WO2022214022A1 - 基于细胞死活表型的信号通路激活剂和抑制剂的新型筛选系统 - Google Patents

基于细胞死活表型的信号通路激活剂和抑制剂的新型筛选系统 Download PDF

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WO2022214022A1
WO2022214022A1 PCT/CN2022/085516 CN2022085516W WO2022214022A1 WO 2022214022 A1 WO2022214022 A1 WO 2022214022A1 CN 2022085516 W CN2022085516 W CN 2022085516W WO 2022214022 A1 WO2022214022 A1 WO 2022214022A1
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pathway
cell
cells
signaling pathway
screening
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姜海
陈奭爽
何正金
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中国科学院分子细胞科学卓越创新中心
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the field of biotechnology, in particular to a novel screening system for signal pathway activators and inhibitors based on cell dead and alive phenotypes.
  • the detection and display of the activation levels of different signaling pathways by the luciferase reporter system is mainly achieved by linking together specific reporter elements and luciferase.
  • the transfection of luciferase reporter plasmids and the addition of luciferin all bring a large workload, and it is difficult to achieve high-throughput screening. It is also difficult to control the uniformity of the effects of transfection experiments.
  • the purpose of the present invention is to provide an efficient and sensitive drug screening system acting on signal pathways.
  • a screening system for screening inhibitors against target signaling pathways comprising a culture system and the following components present in the culture system:
  • the detection cell when the target signal pathway in the cell is activated under the activation culture condition, the detection cell expresses the suicide protein element and the protection protein element, and the suicide protein element is present in the presence of the first inducer Inducing the detection cell to undergo apoptosis under the following conditions;
  • test substance to be screened (b) the test substance to be screened
  • the first inducer when the target signaling pathway in the test cell is activated, and the suicide protein element is expressed, and the test substance does not cause the degradation or reduction of the suicide protein element, the first inducer will inducing apoptosis in the detection cells;
  • the first inducer does not induce apoptosis in the test cell.
  • test substance when the test substance causes the target signaling pathway in the detection cell to be inactivated, and causes the detection cell not to express the suicide protein element, the test substance is deactivated by considered a candidate inhibitor.
  • the candidate inhibitor inhibits the activation of the target signaling pathway.
  • the "activating culture condition” refers to a culture condition capable of activating the target signaling pathway in the detection cell.
  • the screening system further includes:
  • the suicide protein element in the presence of the first inducer, forms a dimer, thereby inducing apoptosis.
  • the suicide protein element in the absence of the first inducer, maintains a monomeric form and does not induce apoptosis.
  • the first inducer is AP1903.
  • a screening system for screening an activator against a target signal pathway comprising a culture system and the following components present in the culture system:
  • the detection cell when the detection cell is under conventional culture conditions, the target signaling pathway in the cell is not activated, and the detection cell does not express the suicide protein element and the protection protein element; the detection cell is in the second induction apoptosis in the presence of the agent;
  • test substance to be screened (b) the test substance to be screened
  • the second inducer will induce the detection cell to undergo apoptosis
  • the test substance causes the activation of the target signaling pathway in the test cell, and the protection protein element is expressed, and the test substance does not cause the protection protein element to be degraded or reduced, then the protection protein element is expressed by the test substance.
  • the second inducer does not induce apoptosis in the test cells.
  • test substance when the test substance causes activation of the target signaling pathway in the detection cell and expresses the protective protein element, and the test substance does not cause degradation of the protective protein element or When reduced, the test article is considered a candidate activator.
  • the candidate activator activates the target signaling pathway.
  • the "activation of the target signaling pathway” means that the reporter element of the target signaling pathway binds to the most downstream transcription factor of the target signaling pathway, and induces the detection cells to express suicide protein elements and protective proteins element.
  • the "normal culture conditions" refer to the culture conditions in which the target signaling pathway in the detection cells is not activated.
  • the reporter element of the target signal pathway comprises the binding site of the most downstream transcription factor in the target signal pathway and the downstream promoter.
  • downstream promoter promotes the expression of downstream suicide protein elements and protection protein elements.
  • the screening system further includes:
  • a second inducer which induces the detection cell to undergo apoptosis when the detection cell does not express the protective protein element.
  • the second inducer is puromycin.
  • the target signaling pathway is selected from Wnt pathway, TGF ⁇ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1 ⁇ pathway, JAK-STAT1/2 pathway, MAP/ERK pathway, cAMP/PKA pathway, NF ⁇ B pathway, p53 pathway.
  • the expression cassette integrated in the genome of the detection cell has the structure shown in formula I from 5' to 3':
  • each "-" is independently a bond or a nucleotide linking sequence
  • Z1 is the reporting element
  • Z2 is the coding sequence for the suicide protein element
  • Z3 is no or self-cutting sequence
  • Z4 is the coding sequence of the protective protein element
  • the reporter element comprises the binding site Y1 of the most downstream transcription factor in the target signaling pathway, and the 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 is used
  • the downstream promoter Y2 drives the expression of suicide and protector elements when bound to the most downstream transcription factors in the target signaling pathway.
  • the expression cassette integrated in the genome of the detection cell has the structure shown in formula II from 5' to 3':
  • each "-" is independently a bond or a nucleotide linking sequence
  • Y1 is the binding site of the most downstream transcription factor in the target signaling pathway
  • Y2 is the downstream promoter
  • Z2 is the coding sequence for the suicide protein element
  • Z3 is no or self-cutting sequence
  • Z4 is the coding sequence for the protective protein element.
  • downstream promoter Y2 is the minimal promoter of the TATA box.
  • the site Y1 comprises the binding site of the most downstream transcription factor in the target signaling pathway repeated n times, wherein n is any positive integer from 1 to 20, preferably, n is 1- 10.
  • the target signaling pathway is selected from Wnt pathway, TGF ⁇ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1 ⁇ pathway, JAK-STAT1/2 pathway, MAP/ERK pathway, cAMP/PKA pathway, NF ⁇ B pathway, p53 pathway.
  • the most downstream transcription factor in the target signaling pathway is selected from TCF/LEF complex, SMAD3/4 complex, YAP-TEAD/TEF complex, Nrf2, HIF1 ⁇ , STAT1/2, AP1, CRE Binding protein, NF ⁇ B, p53.
  • the coding sequence of the protective protein element is a puromycin resistance gene.
  • the target signaling pathway is selected from Wnt pathway, TGF ⁇ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1 ⁇ pathway, JAK-STAT1/2 pathway, MAP/ERK pathway, cAMP/PKA pathway, NF ⁇ B pathway, p53 pathway.
  • the self-cleaving sequence is shown in SEQ ID NO:3.
  • the coding sequence of the protective protein element is a puromycin (Puromycin) resistance gene, and the sequence is shown in SEQ ID NO: 2.
  • sequence of the reporter element is selected from: SEQ ID NO: 4, 5, 6, 7, 8, 9, 11, 12, 13.
  • the structure of the suicide protein element is shown in the following formula III:
  • each "-" is independently a linking peptide or peptide bond
  • F is suicide gene inducible element
  • L2 is no or flexible joint
  • C is a suicide gene element.
  • the C is a gene encoding caspase-9 (Caspase9 gene).
  • the F is the FKBP12-F36V domain.
  • the FKBP12-F36V domain comprises a FKBP domain, and the 36th amino acid of the FKBP domain is mutated from phenylalanine to valine.
  • the suicide protein element is FKBP12(F36V)- ⁇ CASP9, and its coding sequence is shown in SEQ ID NO:1.
  • the cells are genetically engineered cells.
  • the cells are mammalian cells.
  • 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.
  • a method for screening target signaling pathway inhibitors comprising the steps of:
  • the culture system with the test substance to be screened is used as the experimental group; the culture system without the test compound is used as the blank control group, wherein the culture system contains the cultured live detection A cell, an expression cassette is integrated into the genome of the detection cell, and in the expression cassette, the reporter element, the suicide protein element and the protection protein element of the target signaling pathway are operably linked, wherein, when the detection cell is activated and cultured Under conditions, when the target signaling pathway in the cell is activated, the detection cell expresses the suicide protein element and the protective protein element, and the suicide protein element can induce the detection cell to undergo apoptosis in the presence of the first inducer ;and
  • the survival number of the detected 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 "significantly higher than” refers to the number of apoptotic cells detected in the experimental group
  • the method further comprises: (c1) testing the inhibitory effect of the candidate inhibitor on the target signaling pathway, and/or the preventive or therapeutic effect on the disease related to the target signaling pathway.
  • a method for screening target signaling pathway activators comprising the steps of:
  • the culture system with the test substance to be screened is used as the experimental group; the culture system without the test compound is used as the blank control group, wherein the culture system contains the cultured live detection A cell; an expression cassette is integrated into the genome of the detection cell, and in the expression cassette, the reporter element, the suicide protein element and the protection protein element of the target signaling pathway are operably linked,
  • the detection cell when the detection cell is under conventional culture conditions and the target signaling pathway in the cell is not activated, the detection cell does not express the suicide protein element and the protection protein element; the detection cell is in the second induction apoptosis in the presence of the agent; and
  • the survival number of the tested 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 "significantly higher” refers to the ratio (F1/F0) of the number of apoptotic cells detected in the experimental group F1 to the number of apoptotic cells detected in the blank experimental group F0 (F1/F0) ⁇ 1.5, preferably Ground ⁇ 3.0, more preferably ⁇ 5.
  • the method further comprises: (c1) testing the activation effect of the candidate activator on the target signaling pathway, and/or the preventive or therapeutic effect on diseases related to the target signaling pathway.
  • the screening system according to the first aspect or the second aspect of the present invention, for screening target signaling pathway activators and/or inhibitors.
  • a screening device for screening inhibitors or activators against a target signaling pathway comprising:
  • an apoptosis screening module includes one or more culture units, and the culture unit is provided with n culture chambers (or wells) (compartments) for culturing live detection cells, wherein the culture chamber contains the screening system for screening inhibitors or activators for target signaling pathways according to the first aspect or the second aspect of the present invention; n is a positive integer ⁇ 2;
  • a data collection module configured to collect data on the apoptosis of the detected cells in each culture chamber in the apoptosis screening module;
  • the screening analysis module is configured to analyze the apoptosis situation from the data acquisition module to obtain an analysis of whether the test substance to be screened is an inhibitor or activator for the target signaling pathway results;
  • the number of the culture units is 1-200, preferably 4-100, more preferably 8-50, and most preferably 10-20.
  • n is ⁇ 16, preferably ⁇ 48, more preferably ⁇ 96, such as 16-100000, 48-10000, or 96-5000.
  • the culture unit is a multi-well plate, such as a 1536-well plate, a 384-well plate, and a 96-well plate.
  • the volume of the culture chamber (or well) for detecting cells is 5 ⁇ l-5 ml.
  • the screening system is a high-throughput screening system.
  • the culture unit is provided with a culture room for the blank control group and a culture room for the experimental group, wherein the culture system to which the test substance to be screened is added is used as the experimental group, and the test compound is not added.
  • the culture system was used as blank control group. (That is, except for adding or not adding the test substance to be screened, the other conditions of the experimental group and the blank control group are the same).
