMEM16A ion channel specificity correctives can be CaCCs relevant disease provides new drug target.
The Invitrogen of Life Sci-Tech company of the U.S.
tMkit Premo
tMthe fluorescence of the Trimutant protein YFP-F46L/H148Q/I152L of halogenide sensor utilization yellow fluorescence protein (Yellow Fluorescence Protein, YFP), as index, screens the correctives of CaCCs/TMEM16A ion channel.Use the method, 2010, Namkung etc. (Namkung W etc., The FASEB J., 2010,24:4178-4186) applied Premo
tMhalogenide sensor reagent box screening obtains the inhibitor that tannic acid (tannic acid) and relevant nutgall tannin (gallotannin) are TMEM16A/CaCCs.
Regrettably, at present lack simple and easy to do TMEM16A calcium and activate the screening technique of chloride channel inhibitor, cause the applied research slower development in the disease such as tumour of hypertension, CF disease, asthma, stomach and intestine dynamics obstacle and some type take TMEM16A/CaCCs as drug target.
Summary of the invention
The object of this invention is to provide the screening technique that a kind of TMEM16A calcium activates chloride channel inhibitor, the TMEM16A calcium filtering out by this screening technique activates chloride channel inhibitor and provides basis for the application of research TMEM16A/CaCCs in the diseases such as the tumour of hypertension, CF disease, asthma, stomach and intestine dynamics disorder disease and some type.
The invention provides the screening technique that a kind of TMEM16A calcium activates chloride channel inhibitor, use TMEM16A calcium to activate the positive control of chloride channel activator chitosan oligosaccharide as screening inhibitor, comprise the steps:
(a) TMEM16A plasmid stable transfection is entered to mammalian cell HEK293 cell;
(b) by the HEK293 cell of the stable transfection TMEM16A calcium activation chloride channel in yellow fluorescence protein YFP Trimutant YFP-F46L/H148Q/I152L importing step (a);
(c) cell after drug candidate and the described importing of step (b) is hatched, add again the solution that contains iodide ion to observe, discovery fluorescence intensity is unchanged, now add again chitosan oligosaccharide fluorescence intensity still unchanged, determine that drug candidate may be the inhibitor of TMEM16A passage, and adopt step (d) further to verify;
(d) measure the impact of drug candidate on electric current in step (a) with electrophysiological method, if wherein add drug candidate after-current to reduce than contrast, determine that drug candidate is the inhibitor that TMEM16A calcium activates chloride channel, if current ratio contrast does not reduce, determine that this drug candidate is not the inhibitor that TMEM16A calcium activates chloride channel.
Further preferably, in described step (b), YFP Trimutant YFP-F46L/H148Q/I152L is by baculovirus vector transfered cell.
Further preferably, the fluorescence intensity described in step (d) is detected by laser microscope, and wherein excitation wavelength is 470-540nm, and detection wavelength is 540-590nm.
Further preferably, described drug candidate is tannic acid or other inhibitor molecules.
Adopt screening technique of the present invention can filter out TMEM16A calcium and activate chloride channel inhibitor, can be developed as the medicine of the disease such as tumour for the treatment of hypertension, CF disease, asthma, stomach and intestine dynamics disorder disease and some type.
Accompanying drawing explanation
Fig. 1: according to TMEM16A calcium of the present invention activate chloride channel inhibitor tannic acid to the fluorescence intensity of yellow fluorescence protein without the obvious trend map of humidification.
Fig. 2: activating chloride channel inhibitor tannic acid according to TMEM16A calcium of the present invention has inhibiting trend map for TMEM16A electric current.
