Novel screening system for activators and inhibitors of signaling pathways based on cell death and life phenotypes

Abstract

This invention provides a novel screening method for signaling pathway activators and inhibitors based on cell viability / death phenotypes. Specifically, this invention provides genetically engineered cells that enable large-scale screening of signaling pathway activators and inhibitors based on the expression of protective and suicide genes driven by signaling pathway transcription factors. The viability / death phenotype reporter system is effective and convenient for screening drugs affecting signaling pathways and can be applied to high-throughput screening in the early stages of drug development.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a novel screening system for signal pathway activators and inhibitors based on cell death and live phenotypes. BACKGROUND

[0002] Many important physiological processes of organisms are closely related to signal pathways, such as Wnt signal in development process, Hippo signal in organ size control, and NFκB signal in immune response. Abnormalities of these signal pathways can cause some diseases, for example, over-activation of EGFR and Wnt pathways can cause abnormal regulation of cell growth and proliferation, and further cause tumor; abnormal transduction of TGFβ signal pathway can cause bone metabolic disorder, and further cause osteoporosis. From the perspective of drug development, the progress of diseases can be controlled, and even cured, by regulating the related signal pathways.

[0003] The detection and display of different signal pathway activation levels by luciferase reporter system are mainly achieved by specific reporter elements and luciferase connected together. However, in large-scale screening of signal pathway-related activators or inhibitors, the transfection of luciferase reporter plasmid and the addition of luciferin bring a large amount of work, which is difficult to achieve high-throughput screening, and the uniformity of each transfection experiment is also difficult to control.

[0004] Therefore, in order to facilitate and intuitively study the drugs acting on a certain signal pathway, a high-efficiency and sensitive screening system needs to be developed. SUMMARY

[0005] The present application aims to provide a high-efficiency and sensitive drug screening system acting on signal pathways.

[0006] In the first aspect of the present application, a screening system for screening inhibitors for a target signal pathway is provided, the screening system comprising a culture system and the following components present in the culture system:

[0007] (a) live detection cells, the genome of the detection cells having integrated an expression cassette, in which a reporter element of a target signal pathway, a suicide protein element and a protective protein element are operably linked,

[0008] wherein when the target signal pathway in the detection cells is activated under activated culture conditions, the detection cells express the suicide protein element and the protective protein element, and the suicide protein element induces apoptosis of the detection cells in the presence of a first inducer;

[0009] (b) a test substance to be screened;

[0010] wherein, when the target signaling pathway in the detection cell is activated and the detection cell expresses the suicide protein element, and the test substance does not cause the suicide protein element to degrade or decrease, the first inducer induces the detection cell to undergo apoptosis;

[0011] and, when the test substance causes the target signaling pathway in the detection cell to be not activated and the detection cell does not express the suicide protein element, the first inducer does not induce the detection cell to undergo apoptosis.

[0012] In another preferred embodiment, when the test substance causes the target signaling pathway in the detection cell to be not activated and the detection cell does not express the suicide protein element, the test substance is considered to be a candidate inhibitor.

[0013] In another preferred embodiment, the candidate inhibitor inhibits the activation of the target signaling pathway.

[0014] In another preferred embodiment, the "activating culture condition" refers to a culture condition capable of activating the target signaling pathway in the detection cell.

[0015] In another preferred embodiment, the screening system further comprises:

[0016] (c) a first inducer, which induces the detection cell to undergo apoptosis through the suicide protein element.

[0017] In another preferred embodiment, when the first inducer is present, the suicide protein element forms a dimer, thereby inducing cell apoptosis.

[0018] In another preferred embodiment, when the first inducer is absent, the suicide protein element maintains a monomer form and does not induce cell apoptosis.

[0019] In another preferred embodiment, the first inducer is AP1903.

[0020] In a second aspect of the present application, a screening system for screening an activator for a target signaling pathway is provided, which comprises a culture system and the following components present in the culture system:

[0021] (a) a living detection cell, wherein a target signaling pathway reporter element, a suicide protein element and a protector protein element are operably linked in an expression cassette integrated in the genome of the detection cell,

[0022] wherein, when the detection cell is under a normal culture condition, the target signaling pathway in the cell is not activated, and the detection cell does not express the suicide protein element and the protector protein element; the detection cell undergoes apoptosis in the presence of a second inducer.

[0023] (b) Test items to be screened;

[0024] Specifically, when the target signaling pathway in the detection cells is not activated and the protective protein element is not expressed, the second inducer induces apoptosis in the detection cells.

[0025] Furthermore, when the test substance causes activation of the target signaling pathway in the detection cells and expression of the protective protein element, and the test substance does not cause degradation or reduction of the protective protein element, the second inducer does not induce apoptosis in the detection cells.

[0026] In another preferred embodiment, the test substance is considered a candidate activator when it causes activation of the target signaling pathway in the detection cell and expression of the protective protein element, and the test substance does not cause degradation or reduction of the protective protein element.

[0027] In another preferred embodiment, the candidate activator activates the target signaling pathway.

[0028] In another preferred embodiment, "activating the target signaling pathway" means that the reporter element of the target signaling pathway binds to the transcription factor most downstream of the target signaling pathway and induces the detection cell to express suicide protein elements and protective protein elements.

[0029] In another preferred embodiment, the “normal culture conditions” refer to culture conditions in which the target signaling pathway in the detection cells is not activated.

[0030] In another preferred embodiment, the reporter element of the target signaling pathway includes the binding site of the most downstream transcription factor in the target signaling pathway and the downstream promoter.

[0031] In another preferred embodiment, the downstream promoter initiates the expression of downstream suicide protein elements and protective protein elements.

[0032] In another preferred embodiment, the screening system further includes:

[0033] (c) A second inducer that induces apoptosis in the test cells when the test cells do not express protective protein elements.

[0034] In another preferred embodiment, the second inducer is puromycin.

[0035] In another preferred embodiment, the expression cassette integrated into the genome of the detection cell has a structure from 5'-3' as shown in Formula I:

[0036] Z1-Z2-Z3-Z4 Formula I

[0037] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0038] Z1 is the reporting element;

[0039] Z2 is the coding sequence for the suicide protein element; and

[0040] Z3 is a sequence with no or no self-cutting;

[0041] Z4 is the coding sequence for a protective protein element;

[0042] The reporter element includes a binding site Y1 for the downstream transcription factor in the target signaling pathway and a downstream promoter Y2, wherein Y1 and Y2 are operatively connected so that when the binding site Y1 for the downstream transcription factor in the target signaling pathway binds to the downstream transcription factor in the target signaling pathway, the downstream promoter Y2 drives the expression of suicide protein elements and protective protein elements.

[0043] In another preferred embodiment, the expression cassette integrated into the genome of the detection cell has a structure from 5'-3' as shown in Formula II:

[0044] Y1-Y2-Z2-Z3-Z4 Formula II

[0045] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0046] Y1 is the binding site of the most downstream transcription factor in the target signaling pathway;

[0047] Y2 is the downstream promoter;

[0048] Z2 is the coding sequence for the suicide protein element; and

[0049] Z3 is a sequence with no or no self-cutting;

[0050] Z4 is the coding sequence for a protective protein element.

[0051] In another preferred embodiment, the downstream promoter Y2 is the minimal promoter of the TATA box.

[0052] In another preferred embodiment, the site Y1 contains the binding site of the most downstream transcription factor in the target signaling pathway repeated n times, where n is any positive integer from 1 to 20, preferably n is from 1 to 10.

[0053] In another preferred embodiment, the target signal pathway is selected from the Wnt pathway, TGFβ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1α pathway, JAK-STAT1 / 2 pathway, MAP / ERK pathway, cAMP / PKA pathway, NFκB pathway, and p53 pathway.

[0054] In another preferred embodiment, the most downstream transcription factor in the target signaling pathway is selected from the TCF / LEF complex, SMAD3 / 4 complex, YAP-TEAD / TEF complex, Nrf2, HIF1α, STAT1 / 2, AP1, CRE-binding protein, NFκB, and p53.

[0055] In another preferred embodiment, the coding sequence of the protective protein element is the Puromycin resistance gene.

[0056] In another preferred embodiment, the coding sequence of the suicide protein element is as shown in SEQ ID NO:1.

[0057] In another preferred embodiment, the self-cutting sequence is shown in SEQ ID NO:3.

[0058] In another preferred embodiment, the coding sequence of the protective protein element is shown in SEQ ID NO:2.

[0059] In another preferred embodiment, the sequence of the reporting element is selected from: SEQ ID NO:4, 5, 6, 7, 8, 9, 11, 12, 13.

[0060] In another preferred embodiment, the structure of the suicide protein element is shown in Formula II:

[0061] F-L2-C (II)

[0062] in,

[0063] Each "-" independently represents a linking peptide or peptide bond;

[0064] F is a suicide gene inducing element;

[0065] L2 is either a non-connector or a flexible connector;

[0066] C represents the suicide gene element.

[0067] In another preferred embodiment, C is the gene encoding cysteine ​​aspartate protease-9 (Caspase9 gene).

[0068] In another preferred embodiment, F is an FKBP12-F36V structural domain.

[0069] In another preferred embodiment, the FKBP12-F36V domain comprises an FKBP domain, and the 36th amino acid of the FKBP domain is mutated from phenylalanine to valine.