  • m different experimental groups are provided to test m different test substances to be screened or combinations of test substances, m is a positive integer ⁇ 1 (1 -1600).
  • an expression cassette is provided, and the expression cassette has the structure shown in formula I from 5'-3':
  • each "-" is independently a bond or a nucleotide linking sequence
  • Z1 is the reporting element
  • Z2 is the coding sequence for the suicide protein element
  • Z3 is no or self-cutting sequence
  • Z4 is the coding sequence of the protective protein element
  • the reporter element comprises the binding site Y1 of the most downstream transcription factor in the target signaling pathway, and the 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 is used
  • the downstream promoter Y2 drives the expression of suicide and protector elements when bound to the most downstream transcription factors in the target signaling pathway.
  • the expression cassette integrated in the genome of the detection cell has the structure shown in formula II from 5' to 3':
  • each "-" is independently a bond or a nucleotide linking sequence
  • Y1 is the binding site of the most downstream transcription factor in the target signaling pathway
  • Y2 is the downstream promoter
  • Z2 is the coding sequence for the suicide protein element
  • Z3 is no or self-cutting sequence
  • Z4 is the coding sequence for the protective protein element.
  • downstream promoter Y2 is the minimal promoter of the TATA box.
  • the site Y1 comprises the binding site of the most downstream transcription factor in the target signaling pathway repeated n times, wherein n is any positive integer from 1 to 20, preferably, n is 1- 10.
  • the target signaling pathway is selected from Wnt pathway, TGF ⁇ pathway, Hippo pathway, Keap1-Nrf2 pathway, VHL-HIF1 ⁇ pathway, JAK-STAT1/2 pathway, MAP/ERK pathway, cAMP/PKA pathway, NF ⁇ B pathway, p53 pathway.
  • the most downstream transcription factor in the target signaling pathway is selected from TCF/LEF complex, SMAD3/4 complex, YAP-TEAD/TEF complex, Nrf2, HIF1 ⁇ , STAT1/2, AP1, CRE Binding protein, NF ⁇ B, p53.
  • the coding sequence of the protective protein element is a puromycin resistance gene.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a host cell wherein the expression cassette of the seventh aspect of the present invention is integrated into one or more sites in the genome of the host cell, or the host cell contains the present The carrier according to the eighth aspect of the invention.
  • the cells are isolated cells, and/or the cells are genetically engineered cells.
  • the cells are mammalian cells.
  • 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.
  • Figure 1 shows a schematic diagram of the components of the luciferase reporter system; when the signal pathway is activated, the luciferase expression level is high, and the bioluminescence is strong; when the signal pathway is inhibited, the luciferase expression level is low, and the biofluorescence is weak.
  • Figure 2 shows a schematic diagram of the elements of the dead-live phenotype reporter system; when the signaling pathway is activated, the expression levels of "suicide genes” and “protection genes” are high; when the signaling pathway is inhibited, the expression levels of "suicide genes” and “protection genes” are low.
  • Figure 3 shows a detailed diagram of the elements of the dead-live phenotype reporter system; wherein, picture a is the schematic diagram of AP1903-induced FKBP12(F36V)- ⁇ CASP9 dimerization; picture b is the dead-live phenotype gene reporter element: TFs binding sites are the binding sites of transcription factors Dot sequence; minP is the minimal promoter containing the TATA box; FKBP12(F36V)- ⁇ CASP9 is the "suicide gene” expressing suicide protein; PuroR is the "protective gene", which can protect cells from puromycin killing; The cleavage sequence makes the two genes FKBP12(F36V)- ⁇ CASP9 and PuroR be cleaved into two proteins after translation and expression.
  • Figure 4 shows the corresponding map between the status of the signaling pathway and the cell dead and alive phenotype; among them, in Figure a, under conventional culture conditions, the signaling pathway to be screened is inactive, the cells survived after AP1903 treatment, and the cells died after puromycin treatment; Under activation culture conditions (when adding signaling pathway activator to culture), the pathway is activated, cells die after AP1903 treatment, and cells survive after puromycin treatment;
  • Figure b is a schematic diagram of the high-throughput drug screening process, in which Puromycin means puromycin , cells in plate A1 were cultured under conventional culture conditions, and cells in plate A2 were cultured under activated culture conditions.
  • Figure 5 shows the validation diagram of the Wnt pathway reporter cell line; in which, Figure a shows the dead and live phenotype reporter system elements of the Wnt pathway; Figure b shows that the cells were treated with 20 mM LiCl, 10% R-spondin1 for 1 day, and the Wnt signaling pathway was activated ; c figure shows that the cells are knocked out of CK1, the Wnt signaling pathway is activated; d figure shows that the cells are activated by 10% R-spondin1 at the same time, add 100nM WNT-C59 for 1 day, the Wnt pathway is not activated.
  • WT is wild type.
  • PURO and AP1903 represent puromycin treatment and AP1903 treatment, respectively.
  • minP is the minimal promoter containing the TATA box
  • P2A is the self-cutting sequence
  • PuroR is the "protection gene”
  • 7 ⁇ TCF is the binding site of 7-repeat TCF/LEF
  • RSPO1 is R-spondin1.
  • FIG. 6 shows the validation diagram of the TGF ⁇ pathway reporter cell line
  • Figure a shows the elements of the TGF ⁇ pathway dead-live phenotype reporter system
  • Figure b shows the cells were treated with 10ng/ml TGF ⁇ for 1 day, the TGF ⁇ signaling pathway was activated, and the activation of TGF ⁇ simultaneously , adding 500nM LY2109761 or SB525334 for 1 day, the TGF ⁇ pathway was not activated.
  • PURO stands for puromycin
  • minP is the minimal promoter containing the TATA box
  • P2A is the self-cleaving sequence
  • PuroR is the "protection gene”
  • 4 ⁇ SBE is the binding site of the 4-repeat Smad3/4 complex SBE .
  • Figure 7 shows the validation map of the Hippo pathway reporter cell line
  • Figure a shows the elements of the Hippo pathway dead and live phenotype reporter system
  • )- ⁇ CASP9 is not expressed, but under low-density culture conditions, YAP enters the nucleus, and PuroR and FKBP12(F36V)- ⁇ CASP9 are expressed
  • panel c shows that after overexpression of the activated mutant YAP (S127A), YAP is abnormal regardless of density Staying in the nucleus, PuroR and FKBP12(F36V)- ⁇ CASP9 are expressed.
  • PURO stands for puromycin
  • minP is the minimal promoter containing the TATA box
  • P2A is the self-cleaving sequence
  • PuroR is the "protection gene”
  • 3 ⁇ SD is the binding site of 3 repeated TEAD/TEF.
  • Figure 8 shows the validation diagram of the Keap1-Nrf2 pathway reporter cell line; in which, Figure a shows the Keap1-Nrf2 pathway dead-live phenotype reporter system elements; Figure b shows that cells were treated with 50 ⁇ M tBHQ for 1 day, and the Keap1-Nrf2 signaling pathway was activated ; Panel c shows that cells knock down Keap1, and the pathway is activated.
  • WT is wild-type
  • PURO puromycin
  • minP is a minimal promoter containing TATA box
  • P2A is a self-cleaving sequence
  • PuroR is a "protection gene”
  • 2 ⁇ ARE is the 2-repeat Nrf2 binding site ARE.
  • Figure 9 shows the validation diagram of the HIF1 ⁇ pathway reporter cell line;
  • Figure a shows the elements of the HIF1 ⁇ pathway dead-live phenotype reporter system;
  • Figure b shows the activation of the HIF1 ⁇ signaling pathway after cells were treated with 10 ⁇ M 1,10-phenanthroline for 5 h;
  • c Figure shows that cells knocked out VHL, pathway activation.
  • WT is wild-type
  • PURO puromycin
  • minP is a minimal promoter containing TATA box
  • P2A is a self-cutting sequence
  • PuroR is a "protection gene”
  • 4 ⁇ HRE is a four-repeat HIF1 ⁇ -regulated binding site HRE .
  • Figure 10 shows the validation map of the STAT1/2 pathway reporter cell line; panel a shows the dead-live phenotype reporter system elements of the STAT1/2 pathway; panel b shows the activation of the STAT1/2 signaling pathway after cells were treated with 10ng/ml IFN ⁇ for 1 day .
  • PURO stands for puromycin
  • minP is a minimal promoter containing a TATA box
  • P2A is a self-splicing sequence
  • PuroR is a "protection gene”
  • 5 ⁇ ISRE is a five-repeat interferon-stimulated response element ISRE.
  • Figure 11 shows the validation map of the MAPK/ERK pathway reporter cell line; panel a shows the elements of the MAP/ERK pathway dead-live phenotype reporter system; panel b shows that under conventional culture, the MAP/ERK pathway is not activated, with 1% serum After the cells were starved overnight in the culture medium, and then treated with the culture medium containing 20% serum and 10 ng/ml PMA for 6 h, the pathway was activated.
  • PURO stands for puromycin
  • minP is a minimal promoter containing a TATA box
  • P2A is a self-cleaving sequence
  • PuroR is a "protection gene”
  • AP1-RE is an AP1 response element AP1-RE.
  • Figure 12 shows the validation map of the CREB pathway reporter cell line;
  • Figure a shows the cAMP/PKA pathway dead-live phenotype reporter system elements;
  • Figure b shows the activation of the cAMP/PKA signaling pathway after cells were treated with 5 ⁇ g/ml Forskolin for 1 day.
  • PURO stands for puromycin
  • minP is a minimal promoter containing a TATA box
  • P2A is a self-splicing sequence
  • PuroR is a "protection gene”
  • CRE is a cAMP response element CRE.
  • Figure 13 shows the validation diagram of the NF ⁇ B pathway reporter cell line;
  • Figure a shows the elements of the NF ⁇ B pathway dead and live phenotype reporter system;
  • Figure b shows that the cells were treated with 20ng/ml TNF ⁇ for 5h, and the NF ⁇ B signaling pathway was activated.
  • PURO stands for puromycin
  • minP is the minimal promoter containing the TATA box
  • P2A is the self-cleaving sequence
  • PuroR is the "protection gene”
  • 5 ⁇ NF ⁇ B-RE is the binding site of 5 repeated NF ⁇ B NF ⁇ B-RE .
  • Figure 14 shows the validation map of the p53 pathway reporter cell line;
  • Figure a shows the elements of the p53 pathway dead-live phenotype reporter system;
  • Figure b shows that the cells were treated with 100nM DOX for 20h, and the p53 signaling pathway was activated.
  • PURO stands for puromycin
  • minP is the minimal promoter containing the TATA box
  • P2A is the self-cleaving sequence
  • PuroR is the "protection gene”
  • 2 ⁇ NF ⁇ B-RE is the binding site p53-RE of 2 repeats of p53. .
  • Figure 15 shows part of the validation results of drug screening;
  • Figure a shows some of the Wnt pathway inhibitors screened;
  • Figure b shows 293T-TCF7 cells treated with 10% R-spondin1 and screened inhibitors (28-H11, 3 -F7) treated for 24h, the inhibitors 28-H11 and 3-F7 could reduce the stability of ⁇ -catenin and the level of active ⁇ -catenin.
  • RSPO1 is R-spondin1
  • actin is actin.
