阅读说明：本技术 一种基于荧光的早期药物筛选方法 (A kind of fluorescence-based early stage drug screening method ) 是由 郑双佳 魏成喜 张旭 赵明 李英骥 闫励 于 2019-08-20 设计创作，主要内容包括：本发明涉及一种基于荧光的早期药物筛选方法。该方法包括以下步骤：步骤1)提供稳定表达离子通道靶点的目标细胞；步骤2)将电压敏感染料和具有不同浓度的所述药物与所述目标细胞一起孵育；步骤3)利用荧光测量装置测量孵育后的细胞的荧光基线值和随时间变化的数值；和步骤4)对所得的数值进行数据拟合,确定所述药物对所述离子通道靶点是具有激动作用或抑制作用。利用该方法,能够利用成本低廉的酶标仪来以中等通量对数十个离子通道靶点进行药物筛选,尤其适用于早期的药物筛选。(The present invention relates to a kind of fluorescence-based early stage drug screening methods.Method includes the following steps: step 1) provides the target cell for stablizing expression ion channel target spot；Voltage sensitive dye and the drug with various concentration are incubated with by step 2 with the target cell；The numerical value that step 3) is measured the fluorescence baseline value of the cell after being incubated for using fluorescence measuring device and changed over time；Data fitting is carried out to resulting numerical value with step 4), determines that the drug is with agonism or inhibiting effect to the ion channel target spot.Using this method, drug screening can be carried out to dozens of ion channel target spot with moderate fluxes using low-cost microplate reader, be particularly suitable for the drug screening of early stage.)

1. a kind of fluorescence-based early stage drug screening method, the described method comprises the following steps:

Step 1) provides the target cell for stablizing expression ion channel target spot；

Voltage sensitive dye and the drug with various concentration are incubated with by step 2 with the target cell；

The numerical value that step 3) is measured the fluorescence baseline value of the cell after being incubated for using fluorescence measuring device and changed over time；With

Step 4) carries out data fitting to resulting numerical value, determines that the drug is that there is excitement to make to the ion channel target spot With or inhibiting effect.

2. the method according to claim 1, wherein multiple Asias that the ion channel target spot has performance different Type.

3. according to the method described in claim 2, having it is characterized in that, the ion channel target spot is GABAA receptor GABA α 1 ~ 6 six hypotype: 1 β of α, 2 γ 2,2 β of α, 3 γ 2,3 β of α, 3 γ 2,4 β of α, 3 γ 2,5 β of α, 3 γ 2,6 β of α, 2 γ 2.

4. the method according to claim 1, wherein the target cell is to stablize expression GABA α 1 ~ 6 HEK293 cell line.

5. the method according to claim 1, wherein the voltage sensitive dye is cell membrane potential sensitivity dye Material.

6. the method according to claim 1, wherein the fluorescence measuring device is microplate reader.

7. the method according to claim 1, wherein with the drug concentration increase, measured fluorescence Numerical value reduces, then judges that the drug is inhibited to the sensible target spot of the ion.

8. the method according to claim 1, wherein with the drug concentration increase, measured fluorescence Numerical value increases, then judges that the drug has agonism to the sensible target spot of the ion.

9. a kind of early stage drug screening method of fluorescence-based GABA receptor, the described method comprises the following steps:

Step 1) provides the HEK293 cell line for stablizing expression GABA α 1 ~ 6；

Voltage sensitive dye and the drug with various concentration are incubated with by step 2 with the HEK293 cell line；

Step 3) be incubated for using fluorescence measuring device measurement after HEK293 cell line fluorescence baseline value and change over time Numerical value；With

Step 4) to resulting numerical value carry out data fitting, determine the drug to the GABA α 1 ~ 6 have agonism or Inhibiting effect.

10. according to the method described in claim 9, wherein the voltage sensitive dye is the Red derived from AAT Inc., the U.S. The voltage sensitive dye for including in Fluorescence film potential detection kit.

Technical field

The present invention relates to drug screening method, in particular to fluorescence-based early stage drug screening method.