Embodiment
The invention provides the screening technique that a kind of TMEM16A calcium activates chloride channel inhibitor, use TMEM16A calcium to activate the positive control of chloride channel activator chitosan oligosaccharide as screening inhibitor, comprise the steps:
(a) TMEM16A plasmid stable transfection is entered to mammalian cell HEK293 cell;
(b) by the HEK293 cell of the stable transfection TMEM16A calcium activation chloride channel in yellow fluorescence protein YFP Trimutant YFP-F46L/H148Q/I152L importing step (a);
(c) cell after drug candidate and the described importing of step (b) is hatched, add again the solution that contains iodide ion to observe, discovery fluorescence intensity is unchanged, now add again chitosan oligosaccharide fluorescence intensity still unchanged, determine that drug candidate may be the inhibitor of TMEM16A passage, and adopt step (d) further to verify;
(d) measure the impact of drug candidate on electric current in step (a) with electrophysiological method, if wherein add drug candidate after-current to reduce than contrast, determine that drug candidate is the inhibitor that TMEM16A calcium activates chloride channel, if current ratio contrast does not reduce, determine that this drug candidate is not the inhibitor that TMEM16A calcium activates chloride channel.
The cell that can be used for screening technique of the present invention is not particularly limited, and representational example comprises: mammalian cell, and as HEK293 cell, Chinese hamster ovary celI or vertebrate Africa xenopus (Xenopus laevis) egg mother cell.
The electrophysiology assay method that can be used for screening technique of the present invention is not particularly limited.Representational example comprises: patch-clamp method (for mammalian cell) or bipolar electrode voltage clamp method (for Xenopus Oocytes).
The fluorescence intensity detection technique that can be used for screening technique of the present invention is not particularly limited.Representational example comprises: laser confocal microscope (for fluorescence real time imagery) or multi-functional microplate reader (for fluorescence intensity real time record) etc.
Yellow fluorescence protein (YFP) is the fluorescin that one derives from green fluorescent protein (GFP), can under wavelength 515nm, be excited and send yellow fluorescence.Iodide ion can be combined and make fluorescent quenching with YFP, and suddenly change two site H148Q and I152L can make YFP strengthen the susceptibility of iodide ion.CaCCs passage is not only a kind of chloride channel, and it has permeation to the most of negative ion including iodide ion.This drug screening test imports the YFP gene of external source in HEK293 cell by the method for virus infections, makes YFP great expression in born of the same parents; Again drug candidate and cell are hatched itself and passage are fully acted on; Finally observe and add the solution cancellation degree of YFP fluorescence later that contains iodide ion.Can make YFP can be considered to the inhibitor of CaCCs passage without the medicine of obviously cancellation by the method.
Chitosan oligosaccharide (chitosan oligosaccharide, COS) is the linear homogeneous polysaccharide being formed by chitin dehydration.COS is naturally occurring alkaline amino-oligosacchride, there is good water solubility, safety non-toxic, easily by advantages such as animal body absorptions, therefore its biologic activity receives much concern, comprise antibacterial (Choi BK etc., International Journal of Antimicrobial Agents, 2001:18:553 – 557), antiviral (Bacon A etc., Infection and Immunity, 2000, 68:5764 – 5770), antitumor (Xiong C etc., Carbohydrate Research, 2009, 344:1975 – 1983), reduce Blood Cholesterol (Se-Kwon K, Carbohydr.Polym., 2005, 62:357 – 368), immunological regulation (Okamoto Y etc., Macromol.Biosci.2003, 3:587-590), and to asthma (Chung MJ etc., Int.Immunopharmacol., 2012, 12::43-459), diabetes (Lee HW etc., Biol.Pharm.Bull., 2003, the therapeutic action such as 26:1100-1103), but its action site and pharmacological characteristics are still not unclear.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, such as Sambrook etc., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1:
One, first carry out the scalping of inhibitor by fluorescence experiments method, concrete steps are as follows:
First day is transfected into expression plasmid pEGFP-N1-TMEM16A in mammalian cell HEK293, cultivates the HEK293 cell of stable transfection YFP in the burnt special ware of copolymerization; Within second day, the HEK293 cell of transfection YFP overnight incubation is rinsed 3 times with D-PBS, finally leave 800 μ l D-PBS; Add tannic acid to hatch 10min, recording fluorescence intensity is baseline fluorescence intensity; By laser confocal microscope real time record fluorescence intensity, as shown in Figure 1, now add the solution 800 μ l that contain 150mM I-, make I concentration reach 75mM, YFP fluorescence intensity is without significant change, then adds chitosan oligosaccharide, and the fluorescence intensity in this process is always without significant change (Fig. 1 a, 1b), result shows that tannic acid may be the inhibitor that TMEM16A calcium activates chloride channel.