  • Figure 16 shows the effect of TGF ⁇ pathway inhibitors;
  • Figure a shows some of the screened TGF ⁇ pathway inhibitors 35-C11, 35-D10, 33-E10;
  • Figure b shows the screened TGF ⁇ pathway activator 21-H2.
  • PURO represents puromycin.
  • the present inventors have developed a screening system for the first time based on cell dead and alive phenotypes to screen inhibitors or activators of target signaling pathways. Using this screening system, inhibitors or activators of target signaling pathways can be screened with high throughput.
  • the detection cell with the expression cassette as shown in formula I integrated into the genome under the treatment of AP1903 or puromycin, converts the activation or silencing of the target signaling pathway into a complete dead and alive phenotype of the cell, and has pathway specificity .
  • the gene components of the screening system of the present invention are stably integrated into the cell chromosomes, and the dead and live phenotypes are stable, and only a small amount of the compound to be screened can realize large-scale high-pass quantity filter.
  • the present invention has been completed on this basis.
  • operably linked to means that a gene of interest to be transcribed and expressed is linked to its control sequences to be transcribed and/or expressed in a manner conventional in the art. For example, certain portions of a linear DNA sequence can modulate or control the activity of other portions of the same linear DNA sequence.
  • a promoter is operably linked to a coding sequence if it controls transcription of the sequence.
  • the binding site Y1 of the most downstream transcription factor in the target signaling pathway is operatively connected to the promoter Y2, 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 suicide protein elements and protector protein elements.
  • each "-" is independently a bond or a nucleotide linking sequence
  • Z1 is the reporting element
  • Z2 is the coding sequence for the suicide protein element
  • Z3 is no or self-cutting sequence
  • Z4 is the coding sequence of the protective protein element
  • the reporter element comprises the binding site Y1 of the most downstream transcription factor in the target signaling pathway, and the 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 is used
  • the downstream promoter Y2 drives the expression of suicide and protector elements when bound to the most downstream transcription factors in the target signaling pathway.
  • the expression cassette integrated in the genome of the detection cell has the structure shown in formula II from 5' to 3':
  • each "-" is independently a bond or a nucleotide linking sequence
  • Y1 is the binding site of the most downstream transcription factor in the target signaling pathway
  • Y2 is the downstream promoter
  • Z2 is the coding sequence for the suicide protein element
  • Z3 is no or self-cutting sequence
  • Z4 is the coding sequence for the protective protein element.
  • the reporter element contains one or more binding sites for the most downstream transcription factors in the target signaling pathway.
  • the transcription factor of the signaling pathway is the most downstream transcription factor in the target signaling pathway. After binding to the site Y1 of the reporter element, after activating the corresponding signaling pathway, the promoter Y2 in the reporter element is activated, which promotes the transcription and expression of the downstream pathway. corresponding protein.
  • the reporter elements shown are composed of the binding site sequences of transcription factors of each signaling pathway and the minimal promoter containing TATA box, and the reporter elements of each signaling pathway are verified to be regulated and specific. promoter.
  • the promoter when the corresponding signaling pathway in the cell is not activated, the promoter is in a closed state, and the "coding sequence of suicide protein element" (ie “suicide gene”) and the “coding sequence of protective protein element” (ie “protection protein element”) are in a closed state. Gene”) is not expressed; when this signaling pathway activates downstream transcription factors to promote transcription, the promoter initiates the expression of "suicide genes” and "protection genes”.
  • the order of the "suicide gene” and “protection gene” in the expression cassette is not particularly limited, as long as they can be expressed under the control of the reporter element.
  • the reporter element may also contain a coding sequence or a tag sequence (eg, a 6His tag) for a leader or secretory peptide.
  • the "suicide gene” encoding the suicide protein element is a gene expressing the FKBP12(F36V)- ⁇ CASP9 fusion protein (Fig. 3a).
  • the suicide protein element comprises the FKBP12-F36V domain, which can be linked to caspase 9 (Caspase 9) through flexible Ser-Gly-Gly-Gly-Ser, which does not Contains a recruitment domain (denoted as ⁇ CASP9).
  • FKBP12-F36V contains a FKBP domain with phenylalanine instead of valine at the 36th amino acid residue position.
  • AP1903 (Rimiducid, Bellicum Pharmaceutical). Cells expressing this suicide protein element were able to induce apoptosis by the compound AP1903.
  • the basic principle is that the F36V mutant of FKBP12 can bind to the compound AP1903. Since AP1903 is a symmetrical molecule, AP1903 can induce the self-aggregation of the FKBP12(F36V)- ⁇ CASP9 fusion protein. After the self-aggregation of the apoptosis-enforcing protein Caspase9, apoptosis is rapidly induced.
  • FKBP12(F36V)- ⁇ CASP9 has a high expression level in cells, and the cells die after adding AP1903; on the contrary, when the downstream transcription factors of the signaling pathway are silenced, FKBP12(F36V)- ⁇ CASP9 ( F36V)- ⁇ CASP9 was hardly expressed, and the addition of AP1903 had no effect on the cells (Fig. 4).
  • the "protective gene” encoding the protective protein element is the puromycin resistance gene ( Figure 3).
  • Puromycin is an antibiotic that can inhibit protein synthesis. When an appropriate concentration of puromycin is added to the cell culture medium, Cells die and only survive if they express the puromycin resistance gene. Therefore, when the downstream transcription factors of a certain signaling pathway are active, the puromycin resistance gene has a higher expression level in the cells, and the cells survive after adding puromycin; on the contrary, when the downstream transcription factors of the signaling pathway are silenced, the cells The puromycin resistance gene was not expressed, and cells died upon addition of puromycin ( Figure 4).
  • sequence encoding the FKBP12(F36V)- ⁇ CASP9 protein is shown in SEQ ID NO: 1; the nucleotide sequence of the protective protein element is shown in SEQ ID NO: 2; the self-cutting sequence is SEQ ID NO:3.
  • the present invention realizes the stable integration of the expression cassette in the cell genome by means of lentivirus infection, thereby constructing reporter cell lines of different signaling pathways.
  • the target signaling pathway is selected from the Wnt pathway, the TGF ⁇ pathway, the Hippo pathway, the Keap1-Nrf2 pathway, the VHL-HIF1 ⁇ pathway, the JAK-STAT1/2 pathway, the MAP/ERK pathway, the cAMP/PKA pathway, the NF ⁇ B pathway, and the p53 pathway.
  • Wnt signaling pathway is an evolutionarily conserved signaling pathway that plays a key role in embryonic development and the formation of the central nervous system, regulating cell growth, migration, and differentiation.
  • the regulatory complex in the cytoplasm promotes the phosphorylation of ⁇ -catenin, which is degraded by the proteasome; in the presence of Wnt ligand signal, ⁇ -catenin is not degraded, accumulates, and enters the nucleus increased, as a co-activator of the transcription factor TCF/LEF, promoting the expression of downstream target genes, thereby promoting cell proliferation.
  • the pathway's signal transduction will be abnormal.
  • mutations in APC occur frequently in colorectal cancer, and this mutation prevents ⁇ -catenin from being degraded, resulting in persistent activation of the Wnt pathway and canceration of cells. Therefore, if there are suitable drugs that 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.
  • the reporter element of the Wnt pathway consists of seven repetitions of the TCF/LEF binding site (abbreviated as 7 ⁇ TCF).
  • 7 ⁇ TCF TCF/LEF binding site
  • the most downstream transcriptional event of the TGF ⁇ pathway is performed by the SMAD3/4 complex.
  • the reporter element of the TGF ⁇ pathway consists of four repetitions of the SBE at the binding site of the Smad3/4 complex.
  • the target signaling pathway is TGF ⁇
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO:5.
  • the most downstream transcriptional events of the Hippo pathway are carried out by the YAP-TEAD/TEF complex.
  • the reporter element of the Hippo pathway contains 3 repeated TEAD/TEF binding sites.
  • the target signaling pathway is HIPPO
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO: 6.
  • the most downstream transcriptional event of the Keap1-Nrf2 pathway is performed by the transcription factor Nrf2.
  • the reporter element of the Keap1-Nrf2 pathway contains a twice-repeated Nrf2 binding site ARE.
  • the target signaling pathway is Keap1-Nrf2
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO:7.
  • the most downstream transcriptional event of the VHL-HIF1 ⁇ pathway is performed by the transcription factor HIF1 ⁇ .
  • the reporter element of the HIF1 ⁇ pathway contains the HRE for the HIF1 ⁇ -regulated binding site repeated four times.
  • the target signaling pathway is VHL-HIF1 ⁇
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO:8.
  • the most downstream transcriptional events in the JAK-STAT1/2 pathway are performed by the transcription factor STAT1/2.
  • the reporter element of the STAT1/2 pathway contains the interferon-stimulated response element ISRE, which is repeated 5 times.
  • the target signaling pathway is JAK-STAT1/2
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO:9.
  • 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.
  • the target signaling pathway is MAP/ERK
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO: 10.
  • the reporter element of the cAMP/PKA pathway contains the cAMP response element CRE.
  • the target signaling pathway is cAMP/PKA
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO: 11.
  • the reporter element of the NF ⁇ B pathway contains the NF ⁇ B binding site NF ⁇ B-RE, which is repeated five times.
  • the target signaling pathway is NF ⁇ B
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO: 12.
  • the reporter element of the p53 pathway contains the twice-repeated p53 binding site p53-RE.
  • the target signaling pathway is p53
  • the nucleotide sequence of the reporter element Z1 is shown in SEQ ID NO: 13.
  • the vector can be used to transfect host cells to obtain a host cell carrying the expression vector of the expression cassette, or a host cell with the expression cassette integrated into the genome.
  • host cells are generally mammalian cells.
  • Transformation of 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 selected: infection of packaged virus, calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the host cells transformed by the above method and carrying the expression cassette of formula I can be used to screen agonists or inhibitors of target signaling pathways.
  • the specific promoters responding to the activities of different signal pathways are linked with the "suicide gene” FKBP12(F36V)- ⁇ CASP9 and the "protection gene” puromycin resistance gene, and are stably integrated into the cells through the viral vector
  • a series of cells stably expressing the dead and live phenotype reporter system are constructed, that is, the test cells used for screening target signaling pathway activators and/or inhibitors in the present invention.
  • the signaling pathway is inactive, and cells die after the addition of puromycin. If the candidate compound has an activating effect on the signaling pathway, the cells survive after the addition of puromycin; this experimental phenotype can be used for signaling pathway activation High-throughput screening of agents.
  • conditional suicide protein FKBP12(F36V)- ⁇ CASP9 when a certain pathway activator (such as TGF- ⁇ ) is added to the cells in culture, the conditional suicide protein FKBP12(F36V)- ⁇ CASP9 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 adding AP1903; this experimental phenotype can be used for high-throughput screening of signaling pathway inhibitors.
  • a certain pathway activator such as TGF- ⁇
  • the above experimental conditions utilize the expression of protective genes and suicide genes driven by signaling pathway transcription factors, respectively, and can efficiently achieve large-scale screening of activators and inhibitors of signaling pathways.
  • Day 4 Detect cell survival. Most of the cells in the wells die and a small number of wells survive.
  • the test substance to be screened corresponding to the wells of the surviving cells may be an activator of the target signaling pathway.