Background technique

Ion channel is the basis of central nervous system activities, closely related with the various activities of central nervous system etc., Also it is important drug screening target spot.Drug non-specifically acts on central nervous system ion channel, is drug maincenter mind The main reason for through system adverse reaction.Therefore, the nonspecific effect early screening of drug Central nervous system, is maincenter The important content of nervous system secondary pharmacology screening and safety pharmacology, is early detection drug maincenter during new drug development The important tool of nervous system toxicity.

For traditional central nervous system ion channel Sites Screening technology in toxicity Sites Screening, there are certain to lack It falls into, such as patch clamp technique, although data accuracy is higher, flux is low, and it is at high cost, need stronger technical background, it is difficult to Dozens of ion channel target spot is screened simultaneously.Fluorescent technique is widely used in high flux screening, at low cost, but often Need expensive data acquisition instrument (such as FLIPR).And generally directed to an a large amount of compound of Sites Screening, for one A or several compounds, the action target spot of the different ion channel of screening dozens of property are still unable to do what one wishes.

In addition, some target spots (such as gated ion channel receptor) are there are multiple hypotypes, and each hypotype there is likely to be Multiple subunits, this results in the isomers of a large amount of different subunit combinations, and distribution, function and pharmacological property have Very extensive difference.Therefore, while effect of the detection compound to each hypotype, there is also bigger difficulty.

Therefore, this field there is still a need for it is a kind of it is quick, micro, efficient and economical, can be with moderate fluxes logarithm simultaneously The drug screening method that ten ion channel target spots are screened simultaneously especially carries out the side of Effective selection in early stage to drug Method.

Summary of the invention

In view of the deficienciess of the prior art, it is an object of the present invention to provide a kind of fluorescence-based early stage drugs Screening technique.

Another object of the present invention is to provide a kind of early stage drug screening sides of fluorescence-based GABA receptor Method.

Above-mentioned purpose is realized by following scheme:

On the one hand, the present invention provides a kind of fluorescence-based drug screening method, the described method comprises the following steps: step 1) The target cell for stablizing expression ion channel target spot is provided；Step 2) is by voltage sensitive dye and the medicine with various concentration Object is incubated with the target cell；Step 3) using fluorescence measuring device measurement be incubated for after cell fluorescence baseline value and The numerical value changed over time；Data fitting is carried out to resulting numerical value with step 4), determines the drug to the ion channel Target spot is with agonism or inhibiting effect.

It, can be to realize the drug screening of ion channel target spot using fluorescence measurement by the technical solution.With biography System detection detection method means such as patch-clamp etc. is compared, and is influenced according to the detection process that the method for the technical solution carries out by the external world Factor is smaller, and sensitivity is higher.

The drug screening method according to first aspect, it is preferable that the ion channel target spot has performance different Multiple hypotypes.It is further preferred that the ion channel target spot is GABAA receptor, with GABA α 1~6 six hypotype: 1 β of α 2γ2、α2β3γ2、α3β3γ2、α4β3γ2、α5β3γ2、α6β2γ2。

By the technical solution, high efficiency sieve can be carried out to dozens of ion channel target spot simultaneously with moderate fluxes Choosing.

The drug screening method according to first aspect, it is preferable that the target cell is to stablize expression GABA α 1~6 HEK293 cell line.

The drug screening method according to first aspect, it is preferable that the voltage sensitive dye is that cell membrane potential is quick Infection material.

By the technical solution, in screening process, the variation of fluorescence detection cell membrane potential can be used, thus to medicine Object is screened.

The drug screening method according to first aspect, it is preferable that the fluorescence measuring device is microplate reader.

Microplate reader has the characteristics that detection sensitivity is high, practical.Using microplate reader, can be avoided to expensive detection The demand of instrument such as FLIPR fluorescence detecting system, so that it is economic, efficient to have many advantages, such as the method for the present invention.

The drug screening method according to first aspect, it is preferable that measured as the concentration of the drug increases Fluorescence values reduce, then judge that the drug is inhibited to the sensible target spot of the ion.It is further preferred that with institute The concentration for stating drug increases, and measured fluorescence values increase, then judges that the drug has the sensible target spot of the ion and swash Movement is used.