[0070] In another preferred embodiment, the cell is a genetically engineered cell.

[0071] In another preferred embodiment, the cell is a mammalian cell.

[0072] In another preferred embodiment, the cells are selected from: human renal epithelial cells (293A cells), human peripheral blood leukemia T cells (Jurkat T cells), JHH7 cells, and HeLa cells.

[0073] In a third aspect of the invention, a method for screening inhibitors of target signaling pathways is provided, comprising the steps of:

[0074] (a) Under activated culture conditions, a culture system containing the test compound to be screened is used as the experimental group; a culture system without the test compound is used as the blank control group. The culture system contains cultured live detection cells, the genome of which is integrated with an expression cassette in which a reporter element, a suicide protein element, and a protective protein element of the target signaling pathway are operatively linked. When the target signaling pathway in the detection cells is activated under activated culture conditions, the detection cells express the suicide protein element and the protective protein element, and the suicide protein element induces apoptosis in the detection cells in the presence of a first inducer.

[0075] (b) Add the first inducing agent to the experimental group and the blank control group, and observe the cell viability in the experimental group and the blank control group;

[0076] Specifically, if the number of viable cells detected in the experimental group is significantly higher than that in the control group, it indicates that the test substance is a candidate inhibitor.

[0077] In another preferred embodiment, "significantly higher than" means that the ratio of the number of apoptotic cells E1 detected in the experimental group to the number of apoptotic cells E0 detected in the blank experimental group (E1 / E0) is ≥1.5, preferably ≥3.0, and more preferably ≥4.

[0078] In another preferred embodiment, the method further includes: (c1) testing the inhibitory effect of the candidate inhibitor on the target signaling pathway, and / or its preventive or therapeutic effect on diseases related to the target signaling pathway.

[0079] In a fourth aspect of the invention, a method for screening target signaling pathway activators is provided, comprising the steps of:

[0080] (a) Under standard culture conditions, a culture system containing the test compound to be screened is used as the experimental group; a culture system without the test compound is used as the blank control group, wherein the culture system contains cultured live test cells; the genome of the test cells integrates an expression cassette, in which a reporter element, a suicide protein element, and a protective protein element of the target signaling pathway are operatively linked.

[0081] Wherein, when the target signaling pathway in the test cells is not activated under normal culture conditions, the test cells do not express the suicide protein element and the protective protein element; the test cells undergo apoptosis in the presence of the second inducing agent; and

[0082] (b) Add the second inducing agent to the experimental group and the blank control group, and observe the cell viability in the experimental group and the blank control group;

[0083] When the number of viable cells detected in the experimental group is significantly higher than that in the control group, it indicates that the test substance is a candidate activator.

[0084] In another preferred embodiment, "significantly higher than" means that the ratio of the number of apoptotic cells F1 detected in the experimental group to the number of apoptotic cells F0 detected in the blank experimental group (F1 / F0) is ≥1.5, preferably ≥3.0, and more preferably ≥5.

[0085] In another preferred embodiment, the method further includes: (c1) testing the activation effect of the candidate activator on the target signaling pathway, and / or its preventive or therapeutic effect on diseases related to the target signaling pathway.

[0086] In a fifth aspect of the invention, the use of a screening system as described in the first or second aspect of the invention is provided for screening target signaling pathway activators and / or inhibitors.

[0087] In a sixth aspect of the invention, a screening apparatus is provided for screening inhibitors or activators targeting a signaling pathway, the apparatus comprising:

[0088] (d1) Apoptosis screening module, wherein the apoptosis screening module includes one or more culture units, wherein the culture unit is provided with n culture chambers (or wells) for culturing live detection cells, wherein the culture chambers contain the screening system for screening inhibitors or activators targeting the target signaling pathway as described in the first or second aspect of the present invention; n is a positive integer ≥2;

[0089] (d2) Data acquisition module, which is configured to acquire data on the apoptosis status of the cells to be tested in each culture chamber of the apoptosis screening module;

[0090] (d3) A screening analysis module, configured to analyze apoptosis data from the data acquisition module to obtain analysis results on whether the test substance to be screened is an inhibitor or activator of the target signaling pathway; and

[0091] (d4) Output module, which outputs the analysis results of the filtering analysis module.

[0092] In another preferred embodiment, the number of culture units is 1-200, more preferably 4-100, even more preferably 8-50, and most preferably 10-20.

[0093] In another preferred embodiment, n is ≥16, preferably ≥48, more preferably ≥96, such as 16-100000, 48-10000, or 96-5000.

[0094] In another preferred embodiment, the culture unit is a multi-well plate, such as a 1536-well plate, a 384-well plate, or a 96-well plate.

[0095] In another preferred embodiment, the volume of the cell culture chamber (or well) for detection is 5 μl to 5 ml.

[0096] In another preferred embodiment, the screening system is a high-throughput screening system.

[0097] In another preferred embodiment, the culture unit includes a culture chamber for a blank control group and a culture chamber for an experimental group. The culture system containing the test compound to be screened is designated as the experimental group, and the culture system without the test compound is designated as the blank control group. (That is, the experimental group and the blank control group are identical except for the presence or absence of the test compound to be screened).

[0098] In another preferred embodiment, the culture unit is provided with m different experimental groups to test m different test substances or combinations of test substances to be screened, where m is a positive integer ≥1 (1-1600).

[0099] In a seventh aspect of the invention, an expression box is provided, the expression box having a structure from 5'-3' as shown in Formula I:

[0100] Z1-Z2-Z3-Z4 Formula I

[0101] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0102] Z1 is the reporting element;

[0103] Z2 is the coding sequence for the suicide protein element; and

[0104] Z3 is a sequence with no or no self-cutting;

[0105] Z4 is the coding sequence for a protective protein element;

[0106] The reporter element includes a binding site Y1 for the downstream transcription factor in the target signaling pathway and a downstream promoter Y2, wherein Y1 and Y2 are operatively connected so that when the binding site Y1 for the downstream transcription factor in the target signaling pathway binds to the downstream transcription factor in the target signaling pathway, the downstream promoter Y2 drives the expression of suicide protein elements and protective protein elements.

[0107] In another preferred embodiment, the expression cassette integrated into the genome of the detection cell has a structure from 5'-3' as shown in Formula II:

[0108] Y1-Y2-Z2-Z3-Z4 Formula II

[0109] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0110] Y1 is the binding site of the most downstream transcription factor in the target signaling pathway;

[0111] Y2 is the downstream promoter;

[0112] Z2 is the coding sequence for the suicide protein element; and

[0113] Z3 is a sequence with no or no self-cutting;

[0114] Z4 is the coding sequence for a protective protein element.

[0115] In another preferred embodiment, the downstream promoter Y2 is the minimal promoter of the TATA box.

[0116] In another preferred embodiment, the site Y1 contains the binding site of the most downstream transcription factor in the target signaling pathway repeated n times, where n is any positive integer from 1 to 20, preferably n is from 1 to 10.

[0117] In another preferred embodiment, the target signal pathway is selected from the Wnt pathway, TGFβ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1α pathway, JAK-STAT1 / 2 pathway, MAP / ERK pathway, cAMP / PKA pathway, NFκB pathway, and p53 pathway.

[0118] In another preferred embodiment, the most downstream transcription factor in the target signaling pathway is selected from the TCF / LEF complex, SMAD3 / 4 complex, YAP-TEAD / TEF complex, Nrf2, HIF1α, STAT1 / 2, AP1, CRE-binding protein, NFκB, and p53.

[0119] In another preferred embodiment, the coding sequence of the protective protein element is the Puromycin resistance gene.

[0120] In an eighth aspect of the invention, a carrier is provided, the carrier containing an expression cassette as described in the seventh aspect of the invention.

[0121] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0122] In a ninth aspect of the invention, a host cell is provided, wherein one or more sites of the host cell’s genome are integrated with the expression cassette of the seventh aspect of the invention, or wherein the host cell contains the vector of the eighth aspect of the invention.

[0123] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0124] In another preferred embodiment, the cell is a mammalian cell.

[0125] In another preferred embodiment, the cells are selected from: human renal epithelial cells (293A cells), human peripheral blood leukemia T cells (Jurkat T cells), JHH7 cells, and HeLa cells.

[0126] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0127] Figure 1 A schematic diagram of the luciferase reporter system components is shown; when the signaling pathway is activated, the luciferase expression level is high and the biofluorescence is strong; when the signaling pathway is inhibited, the luciferase expression level is low and the biofluorescence is weak.

[0128] Figure 2 A schematic diagram of the components of the live / dead phenotype report system is shown; when the signaling pathway is activated, the expression levels of the "suicide gene" and the "protective gene" are high; when the signaling pathway is inhibited, the expression levels of the "suicide gene" and the "protective gene" are low.

[0129] Figure 3The diagram shows the detailed components of the live / dead phenotype reporter system; Figure a is a schematic diagram of AP1903-induced dimerization of FKBP12(F36V)-ΔCASP9; Figure b shows the live / dead phenotype reporter elements: TFs binding sites are the transcription factor binding site sequences; minP is the minimal promoter containing a TATA box; FKBP12(F36V)-ΔCASP9 is the "suicide gene" expressing a suicide protein; PuroR is the "protective gene" that protects cells from puromycin killing; P2A is the self-cleaving sequence, which causes the FKBP12(F36V)-ΔCASP9 and PuroR genes to be cleaved into two proteins after translation and expression.