  • Day 4 Detect cell survival. Most of the cells in the wells die and a small number of wells survive.
  • the test substance to be screened corresponding to the wells of the surviving cells may be an inhibitor of the target signaling pathway.
  • the test substance to be screened corresponding to the surviving cells is screened against the background of most of the dead cells.
  • Cell phenotypes are easy to observe and can intuitively reflect the regulatory effects of compounds on signaling pathways. The same compound only needs to be screened twice when the cellular target signaling pathway is silenced or activated, and the method is simple.
  • the cell death status can intuitively reflect the regulatory effect of compounds on signaling pathways, and has pathway specificity
  • Each reporter element has been stably integrated into the cell line, and the operation is simple and consistent for high-throughput drug screening;
  • This system can be applied to multiple signaling pathways in a modular manner, and can screen multiple pathways for inhibitors and activators in parallel in the same screening.
  • the dead-alive phenotype reporting system in the present invention is suitable for large-scale high-throughput screening.
  • Plasmids are stably integrated into animal cells
  • the constructed cells are plated and divided into a control group and an experimental group, respectively adding a control solvent (such as DMSO) or 10 ⁇ M (large-scale primary screening is generally set to a concentration of 10 ⁇ M, which can also be determined according to the characteristics of the drug library) drug treatment For a period of time (depending on the characteristics of different drugs and signaling pathways), add puromycin or AP1903 for 1 day
  • a control solvent such as DMSO
  • 10 ⁇ M large-scale primary screening is generally set to a concentration of 10 ⁇ M, which can also be determined according to the characteristics of the drug library
  • Each reporter cell line was constructed by lentiviral infection to achieve stable integration.
  • the experimental process is as follows:
  • the infected cells were plated in a six-well plate, 1 ml of culture solution and 1 ml of virus solution were added, centrifuged at 1000 ⁇ g for 5 min, and cultured in an incubator;
  • FIG. 7a The sequence of Figure 7a was stably integrated into 293A cells to obtain 293A-3SD cells.
  • 293A-3SD cells are at low density, YAP enters the nucleus and promotes the transcription of TEAD/TEF.
  • cells treated with 2 ⁇ g/ml puromycin survive, and cells treated with 10nM AP1903 die; and when cells grow to a high density, After contact inhibition, YAP was degraded without entering the nucleus, and TEAD/TEF was not transcribed.
  • cells treated with puromycin died and cells treated with AP1903 survived (Fig. 7b).
  • FIG. 8a The sequence of Figure 8a was stably integrated into 293A cells to obtain 293A-2ARE cells.
  • the pathway was inactive and gene components were not expressed under conventional culture conditions.
  • the cells died after treatment with 2 ⁇ g/ml puromycin, and 10nM AP1903 treatment had no effect on the cells.
  • 50 ⁇ M tBHQ was added to activate the pathway, Cells in the puromycin-treated group survived, and cells in the AP1903-treated group died (Fig. 8b).
  • Nrf2 was not degraded and continued to perform its transcriptional function, so puromycin-treated cells survived, while AP1903-treated cells died (Fig. 8c).
  • Figure 9a The sequence of Figure 9a was stably integrated into 293A cells to obtain 293A-4HRE cells.
  • this pathway was inactive and gene components were not expressed.
  • the cells died, and 10nM AP1903 treatment had no effect on the cells.
  • the pathway was activated by roroline, cells in the puromycin-treated group survived and cells in the AP1903-treated group died (Fig. 9b).
  • HIF1 ⁇ was not degraded and continued to perform transcriptional function, so puromycin-treated cells still survived, while AP1903-treated cells died (Fig. 9c).
  • Figure 10a The sequence of Figure 10a was stably integrated into 293A cells to obtain 293A-5ISRE cells.
  • this pathway is inactive, and the gene components are not expressed.
  • the cells died, and adding 10nM AP1903 had no effect on the cells; while adding 10ng/ml IFN ⁇ activated this pathway , the cells in the puromycin-treated group survived, and the cells in the AP1903-treated group died (Fig. 10b).
  • Figure 11a The sequence of Figure 11a was stably integrated into 293A cells to obtain 293A-AP1-RE cells.
  • this pathway was inactive and gene components were not expressed.
  • Cells were killed by 2 ⁇ g/ml puromycin treatment, and 10nM AP1903 treatment had no effect on cells; After the cells were starved overnight with 20% serum and 10 ng/ml PMA, the pathway was activated and the gene component was expressed. The cells in the puromycin-treated group survived and the cells in the AP1903-treated group died (Fig. 11b). ).
  • Figure 12a The sequence of Figure 12a was stably integrated into 293A cells to obtain 293A-CRE cells.
  • this pathway was inactive and gene components were not expressed.
  • the cells died, and 10nM AP1903 treatment had no effect on the cells.
  • 5 ⁇ g/ml Forskolin was added to activate the pathway , the cells in the puromycin-treated group survived, and the cells in the AP1903-treated group died (Fig. 12b).
  • Fig. 13a The sequence of Fig. 13a was stably integrated into 293A cells to obtain 293A-5NF ⁇ B-RE cells.
  • this pathway was inactive and gene components were not expressed.
  • the cells died, and 10nM AP1903 treatment had no effect on the cells; and when 20ng/ml TNF ⁇ was added to activate the cells In this pathway, cells in the puromycin-treated group survived, and cells in the AP1903-treated group died (Fig. 13b).
  • Fig. 14a The sequence of Fig. 14a was stably integrated into U2OS cells to obtain U2OS-2p53-RE cells.
  • this pathway is inactive, and the gene components are not expressed.