By the technical solution, drug can intuitively, fast be understood to institute according to measured fluorescence values variation The ion channel target spot of screening is with inhibition or agonism, and the high sensitivity of detection.

In second aspect, the present invention provides a kind of early stage drug screening sides of fluorescence-based GABA receptor Method the described method comprises the following steps:

Step 1) provides the HEK293 cell line for stablizing expression GABA α 1~6；

Voltage sensitive dye and the drug with various concentration are incubated with by step 2) with the HEK293 cell line；

Step 3) be incubated for using fluorescence measuring device measurement after HEK293 cell line fluorescence baseline value and change over time Numerical value；With

Step 4) to resulting numerical value carry out data fitting, determine the drug to the GABA α 1~6 have agonism or Inhibiting effect.

The drug screening method according to second aspect, the voltage sensitive dye are the Red derived from AAT Inc., the U.S. The voltage sensitive dye for including in Fluorescence film potential detection kit.

The cell membrane potential sensitive dye is able to carry out step detection, and has the advantages that cheap, disposable and rapid sensitive.

Through the above technical solutions, present invention obtains following the utility model has the advantages that

1) it is compared with traditional detection detection method means, such as patch-clamp, detection process according to the method for the present invention is by the external world Influence factor is smaller；When cell membrane potential is changed by environmental stimuli, i.e., ion channel opens and closes, ion, which flows into or out, is led Film potential change of cause etc., voltage sensitive dye can also detect this changing value by exciting light and transmitting light irradiation；

2) screening technique step according to the present invention is few, simple, quick, reliable, and the equipment cost needed is cheap, very economical, And dozens of ion channel target spot can be screened simultaneously simultaneously with moderate fluxes, it is high-efficient；

3) according to resulting data stabilization is tested, reproducible, extensive High Throughput Screening Assay is arrived in suitable implementation；

4) according to the method for the present invention, in same screening system, pass through fluorescence-based central nervous system early screening skill Art detects all possible target spots, and then filters out the secondary target of lead compound, avoids in subsequent R&D process Some adverse reactions and undershooting-effect, this has very great significance for the screening tool of drug.

Detailed description of the invention

Some preferred embodiments of the invention are specifically described below with reference to attached drawing.Those skilled in the art It will be understood that these attached drawings are only used for the purpose illustrated, and have no intention to limit the scope of the invention in any way.

Fig. 1 is shown in the GABA dose-dependence measured in microplate reader.The dose-dependence is examined using film potential What test agent box (ATT) measured under different GABA concentration；

Fig. 2 shows the EC50 value for the GABAA recipient cell system using GABA detected in microplate reader: 1 β of A:GABA α, 2 γ 2- HEK293, EC50=14.85 ± 4.33 μM；2 β 3-HEK293 of B:GABA α, EC50=7.40 ± 0.92 μM；C:GABA α3β3- HEK293, EC50=1.53 ± 0.47 μM；4 β of D:GABA α, 3 γ 2-HEK293, EC50=2.51 ± 0.06 μM；E:GABAα5β 3-HEK293, EC50=0.54 ± 0.05 μM；6 β of F:GABA α, 2 γ 2-HEK293, EC50=0.50 ± 0.23 μM；

Fig. 3 is shown in the EC50 value for the GABAA recipient cell system using Bicuculline detected in microplate reader: A:GABA α 1 β 2 γ 2-HEK293, IC50=14.29 μM；B:GABA α 2 β 3-HEK293, IC50=24.99 μM；C:GABAα3β 3- HEK293, IC50=5.92 μM；4 β of D:GABA α 3 γ 2-HEK293, IC50=20.48 μM；5 β 3-HEK293 of E:GABA α, IC50=8.25 μM；6 β of F:GABA α 2 γ 2-HEK293, IC50=8.52 μM；

Fig. 4 A-H shows a series of compounds to the screening schematic diagram of GABAA2 target spot validity.

Specific embodiment

Certain specific embodiments of the invention are described below with reference to drawings and examples.The ordinary skill of this field Personnel it will be appreciated that provide the purposes of these embodiments just for the sake of illustrating how to that the solution of the present invention can be implemented, and It is non-to limit the scope of the invention in any way.

