CN105087644A - Method for high-throughput screening of compound with Nrf2 activating activity - Google Patents

Abstract

The invention relates to a method for high-throughput screening of a compound with Nrf2 activating activity. The method comprises the following steps: (1) establishing a reporter gene containing three Nrf2 binding sites; (2) inserting the reporter gene into plasmids, and thus obtaining reporter gene plasmids; (3) amplifying the reporter gene plasmids by adopting escherichia coli, and purifying the plasmids; and (4) transfecting HeLa cells by adopting the purified reporter gene plasmids, and selecting stable expression cell strains for the selection of an unknown compound. With the adoption of the technical scheme provided by the invention, the method for detecting the activating effect of Nrf2 in the prior art is enabled to adopt to the demand of high-throughput screening at the initial development stage of the new medicine. The traditional method generally needs half a day to two days for completely once of detection, while the quantity of the samples detected by the once experiment does not beyond a dozen. For the method provided by the invention, each 96-pore board can detect at least 10 samples, and the time needed for completing the detection by the 96-pore board is less than 30 minutes.

Description

A kind of high flux screening has the method for the compound of Nrf2 Activation Activity

Technical field

The invention belongs to biomedicine field, be specifically related to a kind of method that high flux screening has the compound of Nrf2 Activation Activity.

Background technology

Nrf2 albumen is intracellular a kind of transcription factor, the expression of controlling gene.Nrf2 is activated after cell is subject to extraneous destructive stimulus, and the Nrf2 of activation increases the gene expression dose of protective factors in various kinds of cell, produces cell protective effect, the damage of T suppression cell.

Utilizing micromolecular compound (medicine) as the activator of Nrf2, activating Nrf2, playing cytoprotection when not having extraneous destructive stimulus by drug effect, is a direction of current new drug development.The small molecules Nrf2 activator of at present external exploitation some enter clinical investigation phase.

At the preliminary stage of new drug development, screening from numerous compounds is needed to have the compound of Nrf2 activation.Research method conventional at present comprises:

(1) expression level (as: TanKP of Nrf2 target gene is detected with molecular biology method, YangM, ItoS:Activationofnuclearfactor (erythroid-2like) factor2bytoxicbileacidsprovokesadaptivedefenseresponsest oenhancecellsurvivalattheemergenceofoxidativestress.MolP harmacol2007,72 (5): 1380-1390.).Main drawback: experimental procedure is complicated, cannot meet the needs that high-throughput (high-level efficiency) screens; Result random error is large.

(2) functionally active (as: Dinkova-KostovaAT of Nrf2 target gene (mainly NAD (P) H: quinone oxidoreductase-1) is detected with biochemical method, LibyKT, StephensonKK, HoltzclawWD, GaoX, SuhN, WilliamsC, RisingsongR, HondaT, GribbleGWetal:Extremelypotenttriterpenoidinducersoftheph ase2response:correlationsofprotectionagainstoxidantandin flammatorystress.ProcNatlAcadSciUSA2005, 102 (12): 4584-4589.).Main drawback: experimental result directly can not reflect the functionally active of Nrf2, and specificity is poor; More difficultly realize high flux screening.

(3) accumulating level (as: WondrakGT of Nrf2 albumen in cell is detected by biochemical method, CabelloCM, VilleneuveNF, ZhangS, LeyS, LiY, SunZ, ZhangDD:Cinnamoyl-basedNrf2-activatorstargetinghumanskin cellphoto-oxidativestress.FreeRadicBiolMed2008,45 (4): 385-395.).Main drawback: the needs that cannot meet high flux screening.

(4) in cell transient expression with the reporter gene of Nrf2 binding site, detect the functional level (WondrakGT of Nrf2 reinforcing gene expression, CabelloCM, VilleneuveNF, ZhangS, LeyS, LiY, SunZ, ZhangDD:Cinnamoyl-basedNrf2-activatorstargetinghumanskin cellphoto-oxidativestress.FreeRadicBiolMed2008,45 (4): 385-395.).The advantage main compared with additive method of this method detects data directly to reflect that the functional transcription factor of Nrf2 is active.Main drawback: the needs that cannot meet high flux screening.

Summary of the invention

Object of the present invention is just to provide a kind of method that high flux screening has the compound of Nrf2 Activation Activity.

To achieve these goals, the present invention adopts following technical scheme:

High flux screening has a method for the compound of Nrf2 Activation Activity, and step is as follows:

(1) reporter gene containing 3 Nrf2 binding sites is built;

(2) reporter gene is inserted in the middle of pGL4.26 [luc2/minP/Hygro] plasmid, obtains reporter plasmid;

(3) with intestinal bacteria (or competence DH5 α bacterial strain), reporter plasmid is increased and plasmid of purifying;

(4) with the reporter plasmid transfection HeLa cell of purifying.