  • the cells died, and 10nM AP1903 treatment had no effect on the cells; and when 100nM DOX was added to activate the pathway , the cells in the puromycin-treated group survived, and the cells in the AP1903-treated group died (Fig. 14b).
  • the screened Wnt pathway inhibitors 28-H11, 3-F7 and R-spondin1 were simultaneously treated with cells, and the levels of ⁇ -catenin and active ⁇ -catenin were detected by western blot.
  • Example 3 Activators of the TGF ⁇ pathway for phenotypic screening of dead and alive cells
  • TGF ⁇ -activated 293A-4SBE cells some natural small-molecule compounds (35-C11, 35-D10, 33-E10) were found to make the cells survive after AP1903 treatment, indicating that these compounds can inhibit the activation of TGF ⁇ pathway (Fig. 16a).
  • a natural small molecule compound 21-H2 was found to enable the cells to survive puromycin treatment, indicating that this compound may be an activator of the TGF ⁇ pathway (Fig. 16b). Small molecule activators of the TGF pathway have not been reported before, so these results demonstrate the unique efficacy of the screening system of the present invention.
  • the detection and display of activation levels of different signaling pathways by the luciferase reporter system is mainly achieved by linking together specific reporter elements and luciferase ( Figure 1).
  • the reporter element here is composed of multiple repetitions of the binding site sequence of the downstream transcription factor of the signaling pathway and a minimal promoter containing the TATA box. Therefore, the expression level of luciferase reflects the activation level of the corresponding signaling pathway.
  • the luciferase reporter plasmid needs to be transfected into the cells first. After adding luciferin, luciferase can catalyze the oxidation of luciferin.
  • the oxidation process During the oxidation process, it emits biological fluorescence, and the corresponding signal pathway can be judged by the fluorescence intensity. activation level. When the activation of the signaling pathway is high, the transcription factor is active, the expression level of luciferase is high, and the bioluminescence emitted is strong; when the signaling pathway is in a state of inhibition, the transcription factor is silent, the expression level of luciferase is low, and the emitted bioluminescence is strong. Biofluorescence is weak.
  • the reporter cell line constructed in the present invention which can be directly screened and identified by the dead and live phenotype, can be efficiently, sensitively and stably used for high-throughput compounds filter.

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Abstract

本发明提供一种基于信号通路转录因子驱动的保护基因和自杀基因的表达，以实现信号通路的激活剂和抑制剂的大规模筛选的基因工程化细胞。本发明还提供一种基于细胞死活表型的信号通路激活剂和抑制剂的新型筛选方法。

Description

基于细胞死活表型的信号通路激活剂和抑制剂的新型筛选系统 技术领域
本发明涉及生物技术领域，具体涉及一种基于细胞死活表型的信号通路激活剂和抑制剂的新型筛选系统。
背景技术
生物体的许多重要的生理过程与信号通路密切相关，比如发育过程中的Wnt信号，控制器官大小的Hippo信号，以及免疫反应中的NFκB信号。这些信号通路的异常会导致一些疾病的发生，比如EGFR、Wnt通路的过度激活，会引起细胞的生长、增殖调控异常，进而导致肿瘤的发生；TGFβ信号通路的转导异常，会造成骨代谢紊乱，导致骨质疏松症。从制药的角度讲，可以通过调控相关的信号通路，来控制疾病的进展，甚至治愈。
荧光素酶报告系统对不同信号通路激活水平的检测和展现，主要是通过连接在一起的特异性报告元件与荧光素酶来实现。然而，在大规模筛选信号通路相关的激活剂或抑制剂时，荧光素酶报告质粒的转染以及荧光素的加入，都带来较大的工作量，难以实现高通量筛选，并且每次转染实验效果的均一性也很难控制。
因此，为了方便、直观地研究作用于某个信号通路的药物，需要开发一种能够高效、灵敏的筛选系统。
发明内容
本发明目的就是提供了一种高效、灵敏的、作用于信号通路的药物筛选系统。
在本发明的第一方面，提供了一种用于筛选针对目标信号通路的抑制剂的筛选体系，所述筛选体系包括一培养体系以及存在于所述培养体系中的以下组分：
(a)活的检测细胞，所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，
其中，当所述检测细胞在激活培养条件下，细胞中的所述目标信号通路激活 时，所述检测细胞表达所述自杀蛋白元件和保护蛋白元件，所述自杀蛋白元件在第一诱导剂存在下会诱导所述检测细胞发生凋亡；
(b)待筛选的测试物；
其中，当所述检测细胞中的所述目标信号通路激活，并且表达所述的自杀蛋白元件，且所述的测试物不导致所述自杀蛋白元件降解或减少时，所述第一诱导剂会诱导所述检测细胞发生凋亡；
并且，当所述测试物导致所述检测细胞中的所述目标信号通路不激活，并且不表达所述的自杀蛋白元件时，则所述第一诱导剂不诱导所述检测细胞发生凋亡。
在另一优选例中，当所述测试物导致所述检测细胞中的所述目标信号通路不激活，并且导致所述检测细胞不表达所述的自杀蛋白元件时，则所述的测试物被认为是候选抑制剂。
在另一优选例中，所述候选抑制剂抑制所述目标信号通路的激活。
在另一优选例中，所述“激活培养条件”指能够激活检测细胞中的目标信号通路的培养条件。
在另一优选例中，所述筛选体系还包括：
(c)第一诱导剂，所述第一诱导剂通过所述的自杀蛋白元件诱导所述检测细胞发生凋亡。
在另一优选例中，当第一诱导剂存在下，所述的自杀蛋白元件形成二聚体，从而诱发细胞凋亡。
在另一优选例中，当第一诱导剂不存在下，所述的自杀蛋白元件维持单体形式，不诱发细胞凋亡。
在另一优选例中，所述第一诱导剂为AP1903。
在本发明的第二方面，提供了一种用于筛选针对目标信号通路的激活剂的筛选体系，所述筛选体系包括一培养体系以及存在于所述培养体系中的以下组分：
(a)活的检测细胞，所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，
其中，当所述检测细胞在常规培养条件下时，细胞中的所述目标信号通路未激活，所述检测细胞不表达所述的自杀蛋白元件和保护蛋白元件；所述检测细胞 在第二诱导剂存在下发生凋亡；
(b)待筛选的测试物；
其中，当所述检测细胞中的所述目标信号通路未激活，并且不表达所述的保护蛋白元件时，所述第二诱导剂会诱导所述检测细胞发生凋亡；
并且，当所述测试物导致所述检测细胞中的所述目标信号通路激活，并且表达所述的保护蛋白元件，且所述的测试物不导致所述保护蛋白元件降解或减少时，则所述第二诱导剂不诱导所述检测细胞发生凋亡。
在另一优选例中，当所述测试物导致所述检测细胞中的所述目标信号通路激活，并且表达所述的保护蛋白元件，且所述的测试物不导致所述保护蛋白元件降解或减少时，则所述的测试物被认为是候选激活剂。
在另一优选例中，所述候选激活剂激活所述目标信号通路。
在另一优选例中，所述“激活所述目标信号通路”指所述目标信号通路的报告元件与目标信号通路最下游的转录因子结合，并诱导所述检测细胞表达自杀蛋白元件和保护蛋白元件。
在另一优选例中，所述“常规培养条件”指所述检测细胞中的目标信号通路未激活的培养条件。
在另一优选例中，所述目标信号通路的报告元件包含目标信号通路中最下游转录因子的结合位点和下游启动子。
在另一优选例中，所述下游启动子启动下游自杀蛋白元件和保护蛋白元件表达。
在另一优选例中，所述筛选体系还包括：
(c)第二诱导剂，所述第二诱导剂在所述检测细胞不表达保护蛋白元件时，诱导所述检测细胞发生凋亡。
在另一优选例中，所述第二诱导剂为嘌呤霉素。
在另一优选例中，所述目标信号通路选自Wnt通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路、p53通路。
在另一优选例中，所述检测细胞的基因组中整合的表达盒，从5'-3'具有如式I所示的结构：
Z1-Z2-Z3-Z4  式I
式中，各“-”独立地为键或核苷酸连接序列；
Z1为报告元件；
Z2为自杀蛋白元件的编码序列；和
Z3为无或自剪切序列；
Z4为保护蛋白元件的编码序列；
其中，所述报告元件包含目标信号通路中最下游转录因子的结合位点Y1、下游启动子Y2，其中Y1与Y2操作性地连接，从而当目标信号通路的最下游转录因子的结合位点Y1结合目标信号通路最下游的转录因子时，下游启动子Y2驱动自杀蛋白元件和保护蛋白元件的表达。
在另一优选例中，所述检测细胞的基因组中整合的表达盒，从5'-3'具有如式II所示的结构：
Y1-Y2-Z2-Z3-Z4  式II
式中，各“-”独立地为键或核苷酸连接序列；
Y1为目标信号通路中最下游转录因子的结合位点；
Y2为下游启动子；
Z2为自杀蛋白元件的编码序列；和
Z3为无或自剪切序列；
Z4为保护蛋白元件的编码序列。
在另一优选例中，所述下游启动子Y2为TATA盒的最小启动子(minimal promoter)。
在另一优选例中，所述位点Y1包含重复n次的目标信号通路中最下游转录因子的结合位点，其中n为1-20的任一正整数，较佳地，n为1-10。
在另一优选例中，所述目标信号通路选自Wnt通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路、p53通路。
在另一优选例中，所述目标信号通路中最下游转录因子选自TCF/LEF复合物、SMAD3/4复合物、YAP-TEAD/TEF复合物、Nrf2、HIF1α、STAT1/2、AP1、CRE结合蛋白、NFκB、p53。
在另一优选例中，所述保护蛋白元件的编码序列为嘌呤霉素(Puromycin) 抗性基因。
在另一优选例中，所述目标信号通路选自Wnt通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路、p53通路。
在另一优选例中，所述自剪切序列如SEQ ID NO:3所示。
在另一优选例中，所述保护蛋白元件的编码序列为嘌呤霉素(Puromycin)抗性基因，序列如SEQ ID NO:2所示。
在另一优选例中，所述报告元件的序列选自：SEQ ID NO:4、5、6、7、8、9、11、12、13。
在另一优选例中，所述自杀蛋白元件的结构如下式III所示：
F-L2-C  (III)
其中，
各“-”独立地为连接肽或肽键；
F为自杀基因诱导元件；