The screening of conventional medicament is that target spot known to some is synthesized a large amount of compound, interacted, in turn Lead compound is filtered out, subsequent research and development are then carried out.It is relatively unstable for certain pleiotropisms, dose-effect relationship complexity, effect Qualitative compound, this method are difficult to find its adverse reaction to other target spots of human body, especially be difficult in preclinical phase It was found that central nervous system adverse reaction, cause the later period research and development failure.

The present invention studies this.In practice, the inventors discovered that, voltage sensitive dye (Voltage- is utilized Sensitivedyes, VSD) it is incubated for cell to be detected, it is irradiated with exciting light, is changed when cell membrane potential is stimulated When (△ Vm), the dyestuff as molecular probe, which synchronizes, there is change in fluorescence, and reads corresponding fluorescence by multi-function microplate reader Changing value, so as to measure excitement or inhibiting effect of the untested compound to the channel GABAA.The base that the present inventor has found herein The present invention is made that on plinth.

Based on the discovery, the invention proposes a kind of fluorescence-based method for screening compound.The method is mainly wrapped Include following: step 1) provides the target cell for stablizing expression ion channel target spot；Step 2) is by voltage sensitive dye and has not The compound with concentration is incubated with the target cell；Step 3) is measured thin after being incubated for using fluorescence measuring device The fluorescence baseline value of born of the same parents and the numerical value changed over time；Data fitting is carried out to resulting numerical value with step 4), determines describedization Closing object is with agonism or inhibiting effect to the ion channel target spot.

The present invention will be illustrated by taking ion channel target spot γ-aminobutyric acid A receptor as an example below.However, Those skilled in the art are it will be appreciated that these embodiments are used only for the purpose illustrated, and have no intention with any side Formula limits the scope of the invention.

γ-aminobutyric acid A receptor abbreviation GABAA receptor (GABAAR) is ligand-gated ion channel superfamily member One of, it is the most important inhibitory neurotransmitter of central nervous system, the most of rapid synaptics of forebrain is mediated to inhibit.By The receptor-mediated behavior effect of GABAA, other than the antianxiety of stable class induction, of flaccid muscles and sedative-hypnotic effect, GABAA receptors ligand can influence circadian rhythm, fertility, appetite, food absorption, kinesitherapy nerve function, defence, identification and memory Deng.Therefore drug effect can generate the toxicity of some column central nervous systems in GABAA receptor.In addition, GABAA receptor It is the ion channel that a kind of chloride ion mediates, when GABAA channel opener, negative potential is surveyed in cell membrane can more hyperpolarization.

Subunit series is divided into 7 subunit races according to amino acid sequence similarity degree by GABAA receptor subunit series, respectively It is named as α 1~6, β 1~4, γ 1~4, δ 1, ε 1, π 1 and ρ 1~3.These subunits and its different combinations constitute numerous Pentagon isomers hypotype, general combination are 2 alpha subunits, 2 β subunits and 1 γ (or δ, ε etc.) subunit. Wherein, according to the difference of alpha subunit, GABAA receptor is divided into GABAA1-GABAA66 hypotype, with alpha subunit and β and γ The various combination of subunit, GABAA receptor form the isomers of a large amount of different subunit combinations, distribution, function and pharmacology Learning property has very extensive difference.Therefore, to simultaneously effect of the detection compound to each hypotype of GABAA receptor, There is also bigger difficulty.

The present invention is directed to be based on fluorescent screening technology, using the multi-function microplate reader of the existing low cost of common laboratory with And commercial membrane potentiometric detection kit is (for example, be purchased from the ScreenQuestMembranePotentialAss of AAT Inc., the U.S. Ay Kit*RedFluorescence* film potential detection kit or the examination for being purchased from U.S. MolecularDevices (MD) company Agent box FLIPRMembranePotentialRed, Explorer), classify to ion channel target spot of different nature, builds A set of ion channel multiple target point screening technique is found, a compound is screened on 96 orifice plates to multiple or even more than ten of ion The effect in channel, so that a moderate fluxes are set up, the multiple ion channels central nervous system secondary pharmacology of lower cost Drug target Screening Platform.