Two, use electrophysiology assay method to carry out determining of inhibitor, concrete steps are as follows:
Expression plasmid pEGFP-N1-TMEM16A is proceeded in mammalian cell HEK293.After cell transfecting, within 24-72h, carry out electrophysiology detection (utilizing patch clamp technique).Concrete grammar is as follows: the high sugar of the DMEM(that contains 10% hyclone for HEK293 cell) the nutrient solution cultivation (adding penicillin and the 100 μ g/ml streptomysins of 100UI/ml) of going down to posterity.Stable transfection process Lipofectamine2000(Invitrogen company) liposome carries out.Cell, in 37 ℃, is cultured to exponential phase for experiment in 5%CO2 saturated humidity incubator.Electricity physiological detection is at room temperature carried out (approximately 22 ℃), adopts inside-out (Inside-Out) logging mode (EPC-10Amplifier, HEKA company, Germany), and the composition of the outer liquid of interior liquid and basis is (unit: mM): NaCl140, MgCl
26H
2o, HEPES10, EGTA5, is adjusted to pH7.4 with NaOH.Add the concentration of tannic acid in the outer liquid of medicine to be made as 1,10,50,100,500 μ g/ml, whether test TMEM16A passage with 500nM free calcium ions expresses, do negative control without calcium solution with 10mM EGTA, and make comparisons with the electric current under 1 μ M free calcium condition, as shown in Figure 2, add tannic acid after-current to reduce than contrast, determine that tannic acid is the inhibitor that TMEM16A calcium activates chloride channel.
Claims (3)
1. TMEM16A calcium activates a screening technique for chloride channel inhibitor, uses TMEM16A calcium to activate the positive control of chloride channel activator chitosan oligosaccharide as screening inhibitor, it is characterized in that, comprises the steps:
(a) TMEM16A plasmid stable transfection is entered to mammalian cell HEK293 cell;
(b) by the HEK293 cell of the stable transfection TMEM16A calcium activation chloride channel in yellow fluorescence protein YFP Trimutant YFP-F46L/H148Q/I152L importing step (a);
(c) cell after drug candidate and the described importing of step (b) is hatched, add again the solution that contains iodide ion to observe, discovery fluorescence intensity is unchanged, now add again chitosan oligosaccharide fluorescence intensity still unchanged, determine that drug candidate may be the inhibitor of TMEM16A passage, and adopt step (d) further to verify; Described fluorescence intensity is detected by laser microscope, and wherein excitation wavelength is 470-540nm, and detection wavelength is 540-590nm;
(d) measure the impact of drug candidate on electric current in step (a) with electrophysiological method, if wherein add drug candidate after-current to reduce than contrast, determine that drug candidate is the inhibitor that TMEM16A calcium activates chloride channel, if current ratio contrast does not reduce, determine that this drug candidate is not the inhibitor that TMEM16A calcium activates chloride channel.
2. screening technique according to claim 1, is characterized in that, in described step (b), YFP Trimutant YFP-F46L/H148Q/I152L is by baculovirus vector transfered cell.
3. screening technique according to claim 2, is characterized in that, described drug candidate is tannic acid or other inhibitor molecules.
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| CN102854236B (en) * | 2012-07-16 | 2015-01-21 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel activator |
| CN103898059B (en) * | 2014-03-05 | 2016-08-17 | 吉林医药学院 | The cell model of screening calcium-activated potassium current inhibitor and screening technique |
| CN108004198B (en) * | 2016-10-28 | 2021-07-16 | 华中农业大学 | Method for establishing high-throughput drug screening model based on ICAM-1 signal channel |
| WO2018112814A1 (en) * | 2016-12-22 | 2018-06-28 | Nanjing University | Reducing smooth muscle contractile response induced by inflammatory constrictors |
| CN111450237A (en) * | 2020-03-17 | 2020-07-28 | 中国人民解放军63919部队 | Novel target for developing osteoporosis treatment drug |
| CN112359088A (en) * | 2020-10-09 | 2021-02-12 | 吉林医药学院 | FRT cell strain and application thereof in preparation of preparation or kit for detecting concentration of free calcium ions in cells |
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