L2为无或柔性接头；
C为自杀基因元件。
在另一优选例中，所述的C为半胱氨酸天冬氨酸蛋白酶-9的编码基因(Caspase9基因)。
在另一优选例中，所述的F为FKBP12-F36V结构域。
在另一优选例中，所述的FKBP12-F36V结构域包含FKBP结构域，且FKBP结构域的第36位氨基酸由苯丙氨酸突变为缬氨酸。
在另一优选例中，所述自杀蛋白元件为FKBP12(F36V)-△CASP9，其编码序列如SEQ ID NO:1所示。
在另一优选例中，所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞选自：人肾上皮细胞(293A细胞)、人外周血白血病T细胞(Jurkat T细胞)、JHH7细胞、Hela细胞。
在本发明的第三方面，提供了一种筛选目标信号通路抑制剂的方法，包括 步骤：
(a)在激活培养条件下，将加入待筛选的测试物的培养体系作为实验组；将不加入测试化合物的培养体系作为空白对照组，其中，所述的培养体系中含有培养的活的检测细胞，所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，其中，当所述检测细胞在激活培养条件下，细胞中的所述目标信号通路激活时，所述检测细胞表达所述自杀蛋白元件和保护蛋白元件，所述自杀蛋白元件在第一诱导剂存在下会诱导所述检测细胞发生凋亡；和
(b)向所述实验组和空白对照组中加入第一诱导剂，观测实验组和空白对照组中检测细胞的存活情况；
其中，当实验组中检测细胞的存活数量显著高于对照组时，则表明该测试物为候选抑制剂。
在另一优选例中，所述“显著高于”指在实验组中检测细胞的凋亡数量
E1与空白实验组中检测细胞的凋亡数量E0之比(E1/E0)≥1.5，较佳地≥3.0，更佳地≥4。
在另一优选例中，所述方法还包括：(c1)测试所述候选抑制剂对目标信号通路的抑制作用，和/或对目标信号通路相关的疾病的预防或治疗作用。
在本发明的第四方面，提供了一种筛选目标信号通路激活剂的方法，包括步骤：
(a)在常规培养条件下，将加入待筛选的测试物的培养体系作为实验组；将不加入测试化合物的培养体系作为空白对照组，其中，所述的培养体系中含有培养的活的检测细胞；所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，
其中，当所述检测细胞在常规培养条件下，细胞中的所述目标信号通路未激活时，所述检测细胞不表达所述的自杀蛋白元件和保护蛋白元件；所述检测细胞在第二诱导剂存在下发生凋亡；和
(b)向所述实验组和空白对照组中加入第二诱导剂，观测实验组和空白对照组中检测细胞的存活情况；
其中，当实验组中检测细胞的存活数量显著高于对照组时，则表明该测 试物为候选激活剂。
在另一优选例中，所述“显著高于”指在实验组中检测细胞的凋亡数量F1与空白实验组中检测细胞的凋亡数量F0之比(F1/F0)≥1.5，较佳地≥3.0，更佳地≥5。
在另一优选例中，所述方法还包括：(c1)测试所述候选激活剂对目标信号通路的激活作用，和/或对目标信号通路相关的疾病的预防或治疗作用。
在本发明的第五方面，提供了一种如本发明的第一方面或第二方面所述的筛选体系的用途，用于筛选目标信号通路激活剂和/或抑制剂。
在本发明的第六方面，提供了一种用于筛选针对目标信号通路的抑制剂或激活剂的筛选装置，所述装置包括：
(d1)凋亡筛选模块，所述的凋亡筛选模块包括一个或多个培养单元，所述培养单元中设有n个用于培养活的检测细胞的培养室(或孔)(compartment)，其中在所述培养室中含有本发明的第一方面或第二方面所述的用于筛选针对目标信号通路的抑制剂或激活剂的筛选体系；n为≥2的正整数；
(d2)数据采集模块，所述数据采集模块被配置为对凋亡筛选模块中各个培养室中的所述检测细胞的凋亡情况进行数据采集；
(d3)筛选分析模块，所述筛选分析模块被配置为对来自所述数据采集模块的细胞凋亡情况进行分析，获得待筛选的测试物是否是针对目标信号通路的抑制剂或激活剂的分析结果；和
(d4)输出模块，所述输出模块输出筛选分析模块的分析结果。
在另一优选例中，所述培养单元的数量为1-200个，较佳地4-100个，更佳地8-50个，最佳地10-20个。
在另一优选例中，n为≥16，较佳地≥48，更佳地≥96，如16-100000，48-10000，或96-5000。
在另一优选例中，所述的培养单元为多孔板，如1536孔板，384孔板，96孔板。
在另一优选例中，所述的检测细胞的培养室(或孔)的体积为5μl-5ml。
在另一优选例中，所述的筛选系统为高通量筛选系统。
在另一优选例中，所述的培养单元中设有空白对照组的培养室和实验组的培 养室，其中，将加入待筛选的测试物的培养体系作为实验组，并将不加入测试化合物的培养体系作为空白对照组。(即除了加入或不加入待筛选的测试物之外，实验组和空白对照组的其他条件相同)。
在另一优选例中，在所述的培养单元中，设有m个不同的实验组，以测试m种不同的待筛选的测试物或测试物的组合，m为≥1的正整数(1-1600)。
在本发明的第七方面，提供了一种表达盒，所述表达盒从5'-3'具有如式I所示的结构：
Z1-Z2-Z3-Z4  式I
式中，各“-”独立地为键或核苷酸连接序列；
Z1为报告元件；
Z2为自杀蛋白元件的编码序列；和
Z3为无或自剪切序列；
Z4为保护蛋白元件的编码序列；
其中，所述报告元件包含目标信号通路中最下游转录因子的结合位点Y1、下游启动子Y2，其中Y1与Y2操作性地连接，从而当目标信号通路的最下游转录因子的结合位点Y1结合目标信号通路最下游的转录因子时，下游启动子Y2驱动自杀蛋白元件和保护蛋白元件的表达。
在另一优选例中，所述检测细胞的基因组中整合的表达盒，从5'-3'具有如式II所示的结构：
Y1-Y2-Z2-Z3-Z4  式II
式中，各“-”独立地为键或核苷酸连接序列；
Y1为目标信号通路中最下游转录因子的结合位点；
Y2为下游启动子；
Z2为自杀蛋白元件的编码序列；和
Z3为无或自剪切序列；
Z4为保护蛋白元件的编码序列。
在另一优选例中，所述下游启动子Y2为TATA盒的最小启动子(minimal promoter)。
在另一优选例中，所述位点Y1包含重复n次的目标信号通路中最下游转录 因子的结合位点，其中n为1-20的任一正整数，较佳地，n为1-10。
在另一优选例中，所述目标信号通路选自Wnt通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路、p53通路。
在另一优选例中，所述目标信号通路中最下游转录因子选自TCF/LEF复合物、SMAD3/4复合物、YAP-TEAD/TEF复合物、Nrf2、HIF1α、STAT1/2、AP1、CRE结合蛋白、NFκB、p53。
在另一优选例中，所述保护蛋白元件的编码序列为嘌呤霉素(Puromycin)抗性基因。
在本发明的第八方面，提供了一种载体，所述载体中含有如本发明的第七方面所述的表达盒。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第九方面，提供了一种宿主细胞，所述宿主细胞的基因组的一个或多个位点整合有本发明的第七方面所述的表达盒，或者所述宿主细胞中含有本发明的第八方面所述的载体。
在另一优选例中，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞选自：人肾上皮细胞(293A细胞)、人外周血白血病T细胞(Jurkat T细胞)、JHH7细胞、Hela细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了荧光素酶报告系统元件示意图；其中，信号通路激活时，荧光素酶表达水平高，发出生物荧光强；信号通路抑制时，荧光素酶表达水平低， 发出生物荧光弱。
图2显示了死活表型报告系统元件示意图；其中，信号通路激活时，“自杀基因”和“保护基因”表达水平高；信号通路抑制时，“自杀基因”和“保护基因”表达水平低。
图3显示了死活表型报告系统元件详图；其中，a图为AP1903诱导FKBP12(F36V)-△CASP9二聚示意图；b图为死活表型基因报告元件：TFs binding sites为转录因子的结合位点序列；minP为含有TATA盒的最小启动子；FKBP12(F36V)-△CASP9为表达自杀蛋白的“自杀基因”；PuroR为“保护基因”，可保护细胞免受嘌呤霉素杀伤；P2A为自剪切序列，使FKBP12(F36V)-△CASP9和PuroR两个基因翻译表达后，被切割成两个蛋白。
图4显示了信号通路状态与细胞死活表型对应图；其中，a图中，在常规培养条件下，待筛选的信号通路无活性，AP1903处理后细胞存活，嘌呤霉素处理后细胞死亡；在激活培养条件下(加入信号通路激活因子培养时)，通路被激活，AP1903处理后细胞死亡，嘌呤霉素处理后细胞存活；b图为高通量药物筛选流程示意图，其中，Puromycin表示嘌呤霉素，板A1中细胞在常规培养条件下培养，板A2中的细胞在激活培养条件下培养。
图5显示了Wnt通路报告细胞系的验证图；其中，a图显示了Wnt通路死活表型报告系统元件；b图显示了细胞经20mM LiCl、10％R-spondin1处理1天，Wnt信号通路激活；c图显示了细胞敲除CK1，Wnt信号通路激活；d图显示了细胞经10％R-spondin1激活的同时，加入100nM WNT-C59处理1天，Wnt通路不激活。WT为野生型。PURO、AP1903分别表示经嘌呤霉素处理、AP1903处理。其中，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，7×TCF为7个重复的TCF/LEF的结合位点，RSPO1为R-spondin1。
图6显示了TGFβ通路报告细胞系的验证图；a图显示了TGFβ通路死活表型报告系统元件；b图显示了细胞经10ng/ml TGFβ处理1天，TGFβ信号通路激活，经TGFβ激活的同时，加入500nM LY2109761或SB525334处理1天，TGFβ通路不激活。其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，4×SBE为4个重复的Smad3/4复合体的结合位点SBE。
图7显示了Hippo通路报告细胞系的验证图；a图显示了Hippo通路死活表型报告系统元件；b图显示了细胞在高密度培养条件下，YAP被降解不入核，PuroR 和FKBP12(F36V)-△CASP9不表达，在低密度培养条件下，YAP入核，PuroR和FKBP12(F36V)-△CASP9表达；c图显示了过表达激活突变体YAP(S127A)后，无论密度，YAP均异常停留于核内，PuroR和FKBP12(F36V)-△CASP9表达。其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，3×SD为3个重复的TEAD/TEF的结合位点。
图8显示了Keap1-Nrf2通路报告细胞系的验证图；其中，a图显示了Keap1-Nrf2通路死活表型报告系统元件；b图显示了细胞经50μM tBHQ处理1天，Keap1-Nrf2信号通路激活；c图显示了细胞敲除Keap1，通路激活。WT为野生型，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，2×ARE为2个重复的Nrf2的结合位点ARE。
图9显示了HIF1α通路报告细胞系的验证图；a图显示了HIF1α通路死活表型报告系统元件；b图显示了细胞经10μM 1,10-邻菲咯啉处理5h，HIF1α信号通路激活；c图显示了细胞敲除VHL，通路激活。WT为野生型，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，4×HRE为4个重复的HIF1α调控的结合位点HRE。
图10显示了STAT1/2通路报告细胞系的验证图；a图显示了STAT1/2通路死活表型报告系统元件；b图显示了细胞经10ng/ml IFNβ处理1天，STAT1/2信号通路激活。其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，5×ISRE为5个重复的干扰素刺激响应元件ISRE。
图11显示了MAPK/ERK通路报告细胞系的验证图；a图显示了MAP/ERK通路死活表型报告系统元件；b图显示了常规培养下，MAP/ERK通路不激活，用含1％血清的培液对细胞饥饿过夜后，再用含20％血清和10ng/ml PMA的培液处理6h后，通路激活。其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，AP1-RE为AP1响应元件AP1-RE。
图12显示了CREB通路报告细胞系的验证图；a图显示了cAMP/PKA通路死活表型报告系统元件；b图显示了细胞经5μg/ml Forskolin处理1天，cAMP/PKA信号通路激活。其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，CRE为cAMP响应元件CRE。
图13显示了NFκB通路报告细胞系的验证图；a图显示了NFκB通路死活表型报告系统元件；b图显示了细胞经20ng/ml TNFα处理5h，NFκB信号通路激活。 其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，5×NFκB-RE为5个重复的NFκB的结合位点NFκB-RE。
图14显示了p53通路报告细胞系的验证图；a图显示了p53通路死活表型报告系统元件；b图显示了细胞经100nM DOX处理20h，p53信号通路激活。其中，PURO表示嘌呤霉素，minP为含有TATA盒的最小启动子，P2A为自剪切序列，PuroR为“保护基因”，2×NFκB-RE为2个重复的p53的结合位点p53-RE。