[0130] Figure 4 The diagram shows the correspondence between signaling pathway status and cell viability / death phenotypes. In Figure a, under normal culture conditions, the signaling pathway to be screened is inactive; cells survive after AP1903 treatment and die after puromycin treatment. Under activated culture conditions (when a signaling pathway activating factor is added), the pathway is activated; cells die after AP1903 treatment and survive after puromycin treatment. Figure b is a schematic diagram of the high-throughput drug screening process, where Puromycin represents puromycin. Cells in plate A1 are cultured under normal culture conditions, and cells in plate A2 are cultured under activated culture conditions.

[0131] Figure 5 The diagram shows the validation of the Wnt pathway reporter cell line. Figure a shows the elements of the Wnt pathway live / dead phenotypic reporter system; Figure b shows the activation of the Wnt signaling pathway after treatment with 20 mM LiCl and 10% R-spondin1 for 1 day; Figure c shows the activation of the Wnt signaling pathway after knocking out CK1; Figure d shows that the Wnt pathway is not activated after treatment with 100 nM WNT-C59 for 1 day while cells are activated with 10% R-spondin1. WT represents wild-type. PURO and AP1903 represent treatment with puromycin and AP1903, respectively. Here, minP is the minimal promoter containing the TATA box, P2A is the self-splicing sequence, PuroR is the "protective gene," 7×TCF is the binding site for 7 repeats of TCF / LEF, and RSPO1 is R-spondin1.

[0132] Figure 6The diagram shows the validation of the TGFβ pathway reporter cell line; Figure a shows the TGFβ pathway live / dead phenotype reporter system elements; Figure b shows that after treatment with 10 ng / ml TGFβ for 1 day, the TGFβ signaling pathway was activated. Simultaneously with TGFβ activation, treatment with 500 nM LY2109761 or SB525334 for 1 day did not activate the TGFβ pathway. In the diagram, PURO represents puromycin, minP is the minimal promoter containing the TATA box, P2A is the self-cleaving sequence, PuroR is the "protective gene," and 4×SBE is the binding site SBE for four repeats of the Smad3 / 4 complex.

[0133] Figure 7 The diagram shows the validation of the Hippo pathway reporter cell line; Figure a shows the Hippo pathway live / dead phenotype reporter system elements; Figure b shows that under high-density culture conditions, YAP is degraded and does not enter the nucleus, and PuroR and FKBP12(F36V)-ΔCASP9 are not expressed; under low-density culture conditions, YAP enters the nucleus, and PuroR and FKBP12(F36V)-ΔCASP9 are expressed; Figure c shows that after overexpression of the activated mutant YAP (S127A), regardless of density, YAP abnormally remains in the nucleus, and PuroR and FKBP12(F36V)-ΔCASP9 are expressed. In this diagram, PURO represents puromycin, minP is the minimal promoter containing a TATA box, P2A is the self-splicing sequence, PuroR is the "protective gene," and 3×SD represents the binding sites of three repeated TEAD / TEF sequences.

[0134] Figure 8 The validation diagram of the Keap1-Nrf2 pathway reporter cell line is shown. Figure a shows the live / dead phenotype reporter system elements of the Keap1-Nrf2 pathway; Figure b shows the activation of the Keap1-Nrf2 signaling pathway after treatment with 50 μM tBHQ for 1 day; Figure c shows the activation of the pathway after Keap1 knockout. WT represents wild-type, PURO represents puromycin, minP is the minimal promoter containing a TATA box, P2A is the self-splicing sequence, PuroR is the "protective gene," and 2×ARE represents the binding site ARE for two duplicated Nrf2 molecules.

[0135] Figure 9The diagram shows the validation of the HIF1α pathway reporter cell line; Figure a shows the HIF1α pathway live / dead phenotype reporter system elements; Figure b shows the activation of the HIF1α signaling pathway after cells were treated with 10 μM 1,10-phenanthroline for 5 h; Figure c shows the activation of the pathway after VHL knockout. WT represents wild-type, PURO represents puromycin, minP is the minimal promoter containing a TATA box, P2A is the self-splicing sequence, PuroR is the "protective gene," and 4×HRE represents four repeated HIF1α-regulated binding sites (HREs).

[0136] Figure 10 The validation diagram of the STAT1 / 2 pathway reporter cell line is shown; Figure a shows the STAT1 / 2 pathway live / dead phenotype reporter system elements; Figure b shows the activation of the STAT1 / 2 signaling pathway after cells were treated with 10 ng / ml IFNβ for 1 day. In the diagram, PURO represents puromycin, minP is the minimal promoter containing a TATA box, P2A is the self-cleaving sequence, PuroR is the "protective gene," and 5×ISRE represents five repeats of the interferon stimulation response element ISRE.

[0137] Figure 11 The validation diagram of the MAPK / ERK pathway reporter cell line is shown. Figure a shows the MAP / ERK pathway live / dead phenotype reporter system elements. Figure b shows that the MAP / ERK pathway is not activated under normal culture conditions, but is activated after overnight starvation with medium containing 1% serum, followed by treatment with medium containing 20% ​​serum and 10 ng / ml PMA for 6 h. In the diagram, PURO represents puromycin, minP is the minimal promoter containing the TATA box, P2A is the self-cleaving sequence, PuroR is the "protective gene," and AP1-RE is the AP1 response element AP1-RE.

[0138] Figure 12 The validation diagram of the CREB pathway reporter cell line is shown; Figure a shows the cAMP / PKA pathway live / dead phenotype reporter system elements; Figure b shows the activation of the cAMP / PKA signaling pathway after cells were treated with 5 μg / ml Forskolin for 1 day. In the diagram, PURO represents puromycin, minP is the minimal promoter containing the TATA box, P2A is the self-cleaving sequence, PuroR is the "protective gene", and CRE is the cAMP response element CRE.

[0139] Figure 13The diagram shows the validation of the NFκB pathway reporter cell line; Figure a shows the elements of the NFκB pathway live / dead phenotype reporter system; Figure b shows the activation of the NFκB signaling pathway after treatment with 20 ng / ml TNFα for 5 h. In the diagram, PURO represents puromycin, minP is the minimal promoter containing a TATA box, P2A is the self-cleaving sequence, PuroR is the "protective gene," and 5×NFκB-RE represents the NFκB-RE binding site with five repeats.

[0140] Figure 14 The diagram shows the validation of the p53 pathway reporter cell line; Figure a shows the p53 pathway live / dead phenotype reporter system elements; Figure b shows the activation of the p53 signaling pathway after cells were treated with 100 nM DOX for 20 h. In the diagram, PURO represents puromycin, minP is the minimal promoter containing a TATA box, P2A is the self-cleaving sequence, PuroR is the "protective gene," and 2×NFκB-RE is the p53-RE binding site for two repeats of p53.

[0141] Figure 15 The results of drug screening are partially validated. Figure a shows some of the Wnt pathway inhibitors obtained through screening. Figure b shows that 293T-TCF7 cells treated with 10% R-spondin1 and the screened inhibitors (28-H11, 3-F7) for 24 h showed that the inhibitors 28-H11 and 3-F7 reduced β-catenin stability and decreased the level of active β-catenin. RSPO1 is R-spondin1, and actin is actin.

[0142] Figure 16 The effects of TGFβ pathway inhibitors are shown; Figure a shows some of the screened TGFβ pathway inhibitors 35-C11, 35-D10, and 33-E10; Figure b shows the screened TGFβ pathway activator 21-H2. PURO represents puromycin. Detailed Implementation

[0143] Through extensive and in-depth research, the inventors have developed for the first time a screening system based on cell viability / inactivation phenotype for inhibitors or activators of target signaling pathways. Using this system, high-throughput screening of inhibitors or activators of target signaling pathways can be achieved. Specifically, detection cells with expression cassettes integrated into their genomes, treated with AP1903 or puromycin, convert the activation or silencing of the target signaling pathway into a complete viability / inactivation phenotype with pathway specificity. Compared to traditional experimental methods involving luciferase transfection and luciferin addition, the gene components of this invention's screening system are stably integrated into the cell chromosome, resulting in a stable viability / inactivation phenotype. Only a trace amount of the compound to be screened is required to achieve large-scale, high-throughput screening. This invention is based on this foundation.

[0144] the term

[0145] As used herein, "operably linked to" means linking a target gene intended for transcription and expression to its control sequence in a manner conventional in the art for transcription and / or expression. For example, certain portions of a linear DNA sequence can regulate or control the activity of other portions of the same linear DNA sequence. For instance, if a promoter controls the transcription of a sequence, then it is operably linked to a coding sequence.

[0146] In this invention, the binding site Y1 of the downstream transcription factor in the target signaling pathway is operatively connected to the promoter Y2, so that when the binding site Y1 of the downstream transcription factor in the target signaling pathway binds to the downstream transcription factor in the target signaling pathway, the downstream promoter Y2 drives the expression of suicide protein elements and protective protein elements.