Whether can obtain reactive good cell strain after stable transfection and depend on reporter gene DNA sequence dna used, combined effect between pUC pUC used and cell used, whether final result is unpredictable, can only be determined by experiment the finished product and can use.The present invention pGL4.26 [luc2/minP/Hygro] plasmid and HeLa cell are arranged in pairs or groups the stable expression cell strain prepared, through with known Nrf2 activator irritation cell with by the positive and negative two kinds of methods of Nrf2 gene silencing (suppressing the activity of original Nrf2), all confirm that there is the Sensitivity and Specificity that can meet test needs.

The reactivity identifying each cell strain with known Nrf2 activator is also comprised in described step (4), choose reactive good (that is: the Luciferase that induces of medicine Sulforaphane or Tert-butylhydroquinone active with contrast (drug solvent of respective concentration, as DMSO) process compare increase by more than 2 times) cell strain.

The application of above-mentioned method in new drug development.

Above-mentioned method screens the compound obtained, and described compound is preparing the application had in Nrf2 Activation Activity medicine

Beneficial effect of the present invention:

High-throughput is required for pharmacy corporation research and development, whether can obtain reactive good cell strain after stable transfection and depend on combined effect between reporter gene DNA sequence dna used and cell used, net result is unpredictable, can only grope by experiment, and this is one of difficult point of success or not.

Present invention improves over the method for original detection Nrf2 activation, the demand of new drug early development stage high flux screening can be adapted to.Original method completes one-time detection needs half a day by two days not etc. usually, and once tests the sample size that can detect and be no more than tens.Use method of the present invention, each 96 orifice plates can detect at least 10 samples, and the time completing 96 orifice plates detections is less than 30 minutes.

The present invention adopts the pUC pUC containing antibiotics resistance gene of a new generation, not only overcome the defect cannot carrying out stable transfection, and plasmid of new generation adds transcription enhancer element at reporter gene end, more optimize the Sensitivity and Specificity of expressing in mammalian cell.High throughput testing technology is based on the experimental technique of molecular level and cell levels, using microplate format as experimental tool carrier, process of the test is performed with automation operating system, experimental result data is gathered with sensitive detecting instrument fast, with Computer Analysis process experimental data, detect number at short notice with sample necessarily, and with the technical system of the associated databases support obtained running, it has trace, the feature such as quick, sensitive and accurate, is that current pharmacy corporation is at new drug development starting stage requisite instrument.For different biomolecules target spots or different classes of compound, the experimental technique of ready-made molecular level and cell levels must be had just to complete screening operation, namely our method is the experimental technique establishing molecular level for " Nrf2 " and cell levels, is the basis of the new drug development high flux screening flow process being biological targets for Nrf2.

Accompanying drawing explanation

Fig. 1 is schema of the present invention;

Fig. 2 is the form of the stably express cell clone seen under light microscopic after HygromycinB screening.

Embodiment

Below in conjunction with accompanying drawing and embodiment, the invention will be further described.

As shown in Figure 1, a kind of high flux screening has the method for the compound of Nrf2 Activation Activity, comprises the following steps:

(1) the artificial constructed reporter gene containing 3 Nrf2 binding sites

(i) synthetic Single-stranded DNA fragments I (78 bases); Sequence as SEQIDNo.1,

5’---CCCAGTCACAGTGACTCAGCAGAATCCAGTCACAGTGACTCAGCAGAATCCAGTCACAGTGACTCAGCAGAATCGTAA---3’

(ii) synthetic Single-stranded DNA fragments II (86 bases); Sequence as SEQIDNo.2,

5’---GATCTTACGATTCTGCTGAGTCACTGTGACTGGATTCTGCTGAGTCACTGTGACTGGATTCTGCTGAGTCACTGTGACTGGGGTAC---3’

Note: DNA synthesis is completed by commercial company

(iii) fragment I and fragment II is dissolved in ultra-pure water respectively the solution being made into 100 μMs of concentration;

(iv) by two kinds of solution even mixing, be heated to 95 DEG C, then to room temperature, product is reporter gene to Slow cooling (1 DEG C/min).

(2) reporter plasmid is prepared

I () pGL4.26 [luc2/minP/Hygro] plasmid buys (Madison, WI, USA) by Promega company, reclaim linear plasmid by after plasmid KpnI and the cutting of BglII restriction endonuclease through gel electrophoresis;

(ii) link enzyme with T4DNALigase after being mixed in 5:1 ratio with reporter gene by linear plasmid to carry out being connected (22 DEG C, 3 hours), obtain reporter plasmid.

(3) with intestinal bacteria, reporter plasmid is increased, purification plasmid

I () is with the reporter plasmid transform competent E. coli cell connected (can select commercially available competence DH5 α bacterial strain);

(ii) heat shock transformation of E. coli, amplification intestinal bacteria, extraction purification plasmid, obtains the reporter plasmid of q.s.

(4) with the reporter plasmid transfection HeLa cell of purifying, screening stable expression cell strain

I () HeLa cell uses the DMEM in high glucose culture medium culturing (5%CO in ordinary cells incubator containing 10% serum ₂, 37 DEG C of saturated humidity environment);

(ii) the Lipofectamine2000 reagent using LifeTechnologies company (Carlsbad, CA, USA) to buy, by recommended flowsheet reporter plasmid transfectional cell, changes fresh culture after 6 hours, continues cultivation 48 hours;

(iii) cell went down to posterity after 48 hours by transfection, and the density kind be paved with about 25% is in culture plate, and the substratum cultured continuously of the hygromycinB of use containing 400 μ g/ml* 6 weeks, changes once containing the substratum of hygromycinB for every 5 days; The cell of stably express reporter gene can grow under hygromycinB exists situation, can not the cell of stably express reporter gene be killed by hygromycinB.