图15显示了药物筛选的部分验证结果；a图显示了筛选得到的部分Wnt通路抑制剂；b图显示了293T-TCF7细胞经10％R-spondin1和筛选得到的抑制剂(28-H11、3-F7)处理24h，抑制剂28-H11、3-F7会降低β-catenin稳定性，activeβ-catenin水平降低。其中，RSPO1为R-spondin1，actin为肌动蛋白。
图16显示了TGFβ通路抑制剂作用；a图显示了筛选得到的部分TGFβ通路抑制剂35-C11、35-D10、33-E10；b图显示了筛选得到的TGFβ通路激活剂21-H2。其中，PURO表示嘌呤霉素。
具体实施方式
本发明人通过广泛而深入的研究，首次开发了一种基于细胞死活表型筛选目标信号通路的抑制剂或激活剂的筛选体系。使用该筛选体系，可以高通量地筛选目标信号通路的抑制剂或激活剂。具体地，基因组中整合有如式I所示的表达盒的检测细胞，在AP1903或嘌呤霉素的处理下，将目的信号通路的激活或沉默转化为细胞完全的死活表型，并且具有通路特异性。与荧光素酶转染、荧光素添加的传统实验方法比较，本发明筛选系统的基因组件稳定整合于细胞染色体上，死活表型稳定，仅需微量的待筛选化合物即可实现大规模的高通量筛选。在此基础上完成本发明。
术语
如本文所用，所述的“可操作地连于”是指将准备转录表达的目的基因以一种本领域的常规方式连接到它的控制序列以被转录和/或表达。例如，线性DNA序列的某些部分能够调节或控制同一线性DNA序列其它部分的活性。例如，如果启动子控制序列的转录，那么它就是可操作地连于编码序列。
本发明中，目标信号通路中最下游转录因子的结合位点Y1与启动子Y2操作性地连接，从而当目标信号通路的最下游转录因子的结合位点Y1结合目标信号通路最下游的转录因子时，下游启动子Y2驱动自杀蛋白元件和保护蛋白元件的表达。
表达盒
本发明中，提供了一种如式I所述的表达盒，所述表达盒具有从5'-3'的结构：
Z1-Z2-Z3-Z4  式I
式中，各“-”独立地为键或核苷酸连接序列；
Z1为报告元件；
Z2为自杀蛋白元件的编码序列；和
Z3为无或自剪切序列；
Z4为保护蛋白元件的编码序列；
其中，所述报告元件包含目标信号通路中最下游转录因子的结合位点Y1、下游启动子Y2，其中Y1与Y2操作性地连接，从而当目标信号通路的最下游转录因子的结合位点Y1结合目标信号通路最下游的转录因子时，下游启动子Y2驱动自杀蛋白元件和保护蛋白元件的表达。
在另一优选例中，所述检测细胞的基因组中整合的表达盒，从5'-3'具有如式II所示的结构：
Y1-Y2-Z2-Z3-Z4  式II
式中，各“-”独立地为键或核苷酸连接序列；
Y1为目标信号通路中最下游转录因子的结合位点；
Y2为下游启动子；
Z2为自杀蛋白元件的编码序列；和
Z3为无或自剪切序列；
Z4为保护蛋白元件的编码序列。
在本发明中，所述报告元件中含有一个或多个目标信号通路中最下游转录因子的结合位点。
应理解，信号通路转录因子为目标信号通路中最下游转录因子，结合所述报告元件的位点Y1，激活相应的信号通路后，报告元件中的启动子Y2激活，促 进下游通路开启转录，表达相应蛋白。
在本发明中，所示的报告元件是由各个信号通路的转录因子的结合位点序列与含有TATA box的minimal promoter组成，并且各信号通路的报告元件均为验证过的可调控且具有特异性的启动子。
如图2所示，当细胞中相应信号通路未激活时，启动子处于关闭状态，“自杀蛋白元件的编码序列”(即“自杀基因”)和“保护蛋白元件的编码序列”(即“保护基因”)不表达；当该信号通路激活下游转录因子促进转录时，启动子启动“自杀基因”和“保护基因”的表达。本领域技术人员知晓，表达盒中“自杀基因”和“保护基因”在表达盒中的先后位置没有特别限定，只要其能够在报告元件的调控下表达。此外，报告元件还可含有前导肽或分泌肽的编码序列或标签序列(如6His标签)。
编码自杀蛋白元件的“自杀基因”是一个表达FKBP12(F36V)-△CASP9融合蛋白的基因(图3a)。在本发明一优选实施例中，自杀蛋白元件包含FKBP12-F36V结构域，可通过柔性Ser-Gly-Gly-Gly-Ser连接半胱氨酸天冬氨酸蛋白酶9(Caspase 9)，后者不含募集结构域(记为△CASP9)。FKBP12-F36V包含一个FKBP结构域，在第36个氨基酸残基位点上苯丙氨酸替代了缬氨酸。它具有高选择性和亚纳摩尔亲和力，能够结合二聚合成配基，如其他惰性小分子AP1903(Rimiducid，Bellicum Pharmaceutical)。表达该自杀蛋白元件的细胞能够被化合物AP1903诱导凋亡。基本原理是，FKBP12的F36V突变体能与化合物AP1903结合，由于AP1903是一个对称分子，因此AP1903能够诱导FKBP12(F36V)-△CASP9融合蛋白的自聚。细胞凋亡执行蛋白Caspase9发生自聚后，迅速引发细胞凋亡。因此，当某个信号通路下游转录因子作用活跃时，FKBP12(F36V)-△CASP9在细胞中有较高表达水平，加入AP1903后细胞死亡；相反，当信号通路下游转录因子作用沉默时，FKBP12(F36V)-△CASP9几乎不表达，加入AP1903对细胞没有影响(图4)。
编码保护蛋白元件的“保护基因”则为嘌呤霉素抗性基因(图3)，嘌呤霉素是一种抗生素，能够抑制蛋白合成，当向细胞培液中加入适当浓度的嘌呤霉素后，细胞会死亡，只有在细胞表达了嘌呤霉素抗性基因时，细胞才能存活。因此，当某个信号通路下游转录因子作用活跃时，嘌呤霉素抗性基因在细胞中有较高表达水平，加入嘌呤霉素后细胞存活；相反，当信号通路下游转录因子作用沉默时，细胞不表达嘌呤霉素抗性基因，加入嘌呤霉素后细胞死亡(图4)。
在另一优选例中，所述编码FKBP12(F36V)-△CASP9蛋白的序列如SEQ ID NO:1所示；保护蛋白元件的核苷酸序列如SEQ ID NO:2所示；自剪切序列为SEQ ID NO:3。
本发明通过慢病毒感染的方式实现表达盒在细胞基因组中的稳定整合，从而构建了不同信号通路的报告细胞系。所述目标信号通路选自Wnt通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路、p53通路。
以Wnt信号通路为例，这是一条在进化上保守的信号通路，在胚胎发育和中枢神经系统的形成中起关键作用，调控着细胞的生长、迁移和分化。在没有Wnt配体信号时，胞质中的调控复合体促进β-catenin的磷酸化，使其被蛋白酶体降解；在有Wnt配体信号时，β-catenin不被降解，发生积累，入核增多，作为转录因子TCF/LEF的共激活因子，促进下游靶基因表达，从而促进细胞增殖。当Wnt信号通路中的重要成员出现变异时，该通路信号转导会发生异常。例如，在大肠癌中高频发生APC的突变，该突变导致β-catenin不能被降解，从而导致Wnt通路的持续激活和细胞癌变。因此，如果有合适的药物可以抑制APC突变导致的Wnt通路持续激活，那么这种抑制剂可能可以应用于带有APC突变的大肠癌患者。
如前所述，Wnt通路的最下游的转录事件是由β-catenin-TCF/LEF复合物执行的。Wnt通路的报告元件是由TCF/LEF的结合位点重复7次组成(简写为7×TCF)。当目标信号通路为Wnt时，所述报告元件Z1的核苷酸序列如SEQ ID NO:4所示。
TGFβ通路的最下游的转录事件是由SMAD3/4复合物执行的。TGFβ通路的报告元件是由Smad3/4复合体的结合位点SBE重复4次组成。当目标信号通路为TGFβ时，所述报告元件Z1的核苷酸序列如SEQ ID NO:5所示。
Hippo通路的最下游的转录事件是由YAP-TEAD/TEF复合物执行的。Hippo通路的报告元件含有重复3次的TEAD/TEF的结合位点。当目标信号通路为HIPPO时，所述报告元件Z1的核苷酸序列如SEQ ID NO:6所示。
Keap1-Nrf2通路的最下游的转录事件是由转录因子Nrf2执行的。Keap1-Nrf2通路的报告元件含有重复2次的Nrf2的结合位点ARE。当目标信号通路为Keap1-Nrf2时，所述报告元件Z1的核苷酸序列如SEQ ID NO:7所示。
VHL-HIF1α通路的最下游的转录事件是由转录因子HIF1α执行的。HIF1α 通路的报告元件含有重复4次的HIF1α调控的结合位点的HRE。当目标信号通路为VHL-HIF1α时，所述报告元件Z1的核苷酸序列如SEQ ID NO:8所示。
JAK-STAT1/2通路最下游的转录事件是由转录因子STAT1/2执行的。STAT1/2通路的报告元件含有重复5次的干扰素刺激响应元件ISRE组成。当目标信号通路为JAK-STAT1/2时，所述报告元件Z1的核苷酸序列如SEQ ID NO:9所示。
MAP/ERK通路最下游的转录事件是由转录因子AP-1执行的，对应的报告元件含有AP1响应元件AP1-RE。当目标信号通路为MAP/ERK时，所述报告元件Z1的核苷酸序列如SEQ ID NO:10所示。
cAMP/PKA通路的报告元件含有cAMP响应元件CRE。当目标信号通路为cAMP/PKA时，所述报告元件Z1的核苷酸序列如SEQ ID NO:11所示。
NFκB通路的报告元件含有重复5次的NFκB的结合位点NFκB-RE。当目标信号通路为NFκB时，所述报告元件Z1的核苷酸序列如SEQ ID NO:12所示。
p53通路的报告元件含有重复2次的p53的结合位点p53-RE。当目标信号通路为p53时，所述报告元件Z1的核苷酸序列如SEQ ID NO:13所示。
载体和宿主细胞
将本发明的表达盒插入载体，就可获得携带所述报告元件或表达盒的载体。
所述载体可用于转染宿主细胞，从而获得携带所述表达盒的表达载体的宿主细胞，或者基因组中整合有所述表达盒的宿主细胞。
在本发明中，宿主细胞一般是哺乳动物细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。可选用如下的DNA转染方法：包装病毒感染，磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
在本发明中，用上述方法转化的、携带式I所述表达盒的宿主细胞，可用于筛选目标信号通路的激动剂或抑制剂。
本发明的筛选体系及方法
本发明中，将响应不同信号通路活性的特异性启动子，与“自杀基 因”FKBP12(F36V)-△CASP9和“保护基因”嘌呤霉素抗性基因连接在一起，通过病毒载体稳定整合入细胞的染色体，构建一系列稳定表达死活表型报告系统的细胞，即本发明中用于筛选目标信号通路激活剂和/或抑制剂的测试细胞。在常规培养条件下，信号通路无活性，加入嘌呤霉素后细胞死亡，若候选化合物对该信号通路有激活作用，则加入嘌呤霉素后细胞存活；这一实验表型可以用于信号通路激活剂的高通量筛选。
相应的，当在培养细胞时加入某个通路的激活因子(如TGF-β)时，细胞中FKBP12(F36V)-△CASP9这一条件自杀蛋白表达，AP1903可以将细胞杀死。如果候选化合物对该信号通路有抑制作用，则加入AP1903后细胞存活；这一实验表型可以用于信号通路抑制剂的高通量筛选。
上述的实验条件分别利用了信号通路转录因子驱动的保护基因和自杀基因的表达，可以高效地实现信号通路的激活剂和抑制剂的大规模筛选。
表1信号通路的激活剂和抑制剂
Figure PCTCN2022085516-appb-000001
在上述系统的基础上，可以利用细胞死活表型进行各个信号通路激活剂、抑制剂的高通量筛选。同一批化合物可以在多个信号通路的报告细胞系进行筛选，这样可以保证所筛出的激活剂、抑制剂具有相对特异性。
具体地，在本发明的一优选的筛选目标信号通路激活剂的药物筛选流程中：
第一天：细胞接种并在常规培养条件下，在384孔板上培养；
第二天：使用10μM的化合物库药物处理细胞；
第三天：使用嘌呤霉素处理细胞，
第四天：检测细胞存活情况，其中大部分孔细胞死亡，小部分孔细胞存活，则存活细胞孔对应的待筛选的测试物可能为该目标信号通路的激活剂。
在本发明的一优选的筛选目标信号通路抑制剂的药物筛选流程中：
第一天：细胞接种并在激活培养条件下，在384孔板上培养；
第二天：使用10μM的化合物库药物处理细胞；
第三天：使用AP1903处理细胞，
第四天：检测细胞存活情况，其中大部分孔细胞死亡，小部分孔细胞存活，则存活细胞孔对应的待筛选的测试物可能为该目标信号通路的抑制剂。
在本发明的化合物筛选中，以大部分死亡的细胞为背景，筛选存活的细胞对应的待筛选的测试物。细胞表型容易观察，可以直观地反映化合物对信号通路的调控作用。同一化合物在细胞目标信号通路沉默或激活的情况下，仅需要筛选两遍，方法简便。
本发明的主要优点包括：
1、细胞死活情况可以直观地反映化合物对信号通路的调控作用、并且有通路特异性；
2、各报告元件已稳定整合于细胞系中，进行高通量药物筛选时操作简单且一致性高；
3、此系统可模块化地应用于多种信号通路，可以在同一次筛选中平行地对多个通路进行抑制剂、激活剂的筛选。
4、本发明中的死活表型报告系统适用于大规模的高通量筛选。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用方法
1.构建相关信号通路转录因子驱动保护基因和自杀基因表达的质粒
2.质粒稳定整合至动物细胞中
3.构建好的细胞铺板，分为对照组和实验组，分别加对照溶剂(如DMSO)或10μM(大规模初筛一般设置为10μM的浓度，也可根据药物库的特性决定)的药物处理一段时间(根据不同药物及信号通路特性决定)，加入嘌呤霉素或AP1903处理1天
4.观察对照组和实验组细胞存活情况
实施例1不同信号通路的报告细胞系
细胞系的构建
各报告细胞系的构建都采用了慢病毒感染的方式来实现稳定整合。实验过程如下：
1.通过钙转等转染方式将慢病毒质粒和包装质粒共转染293T细胞；
2.转染48h后收集细胞培液上清，过滤(去除细胞)，得到病毒液；
3.感染的细胞铺于六孔板中，加入1ml培液和1ml病毒液，1000×g离心5min，置于培养箱培养；
4.细胞感染1-2天后撤去病毒液，用普通培液培养；
5.通过流式或荧光显微镜检测细胞的感染效率(本系统用的慢病毒载体上带有mCherry荧光标记)，若感染效率达不到100％，可以流式分选获取全阳性细胞。
1.Wnt通路报告细胞系