[0147] Expression Box

[0148] In this invention, an expression box as described in Formula I is provided, the expression box having a structure from 5' to 3':

[0149] Z1-Z2-Z3-Z4 Formula I

[0150] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0151] Z1 is the reporting element;

[0152] Z2 is the coding sequence for the suicide protein element; and

[0153] Z3 is a sequence with no or no self-cutting;

[0154] Z4 is the coding sequence for a protective protein element;

[0155] The reporter element includes a binding site Y1 for the downstream transcription factor in the target signaling pathway and a downstream promoter Y2, wherein Y1 and Y2 are operatively connected so that when the binding site Y1 for the downstream transcription factor in the target signaling pathway binds to the downstream transcription factor in the target signaling pathway, the downstream promoter Y2 drives the expression of suicide protein elements and protective protein elements.

[0156] In another preferred embodiment, the expression cassette integrated into the genome of the detection cell has a structure from 5'-3' as shown in Formula II:

[0157] Y1-Y2-Z2-Z3-Z4 Formula II

[0158] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0159] Y1 is the binding site of the most downstream transcription factor in the target signaling pathway;

[0160] Y2 is the downstream promoter;

[0161] Z2 is the coding sequence for the suicide protein element; and

[0162] Z3 is a sequence with no or no self-cutting;

[0163] Z4 is the coding sequence for a protective protein element.

[0164] In this invention, the reporter element contains binding sites for one or more downstream transcription factors in the target signaling pathway.

[0165] It should be understood that the signaling pathway transcription factor is the most downstream transcription factor in the target signaling pathway. After binding to the Y1 site of the reporter element and activating the corresponding signaling pathway, the promoter Y2 in the reporter element is activated, promoting the downstream pathway to start transcription and express the corresponding protein.

[0166] In this invention, the reporter element is composed of the binding site sequence of the transcription factor of each signaling pathway and a minimal promoter containing a TATA box, and the reporter element of each signaling pathway is a verified regulated and specific promoter.

[0167] like Figure 2As shown, when the corresponding signaling pathway in the cell is not activated, the promoter is in the off state, and the coding sequences of the "suicide protein element" (i.e., the "suicide gene") and the "coding sequences of the protection protein element" (i.e., the "protection gene") are not expressed. When the signaling pathway activates downstream transcription factors to promote transcription, the promoter initiates the expression of the "suicide gene" and the "protection gene." Those skilled in the art will understand that the order in which the "suicide gene" and the "protection gene" are located within the expression cassette is not particularly limited, as long as they can be expressed under the regulation of the reporter element. Furthermore, the reporter element may also contain coding sequences for a leader peptide or secreted peptide, or a tag sequence (such as a 6His tag).

[0168] The "suicide gene," which encodes a suicide protein element, is a gene that expresses the FKBP12(F36V)-ΔCASP9 fusion protein. Figure 3 a) In a preferred embodiment of the invention, the suicide protein element comprises an FKBP12-F36V domain, which can be linked via a flexible Ser-Gly-Gly-Gly-Ser linker to cysteine-aspartic protease 9 (Caspase 9), which does not contain a recruitment domain (denoted as ΔCASP9). FKBP12-F36V comprises an FKBP domain in which phenylalanine replaces valine at the 36th amino acid residue. It exhibits high selectivity and sub-nanomolar affinity, enabling it to bind dimerized ligands such as other inert small molecules like AP1903 (Rimiducid, Bellicum Pharmaceutical).

[0169] Cells expressing this suicide protein element can be induced to undergo apoptosis by the compound AP1903. The basic principle is that the F36V mutant of FKBP12 can bind to AP1903. Since AP1903 is a symmetrical molecule, it can induce the self-aggregation of the FKBP12(F36V)-ΔCASP9 fusion protein. After the apoptosis-executing protein Caspase9 undergoes self-aggregation, it rapidly induces apoptosis. Therefore, when downstream transcription factors in a signaling pathway are active, FKBP12(F36V)-ΔCASP9 is highly expressed in cells, and cell death occurs after the addition of AP1903; conversely, when downstream transcription factors are silenced, FKBP12(F36V)-ΔCASP9 is almost not expressed, and the addition of AP1903 has no effect on the cells. Figure 4 ).

[0170] The "protective gene" encoding the protective protein element is the puromycin resistance gene. Figure 3Puromycin is an antibiotic that inhibits protein synthesis. When an appropriate concentration of puromycin is added to cell culture medium, the cells die. Cells can only survive if they express the puromycin resistance gene. Therefore, when downstream transcription factors in a signaling pathway are active, the puromycin resistance gene is highly expressed in cells, and the cells survive after the addition of puromycin; conversely, when downstream transcription factors are silenced, cells do not express the puromycin resistance gene, and the cells die after the addition of puromycin. Figure 4 ).

[0171] In another preferred embodiment, the sequence encoding the FKBP12(F36V)-ΔCASP9 protein is shown in SEQ ID NO:1; the nucleotide sequence protecting the protein element is shown in SEQ ID NO:2; and the self-cleaving sequence is shown in SEQ ID NO:3.

[0172] This invention achieves stable integration of expression cassettes into the cellular genome via lentiviral infection, thereby constructing reporter cell lines with different signaling pathways. The target signaling pathways are selected from the Wnt pathway, TGFβ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1α pathway, JAK-STAT1 / 2 pathway, MAP / ERK pathway, cAMP / PKA pathway, NFκB pathway, and p53 pathway.

[0173] Taking the Wnt signaling pathway as an example, this is an evolutionarily conserved pathway that plays a crucial role in embryonic development and the formation of the central nervous system, regulating cell growth, migration, and differentiation. In the absence of Wnt ligand signaling, the cytoplasmic regulatory complex promotes the phosphorylation of β-catenin, leading to its degradation by the proteasome. In the presence of Wnt ligand signaling, β-catenin is not degraded, accumulates, and increases its nuclear translocation. As a co-activator of the transcription factors TCF / LEF, it promotes the expression of downstream target genes, thereby promoting cell proliferation. When key members of the Wnt signaling pathway mutate, signal transduction becomes abnormal. For example, APC mutations are frequently observed in colorectal cancer. These mutations prevent the degradation of β-catenin, leading to persistent activation of the Wnt pathway and cell carcinogenesis. Therefore, if suitable drugs can inhibit the persistent activation of the Wnt pathway caused by APC mutations, such inhibitors may be applicable to colorectal cancer patients with APC mutations.

[0174] As previously described, the most downstream transcriptional event in the Wnt pathway is performed by the β-catenin-TCF / LEF complex. The reporter element of the Wnt pathway consists of seven repetitions of the TCF / LEF binding site (abbreviated as 7×TCF). When the target signaling pathway is Wnt, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:4.

[0175] The most downstream transcriptional event in the TGFβ pathway is performed by the SMAD3 / 4 complex. The reporter element of the TGFβ pathway consists of four repetitions of the SBE binding site of the Smad3 / 4 complex. When the target signaling pathway is TGFβ, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:5.

[0176] The most downstream transcriptional event in the Hippo pathway is performed by the YAP-TEAD / TEF complex. The reporter element of the Hippo pathway contains a TEAD / TEF binding site repeated three times. When the target signaling pathway is HIPPO, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:6.

[0177] The most downstream transcriptional event in the Keap1-Nrf2 pathway is performed by the transcription factor Nrf2. The reporter element of the Keap1-Nrf2 pathway contains a Nrf2 binding site ARE repeated twice. When the target signaling pathway is Keap1-Nrf2, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:7.

[0178] The most downstream transcriptional event in the VHL-HIF1α pathway is performed by the transcription factor HIF1α. The reporter element of the HIF1α pathway contains an HRE with a HIF1α-regulated binding site repeated four times. When the target signaling pathway is VHL-HIF1α, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:8.

[0179] The most downstream transcriptional event in the JAK-STAT1 / 2 pathway is performed by the transcription factor STAT1 / 2. The reporter element of the STAT1 / 2 pathway consists of an interferon-stimulated response element (ISRE) repeated five times. When the target signaling pathway is JAK-STAT1 / 2, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:9.

[0180] The most downstream transcriptional event in the MAP / ERK pathway is performed by the transcription factor AP-1, and the corresponding reporter element contains the AP1 response element AP1-RE. When the target signaling pathway is MAP / ERK, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO: 10.

[0181] The reporter element of the cAMP / PKA pathway contains a cAMP response element (CRE). When the target signaling pathway is cAMP / PKA, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:11.

[0182] The reporter element of the NFκB pathway contains an NFκB-RE binding site repeated 5 times. When the target signaling pathway is NFκB, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:12.

[0183] The reporter element of the p53 pathway contains a p53-RE binding site, which is repeated twice. When the target signaling pathway is p53, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO:13.

[0184] Vector and host cell

[0185] By inserting the expression box of the present invention into a carrier, a carrier carrying the reporting element or expression box can be obtained.

[0186] The vector can be used to transfect host cells to obtain host cells carrying the expression cassette or host cells with the expression cassette integrated into their genome.

[0187] In this invention, the host cell is generally a mammalian cell.

[0188] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. The following DNA transfection methods can be used: viral packaging infection, calcium phosphate co-precipitation, and conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

[0189] In this invention, host cells transformed using the above method and carrying the expression cassette of Formula I can be used to screen for agonists or inhibitors of target signaling pathways.