The optimum concn of * note: hygromycinB can be adjusted according to practical situation by experimenter

(iv) picking positive cell clone: find well-grown cell clone (see accompanying drawing 2) under low power light microscopic, mark good position.Remove substratum and use normal saline flushing cell once.Respectively the aseptic pipettor choicest of different cell clones is gone out, be placed in 12 orifice plates to be cultured to and be paved with;

V stable cell line is continued Secondary Culture by (), after appropriate amplification, be stored in liquid nitrogen for subsequent use.

(5) reactivity of each cell strain is identified with known Nrf2 activator

I stable expression cell strain is pressed 3 × 10 by () ⁴the density of individual cells/well is inoculated in 96 orifice plates to cultivate and within 24 hours, allows its adherent growth.Cell difference being erected row is divided into control group and drug treating group.Drug treating can select known Nrf2 activator, such as Sulforaphane (concentration 1 μM); Or Tert-butylhydroquinone (concentration 50 μMs).DMSO (drug solvent) process of control group respective concentration.Cultivate and carry out Luciferase Activity determination (see below) after 24 hours;

(ii) reactive good clone (such as above drug-induced Luciferase activity increases by more than 2 times compared with the control) is selected to carry out follow-up screening experiment.

(7) select reactive good cell strain for the screening of unknown compound

I () each experiment condition can do 4 secondary orifices, i.e. the corresponding a kind of experiment condition in 4 holes of 96 orifice plates;

(ii) cell is pressed 3 × 10 ⁴the density of individual cells/well is inoculated in 96 orifice plates and cultivates 24 hours, continues to cultivate after adding testing compound, correspondingly sets up solvent control group, carries out Luciferase Activity determination after 24 hours;

(iii) viable cell quantity is remained in every hole after evaluation process: select Promega, company (Madison, WI, USA) CellTiter96Aqueous test kit detects (article No. G3580, G3581 or G3582), the standard step of by specification is carried out, record absorption values (needing subtracting background absorbance, see test kit specification sheets).

(iv) Luciferase Activity determination: select the LuciferaseAssay test kit of Promega company (article No. E1500) to detect.Before detecting, the liquid remained by previous step in plate hole is cultivated in exhaustion, and the follow-up standard step by test kit specification sheets is carried out, and records the chemiluminescence intensity numerical value in every hole.

The standardization of (v) data: by the absorption values of step (iv) gained luminous intensity numerical value divided by step (iii) gained, gained ratio is the Luciferase reactivity parameter after stdn.

(vi) precaution: reduce more than 50% as processed rear residue viable cell quantity (absorption values), illustrate that compound on intracellular toxic action is comparatively strong, suitably should reduce concentration.

The specific embodiment of the present invention is described although above-mentioned in conjunction with the embodiments; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.

Claims (10)

1. high flux screening has a method for the compound of Nrf2 Activation Activity, it is characterized in that, step is as follows:

(1) reporter gene containing 3 Nrf2 binding sites is built;

(2) reporter gene is inserted in the middle of plasmid, obtains reporter plasmid;

(3) with intestinal bacteria, reporter plasmid is increased and plasmid of purifying;

(4) with the reporter plasmid transfection HeLa cell of purifying, screening stable expression cell strain is used for the screening of unknown compound.

2. the method for claim 1, is characterized in that, described step (1) is specific as follows:

(1) synthesizing single-stranded DNA fragmentation I, sequence as SEQIDNo.1 and Single-stranded DNA fragments II, sequence as SEQIDNo.2,

(2) fragment I and fragment II is dissolved in ultra-pure water respectively the solution being made into 100 μMs of concentration;

(3) by two kinds of solution even mixing, be heated to 95 DEG C, be then cooled to room temperature, product is reporter gene.

3. method as claimed in claim 2, it is characterized in that, described step is cooled to room temperature with 1 DEG C/min in (3).

4. the method for claim 1, is characterized in that, linear plasmid mixes in 5:1 ratio with reporter gene by described step (2).

5. the method for claim 1, is characterized in that, reporter plasmid increases and plasmid of purifying by described step (3) competence DH5 α bacterial strain.

6. the method for claim 1, it is characterized in that, in described step (4), also comprise the cell strain choosing the active increase by more than 2 times compared with the drug solvent process of respective concentration of Luciferase that medicine Sulforaphane or Tert-butylhydroquinone induces.

7. method as claimed in claim 6, it is characterized in that, described solvent is DMSO.

8. the application of method in new drug development as described in as arbitrary in claim 1-7.

9. the method as described in as arbitrary in claim 1-7 screens the compound obtained.

10. compound as claimed in claim 9 is preparing the application had in Nrf2 Activation Activity medicine.