[0190] The screening system and method of the present invention

[0191] In this invention, specific promoters responding to different signaling pathway activities are linked to the "suicide gene" FKBP12(F36V)-ΔCASP9 and the "protective gene" puromycin resistance gene. These are then stably integrated into the cell's chromosome via a viral vector, constructing a series of cells stably expressing a live / dead phenotype reporter system. These cells are the test cells used in this invention to screen for target signaling pathway activators and / or inhibitors. Under standard culture conditions, the signaling pathway is inactive, and cells die upon the addition of puromycin. If a candidate compound activates the signaling pathway, the cells survive upon the addition of puromycin. This experimental phenotype can be used for high-throughput screening of signaling pathway activators.

[0192] Correspondingly, when an activator of a certain pathway (such as TGF-β) is added during cell culture, the conditional suicide protein FKBP12(F36V)-ΔCASP9 is expressed in the cells, and AP1903 can kill the cells. If the candidate compound has an inhibitory effect on this signaling pathway, the cells survive after the addition of AP1903; this experimental phenotype can be used for high-throughput screening of signaling pathway inhibitors.

[0193] The experimental conditions described above utilize the expression of protective genes and suicide genes driven by signaling pathway transcription factors, which can efficiently enable large-scale screening of activators and inhibitors of signaling pathways.

[0194] Table 1. Activators and inhibitors of signaling pathways

[0195]

[0196] Based on the above system, high-throughput screening of activators and inhibitors of various signaling pathways can be performed using cell viability phenotypes. The same batch of compounds can be screened in reporter cell lines of multiple signaling pathways, thus ensuring that the screened activators and inhibitors have relative specificity.

[0197] Specifically, in a preferred drug screening process for screening target signaling pathway activators according to the present invention:

[0198] Day 1: Cells were seeded and cultured in 384-well plates under standard culture conditions;

[0199] Day 2: Treat cells with 10 μM of a compound library drug;

[0200] Day 3: Treat cells with puromycin.

[0201] Day 4: Cell survival is assessed. Most cells in the pores die, while a small number survive. The test substance corresponding to the surviving cell pores may be an activator of the target signaling pathway.

[0202] In a preferred drug screening process for screening inhibitors of the target signaling pathway according to the present invention:

[0203] Day 1: Cells were seeded and cultured in 384-well plates under activated culture conditions;

[0204] Day 2: Treat cells with 10 μM of a compound library drug;

[0205] Day 3: Treat cells with AP1903.

[0206] Day 4: Cell survival is assessed. Most cells in the pores die, while a small number survive. The test substance corresponding to the surviving cell pores may be an inhibitor of the target signaling pathway.

[0207] In the compound screening of this invention, the test compound corresponding to the surviving cells is screened against a background of mostly dead cells. Cell phenotypes are easily observed and can intuitively reflect the regulatory effect of compounds on signaling pathways. The same compound only needs to be screened twice when the target signaling pathway in the cell is silenced or activated, making the method simple.

[0208] The main advantages of this invention include:

[0209] 1. Cell viability can directly reflect the regulatory effect of compounds on signaling pathways and has pathway specificity;

[0210] 2. Each reporter element has been stably integrated into the cell line, making the operation simple and highly consistent when performing high-throughput drug screening;

[0211] 3. This system can be modularly applied to multiple signaling pathways, and multiple pathways can be screened for inhibitors and activators in parallel in the same screening.

[0212] 4. The dead / live phenotype reporting system in this invention is suitable for large-scale high-throughput screening.

[0213] The following specific embodiments further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0214] General Method

[0215] 1. Construct plasmids that drive the expression of protective and suicide genes by transcription factors in related signaling pathways.

[0216] 2. The plasmid was stably integrated into animal cells.

[0217] 3. After constructing the cell plates, divide them into control and experimental groups. Treat the control group with a control solvent (such as DMSO) or a 10 μM drug (generally set at 10 μM for large-scale initial screening, but can be determined according to the characteristics of the drug library) for a period of time (depending on the characteristics of different drugs and signaling pathways). Then treat with puromycin or AP1903 for 1 day.

[0218] 4. Observe the cell viability in the control group and the experimental group.

[0219] Example 1: Reporter cell lines with different signaling pathways

[0220] Cell line construction

[0221] All reporter cell lines were constructed using lentiviral infection to achieve stable integration. The experimental procedure is as follows:

[0222] 1. Lentiviral plasmids and packaging plasmids were co-transfected into 293T cells via calcium transfection and other transfection methods;

[0223] 2. Collect the cell culture supernatant 48 hours after transfection, filter (to remove cells), and obtain the virus solution;

[0224] 3. Infected cells are seeded in a six-well plate, 1 ml of culture medium and 1 ml of virus solution are added, centrifuged at 1000×g for 5 min, and then incubated in an incubator;

[0225] 4. Remove the virus solution 1-2 days after cell infection and culture in ordinary medium;

[0226] 5. The infection efficiency of cells is detected by flow cytometry or fluorescence microscopy (the lentiviral vector used in this system is labeled with mCherry fluorescence). If the infection efficiency is less than 100%, all positive cells can be obtained by flow cytometry sorting.

[0227] 1. Wnt pathway reporter cell line

[0228] Will Figure 5 The α sequence was stably integrated into 293T cells, resulting in 293T-7TCF cells. Under standard culture conditions, the Wnt pathway in 293T-7TCF cells exhibited low activity. Figure 5 The α gene element was not expressed, and cells died after treatment with 2 μg / ml puromycin, while treatment with 10 nM AP1903 had no effect on the cells; as a positive control, when the Wnt pathway activator 20 mM LiCl or 10% R-Spondin1 was added, Figure 5 Expression of gene components of type α leads to cell acquisition of puromycin resistance, rendering puromycin ineffective in killing cells, while expression of FKBP12(F36V)-ΔCASP9 results in complete cell death by AP1903. Figure 5 b).

[0229] exist Figure 5 In experiment c, the negative regulator of the Wnt pathway, CK1, was knocked out using CRISPR-sgRNA. Under these conditions, β-catenin was not degraded, and TCF / LEF continued to perform its transcriptional function. Figure 5The gene components of α were continuously expressed, and cells treated with puromycin survived, while cells treated with AP1903 died. When the Wnt pathway was activated by R-Spondin1 (10%), treatment with the Wnt pathway inhibitor WNT-C59 (100 nM) could block the activity of the Wnt pathway and reverse the cell death-liveness phenotype. Figure 5 d).

[0230] Table 2. Status of Wnt pathway reporter cell lines under different treatment conditions

[0231] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 20mM LiCl Active Dead Live 10% R-Spondin1 Active Dead Live Knockout CK1 Active Dead Live

[0232] 2. TGFβ pathway reporter cell lines

[0233] Will Figure 6 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-4SBE cells. Under standard culture conditions, this gene component was not expressed in 293A-4SBE cells, and cell death occurred after treatment with 2 μg / ml puromycin, while treatment with 10 nM AP1903 had no effect. However, when 10 ng / ml TGFβ was added to activate this pathway, the gene component was expressed; cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Treatment with the TGFβ receptor inhibitors LY2109761 (500 nM) or SB525334 (500 nM) both blocked the activation of TGFβ, reversing the live / dead phenotype. Figure 6 b).

[0234] Table 3. Status of TGFβ pathway reporter cell lines under different treatment conditions

[0235] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 10ng / ml TGF Active Dead Live 500nM LY2109761 Silenced Live Dead 500nM SB525334 Silenced Live Dead

[0236] 3. Hippo pathway reporter cell lines

[0237] Will Figure 7 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-3SD cells. At low cell density, YAP in 293A-3SD cells enters the nucleus, promoting TEAD / TEF transcription. Cells treated with 2 μg / ml puromycin survive at this density, while those treated with 10 nMAP1903 die. However, when cells grow to high density and contact inhibition occurs, YAP is degraded and does not enter the nucleus, and TEAD / TEF transcription ceases. At this density, cells treated with puromycin die, while those treated with AP1903 survive. Figure 7b). However, when the activating mutant YAP (S127A) is stably expressed in cells, YAP abnormally remains in the nucleus, continuously promoting TEAD / TEF transcription. This results in the survival of cells in the puromycin-treated group and cell death in the AP1903-treated group, regardless of cell density. Figure 7 c).

[0238] Table 4. Status of Hippo pathway reporter cell lines under different treatment conditions

[0239] Treatment condition Signaling pathway status AP1903 Puro Low cell density Active Dead Live High cell density Silenced Live Dead Expression YAP(S127A) Active Dead Live

[0240] 4. Keap1-Nrf2 pathway reporter cell lines

[0241] Will Figure 8 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-2ARE cells. Under standard culture conditions, this pathway was inactive in 293A-2ARE cells, the gene component was not expressed, and cell death occurred after treatment with 2 μg / ml puromycin. Treatment with 10 nM AP1903 had no effect on the cells. However, when 50 μM tBHQ was added to activate this pathway, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 8 b). After KEAP1 was knocked out with sgRNA, Nrf2 was not degraded and continued to perform transcriptional function. Therefore, cells treated with puromycin survived, while cells treated with AP1903 died. Figure 8 c).

[0242] Table 5. Status of Keap1-Nrf2 pathway reporter cell lines under different treatment conditions

[0243] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 50μM tBHQ Active Dead Live Knockout KEAP1 Active Dead Live

[0244] 5. VHL-HIF1α pathway reporter cell lines

[0245] Will Figure 9 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-4HRE cells. Under standard culture conditions, this pathway was inactive in 293A-4HRE cells, the gene component was not expressed, and cell death occurred after treatment with 2 μg / ml puromycin. Treatment with 10 nMAP1903 had no effect on the cells. However, when 10 μM 1,10-phenanthroline was added to activate this pathway, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 9 b). After VHL was knocked out with sgRNA, HIF1α was not degraded and continued to perform transcriptional function. Therefore, cells treated with puromycin survived, while cells treated with AP1903 died. Figure 9 c).

[0246] Table 6. Status of VHL-HIF1α pathway reporter cell lines under different treatment conditions

[0247] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 10μM 1,10-Phenanthroline Active Dead Live Knockout VHL Active Dead Live

[0248] 6. JAK-STAT1 / 2 pathway reporter cell lines

[0249] Will Figure 10 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-5ISRE cells. Under standard culture conditions, this pathway was inactive in 293A-5ISRE cells, the gene component was not expressed, and cell death occurred after treatment with 2 μg / ml puromycin. Treatment with 10 nM AP1903 had no effect on the cells. However, when 10 ng / ml IFNβ was added to activate the pathway, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 10 b).

[0250] Table 7. Status of JAK-STAT1 / 2 pathway reporter cell lines under different treatment conditions

[0251] Treatment condition Signaling pathway status AP1903 Puro Regular Active Dead Live 10ng / ml IFN Silenced Live Dead

[0252] 7. MAP / ERK pathway reporter cell lines

[0253] Will Figure 11 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-AP1-RE cells. Under standard culture conditions, the pathway was inactive and the gene component was not expressed in 293A-AP1-RE cells. Treatment with 2 μg / ml puromycin resulted in cell death, while treatment with 10 nM AP1903 had no effect. However, when cells were starved overnight with culture medium containing 1% serum, followed by treatment with culture medium containing 20% ​​serum and 10 ng / ml PMA, the pathway was activated, the gene component was expressed, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 11 b).

[0254] Table 8. Status of MAP / ERK pathway reporter cell lines under different treatment conditions

[0255] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 20% serum and 10ng / ml PMA Active Dead Live

[0256] 8. cAMP / PKA pathway reporter cell lines

[0257] Will Figure 12The sequence 'a' was stably integrated into 293A cells, resulting in 293A-CRE cells. Under standard culture conditions, this pathway was inactive in 293A-CRE cells, the gene component was not expressed, and cell death occurred after treatment with 2 μg / ml puromycin. Treatment with 10 nMAP1903 had no effect on the cells. However, when 5 μg / ml Forskolin was added to activate the pathway, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 12 b).

[0258] Table 9. Status of TGFβ pathway reporter cell lines under different treatment conditions

[0259] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 5μg / ml Forskolin Active Dead Live

[0260] 9. NFκB pathway reporter cell lines

[0261] Will Figure 13 The sequence 'a' was stably integrated into 293A cells, resulting in 293A-5NFκB-RE cells. Under standard culture conditions, this pathway was inactive in 293A-5NFκB-RE cells, the gene components were not expressed, and cell death occurred after treatment with 2 μg / ml puromycin. Treatment with 10 nM AP1903 had no effect on the cells. However, when 20 ng / ml TNFα was added to activate this pathway, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 13 b).

[0262] Table 10. Status of NFκB pathway reporter cell lines under different treatment conditions

[0263]

[0264]

[0265] 10. p53 pathway reporter cell lines

[0266] Will Figure 14 The sequence 'a' was stably integrated into U2OS cells, resulting in U2OS-2p53-RE cells. Under standard culture conditions, this pathway was inactive in U2OS-2p53-RE cells, and the gene components were not expressed. Treatment with 2 μg / ml puromycin led to cell death, while treatment with 10 nM AP1903 had no effect. However, when 100 nM DOX was added to activate this pathway, cells in the puromycin-treated group survived, while cells in the AP1903-treated group died. Figure 14 b).

[0267] Table 11. Status of p53 pathway reporter cell lines under different treatment conditions

[0268] Treatment condition Signaling pathway status AP1903 Puro Regular Silenced Live Dead 100nM DOX Active Dead Live

[0269] Example 2: Wnt pathway inhibitors for cell viability / death phenotype screening

[0270] Currently, this system has been used for high-throughput compound screening in 384-well plates, and some pathway activators and inhibitors have been identified. In 293T-7TCF cells activated with 10% R-Spondin1, some compounds were found to maintain cell survival after AP1903 treatment, indicating that these compounds (natural small molecule compounds 28-H11 and 3-F7) can inhibit Wnt pathway activation and are potential inhibitors of the Wnt pathway. Figure 15 a).

[0271] To further verify the effects of the screened inhibitors, cells were simultaneously treated with the screened Wnt pathway inhibitors 28-H11, 3-F7 and R-spondin1, and the levels of β-catenin and active β-catenin were detected by Western blot.

[0272] Experimental results show that the inhibitors 28-H11 and 3-F7 can significantly reduce the stability of β-catenin and decrease the level of active β-catenin. Figure 15 b).

[0273] Example 3: Activators of the TGFβ pathway in cell viability phenotype screening

[0274] Furthermore, in TGFβ-activated 293A-4SBE cells, several natural small molecule compounds (35-C11, 35-D10, 33-E10) were found to maintain cell survival after AP1903 treatment, indicating that these compounds can inhibit the activation of the TGFβ pathway. Figure 16 a). In 293A-4SBE cells cultured under standard conditions, a natural small molecule compound, 21-H2, was found to enable the cells to survive after treatment with puromycin, suggesting that this compound may be an activator of the TGFβ pathway. Figure 16 b). There have been no previous reports of small molecule activators of the TGF pathway, therefore these results demonstrate the unique effectiveness of the screening system of this invention.

[0275] discuss

[0276] Other signaling pathways are similar to the Wnt pathway; after stimulation by upstream related signals, the expression of corresponding genes is ultimately regulated by different transcription factors. Because the transcription factors regulated by each signaling pathway are specific, and the DNA sequences bound to these different transcription factors are also specific, different signaling pathways can regulate the expression of different target genes. Researchers have utilized this characteristic of different transcription factors recognizing specific DNA sequences to design experimental methods to detect the activation level of signaling pathways. Among these, the luciferase reporter assay is a common method, which can be used to detect the effect of a specific gene or drug on the activation level of a signaling pathway.

[0277] The detection and visualization of activation levels in different signaling pathways by the luciferase reporter system is mainly achieved through the interconnection of specific reporter elements and luciferase. Figure 1 The reporter element here consists of a binding site sequence of a downstream transcription factor in a signaling pathway, repeated multiple times, and a minimal promoter containing a TATA box. Therefore, the expression level of luciferase reflects the activation level of the corresponding signaling pathway. In experiments, a luciferase reporter plasmid is first transfected into cells. After the addition of luciferin, luciferase catalyzes the oxidation of luciferin, emitting biofluorescence during this process. The activation level of the corresponding signaling pathway can be determined by the fluorescence intensity. When the activation level of the signaling pathway is high, the transcription factor is active, the luciferase expression level is high, and the emitted biofluorescence is strong; when the signaling pathway is inhibited, the transcription factor is silenced, the luciferase expression level is low, and the emitted biofluorescence is weak.

[0278] However, large-scale screening of activators or inhibitors related to signaling pathways involves a large workload and is difficult to operate, making high-throughput screening challenging. Furthermore, controlling the uniformity of results from each transfection experiment is also difficult. Therefore, to more conveniently and intuitively study drugs acting on a specific signaling pathway, the reporter cell line constructed in this invention, which can be directly screened and identified using live / dead phenotypes, can be used efficiently, sensitively, and stably for high-throughput compound screening.

[0279] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences <120> Novel Screening System for Signal Activators and Inhibitors Based on Cell Viability / Activity Phenotypes <130> P2020‑2034 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 1188 <212> DNA <213> Artificial Sequence <400> 1 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaagt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaatctggc ggtggatccg gatttggtga tgtcggtgct 360 cttgagagtt tgaggggaaa tgcagatttg gcttacatcc tgagcatgga gccctgtggc 420 cactgcctca ttatcaacaa tgtgaacttc tgccgtgagt ccgggctccg cacccgcact 480 ggctccaaca tcgactgtga gaagttgcgg cgtcgcttct cctcgctgca tttcatggtg 540 gaggtgaagg gcgacctgac tgccaagaaa atggtgctgg ctttgctgga gctggcgcag 600 caggaccacg gtgctctgga ctgctgcgtg gtggtcattc tctctcacgg ctgtcaggcc 660 agccacctgc agttcccagg ggctgtctac ggcacagatg gatgccctgt gtcggtcgag 720 aagattgtga acatcttcaa tgggaccagc tgccccagcc tgggagggaa gcccaagctc 780 tttttcatcc aggcctgtgg tggggagcag aaagaccatg ggtttgaggt ggcctccact 840 tcccctgaag acgagtcccc tggcagtaac cccgagccag atgccacccc gttccaggaa 900 ggtttgagga ccttcgacca gctggacgcc atatctagtt tgcccacacc cagtgacatc 960 tttgtgtcct actctacttt cccaggtttt gtttcctgga gggaccccaa gagtggctcc 1020 tggtacgttg agaccctgga cgacatcttt gagcagtggg ctcactctga agacctgcag 1080 tccctcctgc ttagggtcgc taatgctgtt tcggtgaaag ggatttataa acagatgcct 1140 ggttgcttta atttcctccg gaaaaaactt ttctttaaaa catcataa 1188 <210> 2 <211> 600 <212> DNA <213> Artificial Sequence <400> 2 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 60 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 120 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 240 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360 cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc 420 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600 <210> 3 <211> 65 <212> DNA <213> Artificial Sequence <400> 3 ggatccggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct 60 ggacc 65 <210> 4 <211> 247 <212> DNA <213> Artificial Sequence <400> 4 cacgagacta gcctcctagc ccgggctcga gcagctgaag cttgcatgcc tgcagggtac 60 cgagctctta cgcgagatca aagggggtaa gatcaaaggg ggtaagatca aaggggcgcg 120 agatcaaagg gggtaagatc aaagggggta agatcaaagg gggtaagatc aaaggggcgc 180 gcccgcgtgc tagcccgggc tcgagatcta gactctagag ggtatataat ggaagctcga 240 attccag 247 <210> 5 <211> 124 <212> DNA <213> Artificial Sequence <400> 5 taagtctaga cggcagtcta gacgtactaa gtctagacgg cagtctagac gtaccgcgcc 60 cgcgtgctag cccgggctcg agatctagac tctagagggt atataatgga agctcgaatt 120 ccag 124 <210> 6 <211> 169 <212> DNA <213> Artificial Sequence <400> 6 ctcgaaccaa actatgccag gaatttaaag ctcgacacca aactatgcca ggaatttaaa 60 gctcgacacc aaactatgcc aggaatttaa agctcgaggc gcgcccgcgt gctagcccgg 120 gctcgagatc tagactctag agggtatata atggaagctc gaattccag 169 <210> 7 <211> 152 <212> DNA <213> Artificial Sequence <400> 7 tagcttggaa atgacattgc taatggtgac aaagcaactt ttagcttgga aatgacattg 60 ctaatggtga caaagcaact ttcgcgcccg cgtgctagcc cgggctcgag atctagactc 120 tagagggtat ataatggaag ctcgaattcc ag 152 <210> 8 <211> 146 <212> DNA <213> Artificial Sequence <400> 8 gtgactacgt gctgcctagg tgactacgtg ctgcctaggt gactacgtgc tgcctaggtg 60 actacgtgct gcctagcgcg cccgcgtgct agcccgggct cgagatctag actctagagg 120 gtatataatg gaagctcgaa ttccag 146 <210> 9 <211> 145 <212> DNA <213> Artificial Sequence <400> 9 tagtttcact ttccctagtt tcactttccc tagtttcact ttccctagtt tcactttccc 60 tagtttcact ttccccgcgc ccgcgtgcta gcccgggctc gagatctaga ctctagaggg 120 tatataatgg aagctcgaat tccag 145 <210> 10 <211> 115 <212> DNA <213> Artificial Sequence <400> 10 gggcactgac tcatcaagca ctgactcatc aagcactgac tcatccgcgc ccgcgtgcta 60 gcccgggctc gagatctaga ctctagaggg tatataatgg aagctcgaat tccag 115 <210> 11 <211> 157 <212> DNA <213> Artificial Sequence <400> 11 gcaccagaca gtgacgtcag ctgccagatc ccatggccgt catactgtga cgtctttcag 60 acaccccatt gacgtcaatg ggagaaccgc gcccgcgtgc tagcccgggc tcgagatcta 120 gactctagag ggtatataat ggaagctcga attccag 157 <210> 12 <211> 122 <212> DNA <213> Artificial Sequence <400> 12 gggaatttcc ggggactttc cgggaatttc cggggacttt ccgggaattt cccgcgcccg 60 cgtgctagcc cgggctcgag atctagactc tagagggtat ataatggaag ctcgaattcc 120 ag 122 <210> 13 <211> 128 <212> DNA[[ID=1】] <213> Artificial Sequence <400> 13 tacagaacat gtctaagcat gctgtgcctt gcctggactt gcctggcctt gccttgggcg 60 cgcccgcgtg ctagcccggg ctcgagatct agactctaga gggtatataa tggaagctcg 120 aattccag 128

Claims

1. A screening system for screening inhibitors against a target signaling pathway, characterized by, The screening system comprises a culture system and the following components present in the culture system: (a) a living detection cell, wherein a genome of the detection cell has integrated therein an expression cassette, and wherein the expression cassette has operatively linked thereto a reporter element of a target signal pathway, a suicide protein element, and a protective protein element, wherein, when the target signal pathway in the detection cell is activated under an activating culture condition, the detection cell expresses the suicide protein element and the protective protein element, and the suicide protein element induces apoptosis of the detection cell in the presence of a first inducer; the expression cassette has a structure as shown in Formula I from 5'-3': Z1-Z2-Z3-Z4 Formula I in which each "-" is independently a bond or a nucleotide connecting sequence; Z1 is a reporter element; Z2 is a coding sequence of a suicide protein element; and Z3 is nothing or a self-cleavage sequence; Z4 is a coding sequence of a protective protein element, and the coding sequence of the protective protein element is a puromycin resistance gene; wherein the reporter element comprises a binding site Y1 of a most downstream transcription factor in the target signal pathway, and a downstream promoter Y2, wherein Y1 and Y2 are operatively linked, so that when the binding site Y1 of the most downstream transcription factor in the target signal pathway binds to the most downstream transcription factor in the target signal pathway, the downstream promoter Y2 drives expression of the suicide protein element and the protective protein element; the structure of the suicide protein element is as shown in Formula III: F-L2-C (III) in which, each "-" is independently a connecting peptide or a peptide bond; F is a suicide gene induction element, and the F is an FKBP12-F36V domain; L2 is nothing or a flexible linker; C is a suicide gene element, and the C is a coding gene of Caspase-9; (b) a test substance to be screened; wherein, when the target signal pathway in the detection cell is activated, and the suicide protein element is expressed, and the test substance does not cause degradation or reduction of the suicide protein element, the first inducer induces apoptosis of the detection cell; and, when the test substance causes the target signal pathway in the detection cell not to be activated, and the suicide protein element not to be expressed, the first inducer does not induce apoptosis of the detection cell; when the test substance causes the target signal pathway in the detection cell not to be activated, and causes the detection cell not to express the suicide protein element, the test substance is considered to be a candidate inhibitor; the target signal pathway is selected from a Wnt pathway, a TGFβ pathway, a Hippo pathway, a Keap1-Nrf2 pathway, a VHL-HIF1α pathway, a JAK-STAT1 / 2 pathway, a MAP / ERK pathway, a cAMP / PKA pathway, a NFκB pathway, or a p53 pathway.

2. A screening system for screening for an activator of a target signal pathway, characterized by, The screening system comprises a culture system and the following components present in the culture system: (a) a living detection cell, wherein a genomic of the detection cell is integrated with an expression cassette, wherein a reporter element of a target signaling pathway, a suicide protein element and a protector protein element are operably linked in the expression cassette, wherein, when the detection cell is under normal culture condition, the target signaling pathway in the cell is not activated, the detection cell does not express the suicide protein element and the protector protein element; the detection cell undergoes apoptosis in the presence of a second inducer; the expression cassette has a structure as shown in Formula I from 5'-3': Z1-Z2-Z3-Z4 Formula I in which each "-" is independently a bond or a nucleotide linker sequence; Z1 is a reporter element; Z2 is a coding sequence of a suicide protein element; and Z3 is nothing or a self-cleavage sequence; Z4 is a coding sequence of a protector protein element, which is a puromycin resistance gene; wherein the reporter element comprises a binding site Y1 of a most downstream transcription factor in the target signaling pathway, a downstream promoter Y2, wherein Y1 and Y2 are operably linked, so that when the binding site Y1 of the most downstream transcription factor in the target signaling pathway binds to the most downstream transcription factor in the target signaling pathway, the downstream promoter Y2 drives the expression of the suicide protein element and the protector protein element; the structure of the suicide protein element is as shown in Formula III: F-L2-C (III) in which, each "-" is independently a linker peptide or a peptide bond; F is a suicide gene inducible element, which is FKBP12-F36V domain; L2 is nothing or a flexible linker; C is a suicide gene element, which is a coding gene of Caspase-9; (b) a test substance to be screened; wherein, when the target signaling pathway in the detection cell is not activated and does not express the protector protein element, the second inducer induces the detection cell to undergo apoptosis; and, when the test substance causes the target signaling pathway in the detection cell to be activated and express the protector protein element, and the test substance does not cause the protector protein element to be degraded or reduced, the second inducer does not induce the detection cell to undergo apoptosis; when the test substance causes the target signaling pathway in the detection cell to be activated and express the protector protein element, and the test substance does not cause the protector protein element to be degraded or reduced, the test substance is considered to be a candidate activator; the target signaling pathway is selected from a Wnt pathway, a TGFβ pathway, a Hippo pathway, a Keap1-Nrf2 pathway, a VHL-HIF1α pathway, a JAK-STAT1 / 2 pathway, a MAP / ERK pathway, a cAMP / PKA pathway, a NFκB pathway or a p53 pathway.

3. The screening system of claim 1 or 2, wherein, when the target signaling pathway is Wnt, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO: 4; when the target signaling pathway is TGFβ, the nucleotides of the reporter element Z1 are as shown in SEQ ID NO: 5; when the target signal pathway is Hippo, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 6; when the target signal pathway is Keap1-Nrf2, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 7; when the target signal pathway is VHL-HIF1α, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 8; when the target signal pathway is JAK-STAT1 / 2, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 9; when the target signal pathway is MAP / ERK, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 10; when the target signal pathway is cAMP / PKA, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 11; when the target signal pathway is NFκB, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 12; or when the target signal pathway is p53, the nucleotide of the reporter element Z1 is shown as SEQ ID NO:

13.

4. A method for screening an inhibitor of a signal pathway which is not a disease diagnosis and / or therapeutic agent, characterized by, comprising the steps of: (a) under activated culture conditions, a culture system to which a test substance to be screened is added is taken as an experimental group; and a culture system to which no test compound is added is taken as a blank control group, wherein the culture system contains a culture of living detection cells, and the genome of the detection cells has integrated therein an expression cassette, and in the expression cassette, a reporter element of a target signal pathway, a suicide protein element and a protective protein element are operably linked, wherein when the detection cells are under activated culture conditions, the target signal pathway in the cells is activated, the detection cells express the suicide protein element and the protective protein element, and the suicide protein element induces apoptosis of the detection cells in the presence of a first inducer; the expression cassette has a structure shown as formula I from 5'-3': Z1-Z2-Z3-Z4 Formula I in which each "-" is independently a bond or a nucleotide connection sequence; Z1 is a reporter element; Z2 is a coding sequence of a suicide protein element; and Z3 is nothing or a self-cleavage sequence; Z4 is a coding sequence of a protective protein element, and the coding sequence of the protective protein element is a puromycin resistance gene; wherein the reporter element comprises a binding site Y1 of a most downstream transcription factor in a target signal pathway, and a downstream promoter Y2, wherein Y1 and Y2 are operably linked, so that when the binding site Y1 of the most downstream transcription factor in the target signal pathway binds the most downstream transcription factor in the target signal pathway, the downstream promoter Y2 drives expression of the suicide protein element and the protective protein element; the structure of the suicide protein element is shown as formula III: F-L2-C (III) in which, each "-" is independently a connection peptide or a peptide bond; F is a suicide gene induction element, and the F is an FKBP12-F36V domain; L2 is nothing or a flexible linker; C is a suicide gene element, and the C is a coding gene of Caspase-9; and (b) adding a first inducer to the experimental group and the blank control group, and observing the survival of the detection cells in the experimental group and the blank control group; wherein, when the number of the survival of the detection cells in the experimental group is significantly higher than that in the control group, it indicates that the test substance is a candidate inhibitor; the target signal pathway is selected from the group consisting of Wnt pathway, TGFβ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1α pathway, JAK-STAT1 / 2 pathway, MAP / ERK pathway, cAMP / PKA pathway, NFκB pathway and p53 pathway.

5. A method for screening an activator of a target signal pathway for non-disease diagnosis and / or therapy, characterized by, comprising the steps of: (a) under normal culture conditions, taking a culture system added with a test substance to be screened as an experimental group, and taking a culture system without the addition of the test substance as a blank control group, wherein the culture system contains cultured living detection cells, and the genome of the detection cells is integrated with an expression cassette, wherein a reporter element of a target signal pathway, a suicide protein element and a protective protein element in the expression cassette are operably linked, wherein, when the target signal pathway in the detection cells is not activated under normal culture conditions, the detection cells do not express the suicide protein element and the protective protein element; and the detection cells undergo apoptosis in the presence of a second inducer; the expression cassette has a structure as shown in Formula I from 5'-3': Z1-Z2-Z3-Z4 Formula I in which each "-" is independently a bond or a nucleotide connection sequence; Z1 is a reporter element; Z2 is a coding sequence of a suicide protein element; and Z3 is nothing or a self-cleavage sequence; Z4 is a coding sequence of a protective protein element, which is a puromycin resistance gene; wherein the reporter element comprises a binding site Y1 of a most downstream transcription factor in the target signal pathway and a downstream promoter Y2, wherein Y1 and Y2 are operably linked, so that when the binding site Y1 of the most downstream transcription factor in the target signal pathway binds to the most downstream transcription factor in the target signal pathway, the downstream promoter Y2 drives the expression of the suicide protein element and the protective protein element; the structure of the suicide protein element is as shown in the following Formula III: F-L2-C (III) in which, each "-" is independently a connection peptide or a peptide bond; F is a suicide gene inducer, and the F is FKBP12-F36V domain; L2 is nothing or a flexible linker; C is a suicide gene element, and the C is a coding gene of Caspase-9; and (b) adding a second inducer to the experimental group and the blank control group, and observing the survival of the detection cells in the experimental group and the blank control group; wherein, when the number of the survival of the detection cells in the experimental group is significantly higher than that in the control group, it indicates that the test substance is a candidate activator; the target signal pathway is selected from the group consisting of Wnt pathway, TGFβ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1α pathway, JAK-STAT1 / 2 pathway, MAP / ERK pathway, cAMP / PKA pathway, NFκB pathway and p53 pathway.

6. Use of the screening system according to claim 1 or 2 for non-disease diagnostic and / or therapeutic purposes, characterized in that, For screening activators and / or inhibitors of a target signal pathway.

7. A screening device for screening for inhibitors or activators of a target signaling pathway, characterized in that, The device comprises: (d1) an apoptosis screening module, wherein the apoptosis screening module comprises one or more culture units, and each culture unit comprises n culture chambers or wells for culturing live detection cells, wherein each culture chamber comprises the screening system for screening inhibitors or activators of a target signal pathway according to claim 1 or 2, and n is a positive integer greater than or equal to 2; (d2) a data acquisition module configured to acquire data of apoptosis of the detection cells in each culture chamber of the apoptosis screening module; (d3) a screening analysis module configured to analyze the apoptosis of the detection cells from the data acquisition module to obtain an analysis result of whether the test substance is an inhibitor or activator of the target signal pathway; and (d4) an output module configured to output the analysis result of the screening analysis module.

8. The screening device of claim 7, wherein, The number of culture units is 4-100.

9. The screening device of claim 7, wherein, The number of culture units is 8-50.

10. The screening device of claim 7, wherein, The number of culture units is 10-20.

11. An expression cassette comprising, The expression cassette has a structure as shown in Formula I from 5'-3': Z1-Z2-Z3-Z4 Formula I In the formula, each "-" is independently a bond or a nucleotide connection sequence; Z1 is a reporter element; Z2 is a coding sequence of a suicide protein element; and Z3 is nothing or a self-cleavage sequence; Z4 is a coding sequence of a protection protein element, and the coding sequence of the protection protein element is a puromycin resistance gene; The reporter element comprises a binding site Y1 of a most downstream transcription factor in the target signal pathway and a downstream promoter Y2, wherein Y1 and Y2 are operatively linked, so that when the binding site Y1 of the most downstream transcription factor in the target signal pathway binds to the most downstream transcription factor in the target signal pathway, the downstream promoter Y2 drives the expression of the suicide protein element and the protection protein element; The structure of the suicide protein element is as shown in the following Formula III: F-L2-C (III) In the formula, Each "-" is independently a connection peptide or a peptide bond; F is a suicide gene induction element, and the F is an FKBP12-F36V domain; L2 is nothing or a flexible linker; C is a suicide gene element, and the C is a coding gene of Caspase-9; The target signal pathway is selected from a Wnt pathway, a TGFβ pathway, a Hippo pathway, a Keap1-Nrf2 pathway, a VHL-HIF1α pathway, a JAK-STAT1 / 2 pathway, a MAP / ERK pathway, a cAMP / PKA pathway, a NFκB pathway, or a p53 pathway.

12. The expression cassette of claim 11, wherein when the target signal pathway is Wnt, the nucleotide of the reporter element Z1 is as shown in SEQ ID NO: 4; when the target signal pathway is TGFβ, the nucleotide of the reporter element Z1 is as shown in SEQ ID NO: 5; when the target signal pathway is Hippo, the nucleotide of the reporter element Z1 is as shown in SEQ ID NO: 6; when the target signal pathway is Keap1-Nrf2, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 7; when the target signal pathway is VHL-HIF1a, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 8; when the target signal pathway is JAK-STAT1 / 2, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 9; when the target signal pathway is MAP / ERK, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 10; when the target signal pathway is cAMP / PKA, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 11; when the target signal pathway is NFKB, the nucleotide of the reporter element Z1 is shown as SEQ ID NO: 12; or when the target signal pathway is p53, the nucleotide of the reporter element Z1 is shown as SEQ ID NO:

13.

13. A vector, characterized in that, The vector contains the expression cassette of claim 11.

14. A host cell, characterized in that, The genome of the host cell has one or more sites integrated with the expression cassette of claim 11, or the host cell contains the vector of claim 13.

15. The host cell of claim 14, wherein The cell is an isolated cell, and / or the cell is a genetically engineered cell.

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