将图5a的序列稳定整合于293T细胞，得到了的293T-7TCF细胞。293T-7TCF细胞在常规培养条件下，Wnt通路处于低活性状态，图5a的基因原件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，而10nM AP1903处理对细胞没有影响；作为正对照，当加入Wnt通路的激活剂20mM LiCl或10％R-Spondin1时，图5a的基因组件表达，细胞获得嘌呤霉素抗性，嘌呤霉素不再能够杀死细胞，而FKBP12(F36V)-△CASP9的表达使细胞被AP1903完全杀死(图5b)。
在图5c的实验中，用CRISPR-sgRNA敲除了Wnt通路的负调控基因CK1，在这种情况下β-catenin不被降解，TCF/LEF持续发挥转录功能，因此图5a的基因组件持续表达，经嘌呤霉素处理的细胞依旧存活，而AP1903处理后细胞死亡。当Wnt通路被R-Spondin1(10％)激活后，用Wnt通路抑制剂WNT-C59(100nM)进行处理，能够阻断Wnt通路的活性，逆转细胞的死-活表型(图5d)。
表2.Wnt通路报告细胞系不同处理条件下的状态
Figure PCTCN2022085516-appb-000002
Figure PCTCN2022085516-appb-000003
2.TGFβ通路报告细胞系
将图6a的序列稳定整合于293A细胞，得到了的293A-4SBE细胞。293A-4SBE细胞在常规培养条件下，该基因组件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，而10nM AP1903处理对细胞没有影响；而当加入10ng/ml TGFβ激活该通路时，该基因组件表达，嘌呤霉素处理组细胞存活，AP1903处理组细胞死亡。用TGFβ受体抑制剂LY2109761(500nM)或SB525334(500nM)处理细胞，均可以阻断TGFβ的激活效果，逆转死活表型(图6b)。
表3.TGFβ通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
10ng/ml TGFβ 活跃 死亡 存活
500nM LY2109761 沉默 存活 死亡
500nM SB525334 沉默 存活 死亡
3.Hippo通路报告细胞系
将图7a的序列稳定整合于293A细胞，得到了的293A-3SD细胞。293A-3SD细胞在低密度时，YAP入核，促进TEAD/TEF的转录作用，此时经2μg/ml嘌呤霉素处理细胞存活，经10nM AP1903处理细胞死亡；而当细胞生长到密度很高产生接触抑制后，YAP被降解未入核，TEAD/TEF不进行转录，此时经嘌呤霉素处理细胞死亡，经AP1903处理细胞存活(图7b)。而当在细胞中稳定表达YAP的激活突变体YAP(S127A)时，YAP异常停留于核内，持续促进TEAD/TEF的转录，导致无论细胞密度高低，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图7c)。
表4.Hippo通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
细胞低密度 活跃 死亡 存活
细胞高密度 沉默 存活 死亡
表达YAP(S127A) 活跃 死亡 存活
4.Keap1-Nrf2通路报告细胞系
将图8a的序列稳定整合于293A细胞，得到了的293A-2ARE细胞。293A-2ARE细胞在常规培养条件下，该通路无活性，基因组件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，10nM AP1903处理对细胞没有影响；而当加入50μM tBHQ激活该通路时，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图8b)。用sgRNA敲除了KEAP1后，Nrf2不被降解，持续发挥转录功能，因此嘌呤霉素处理的细胞依旧存活，而A P1903处理后细胞死亡(图8c)。
表5.Keap1-Nrf2通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
50μM tBHQ 活跃 死亡 存活
敲除KEAP1 活跃 死亡 存活
5.VHL-HIF1α通路报告细胞系
将图9a的序列稳定整合于293A细胞，得到了293A-4HRE细胞。293A-4HRE细胞在常规培养条件下，该通路无活性，基因组件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，10nM AP1903处理对细胞没有影响；而当加入10μM 1,10-邻菲咯啉激活该通路时，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图9b)。用sgRNA敲除了VHL后，HIF1α不被降解，持续发挥转录功能，因此嘌呤霉素处理的细胞依旧存活，而AP1903处理后细胞死亡(图9c)。
表6.VHL-HIF1α通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
10μM 1,10-邻菲咯啉 活跃 死亡 存活
敲除VHL 活跃 死亡 存活
6.JAK-STAT1/2通路报告细胞系
将图10a的序列稳定整合于293A细胞，得到了的293A-5ISRE细胞。293A-5ISRE细胞在常规培养条件下，该通路无活性，基因组件不表达，加2μg/ml嘌呤霉素处理后细胞死亡，加10nM AP1903对细胞没有影响；而当加入10ng/ml IFNβ激活该通路时，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图 10b)。
表7.JAK-STAT1/2通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 活跃 死亡 存活
10ng/ml IFNβ 沉默 存活 死亡
7.MAP/ERK通路报告细胞系
将图11a的序列稳定整合于293A细胞，得到了的293A-AP1-RE细胞。293A-AP1-RE细胞在常规培养条件下，该通路无活性，基因组件不表达，经2μg/ml嘌呤霉素处理细胞死亡，10nM AP1903处理对细胞没有影响；而当用含1％血清的培液对细胞饥饿过夜后，再用含20％血清和10ng/ml PMA的培养液处理细胞时，通路激活，该基因组件表达，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图11b)。
表8.MAP/ERK通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
20％血清和10ng/ml PMA 活跃 死亡 存活
8.cAMP/PKA通路报告细胞系
将图12a的序列稳定整合于293A细胞，得到了的293A-CRE细胞。293A-CRE细胞在常规培养条件下，该通路无活性，基因组件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，10nM AP1903处理对细胞没有影响；而当加入5μg/ml Forskolin激活通路时，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图12b)。
表9.TGFβ通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
5μg/ml Forskolin 活跃 死亡 存活
9.NFκB通路报告细胞系
将图13a的序列稳定整合于293A细胞，得到了的293A-5NFκB-RE细胞。 293A-5NFκB-RE细胞在常规培养条件下，该通路无活性，基因组件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，10nM AP1903处理对细胞没有影响；而当加入20ng/ml TNFα激活该通路时，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图13b)。
表10.NFκB通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
20ng/ml TNFα 活跃 死亡 存活
10.p53通路报告细胞系
将图14a的序列稳定整合于U2OS细胞，得到了的U2OS-2p53-RE细胞。U2OS-2p53-RE细胞在常规培养条件下，该通路无活性，基因组件不表达，经2μg/ml嘌呤霉素处理后细胞死亡，10nM AP1903处理对细胞没有影响；而当加入100nM DOX激活该通路时，嘌呤霉素处理组细胞存活下来，AP1903处理组细胞死亡(图14b)。
表11.p53通路报告细胞系不同处理条件下的状态
处理条件 信号通路状态 AP1903 嘌呤霉素
常规 沉默 存活 死亡
100nM DOX 活跃 死亡 存活
实施例2细胞死活表型筛选的Wnt通路抑制剂
目前，已经利用该系统在384孔板上进行了高通量的化合物筛选，并且已找到一些通路的激活剂和抑制剂。在10％R-Spondin1激活的293T-7TCF细胞中，找到一些化合物使细胞在经AP1903处理后依旧存活，表明这些化合物(天然小分子化合物28-H11、3-F7)可以抑制Wnt通路的激活，是潜在的Wnt通路的抑制剂(图15a)。
为了进一步验证筛选得到的抑制剂的作用，将筛选到的Wnt通路抑制剂28-H11、3-F7与R-spondin1同时处理细胞，western blot检测β-catenin和activeβ-catenin水平。
实验结果表明，这抑制剂28-H11、3-F7可以明显降低β-catenin稳定性，且activeβ-catenin水平降低(图15b)。
实施例3细胞死活表型筛选的TGFβ通路的激活剂
此外，在TGFβ激活的293A-4SBE细胞中，找到一些天然小分子化合物(35-C11、35-D10、33-E10)，使细胞经AP1903处理后依旧存活，表明这些化合物可以抑制TGFβ通路的激活(图16a)。在常规培养条件的293A-4SBE细胞中，找到了一个天然小分子化合物21-H2能够使这个细胞在经嘌呤霉素处理后依旧存活，表明这个化合物可能是TGFβ通路的激活剂(图16b)。此前还未有过TGF通路小分子激活剂的报道，因此这些结果体现了本发明筛选系统的独特效能。
讨论
其他信号通路也与Wnt通路相似，经上游相关信号刺激后，最后由不同的转录因子来调控相应基因的表达。因每个信号通路所调控的转录因子具有特异性，且这些不同的转录因子所结合的DNA序列也是特异的，所以，不同信号通路可以调控不同靶基因的表达。而研究者们也利用了不同转录因子识别特定DNA序列的这个特性，设计了一些实验方法来检测信号通路的激活水平，其中较常见的是荧光素酶报告系统(Luciferase Assay)，可应用于检测某个基因或者某个药物对信号通路激活水平的影响。
荧光素酶报告系统对不同信号通路激活水平的检测和展现，主要是通过连接在一起的特异性报告元件与荧光素酶来实现(图1)。这里的报告元件是由多次重复的信号通路下游转录因子的结合位点序列与含有TATA盒的最小启动子组成，因此，荧光素酶的表达水平反映了相应信号通路的激活水平。实验时，需要先往细胞中转染荧光素酶报告质粒，在加入荧光素后，荧光素酶可以催化氧化荧光素，在氧化的过程中，发出生物荧光，通过荧光强度即可判断相应信号通路的激活水平。当该信号通路激活程度较高时，转录因子作用活跃，荧光素酶表达水平高，发出的生物荧光强；当信号通路处于抑制状态时，转录因子作用沉默，荧光素酶表达水平低，发出的生物荧光弱。
然而，在大规模筛选信号通路相关的激活剂或抑制剂时，荧光素酶报告质粒的转染以及荧光素的加入，工作量大，操作困难，难以实现高通量筛选，并且每次转染实验效果的均一性也很难控制。因此，为了更方便且直观地研究作用于某个信号通路的药物，本发明中构建的能够直接用死活表型来筛选鉴定的 报告细胞系，可高效、灵敏、稳定地用于高通量化合物筛选。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种用于筛选针对目标信号通路的抑制剂的筛选体系，其特征在于，所述筛选体系包括一培养体系以及存在于所述培养体系中的以下组分：
    (a)活的检测细胞，所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，
    其中，当所述检测细胞在激活培养条件下，细胞中的所述目标信号通路激活时，所述检测细胞表达所述自杀蛋白元件和保护蛋白元件，所述自杀蛋白元件在第一诱导剂存在下会诱导所述检测细胞发生凋亡；
    (b)待筛选的测试物；
    其中，当所述检测细胞中的所述目标信号通路激活，并且表达所述的自杀蛋白元件，且所述的测试物不导致所述自杀蛋白元件降解或减少时，所述第一诱导剂会诱导所述检测细胞发生凋亡；
    并且，当所述测试物导致所述检测细胞中的所述目标信号通路不激活，并且不表达所述的自杀蛋白元件时，则所述第一诱导剂不诱导所述检测细胞发生凋亡；
    当所述测试物导致所述检测细胞中的所述目标信号通路不激活，并且导致所述检测细胞不表达所述的自杀蛋白元件时，则所述的测试物被认为是候选抑制剂。
  2. 一种用于筛选针对目标信号通路的激活剂的筛选体系，其特征在于，所述筛选体系包括一培养体系以及存在于所述培养体系中的以下组分：
    (a)活的检测细胞，所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，
    其中，当所述检测细胞在常规培养条件下时，细胞中的所述目标信号通路未激活，所述检测细胞不表达所述的自杀蛋白元件和保护蛋白元件；所述检测细胞在第二诱导剂存在下发生凋亡；
    (b)待筛选的测试物；
    其中，当所述检测细胞中的所述目标信号通路未激活，并且不表达所述的保护蛋白元件时，所述第二诱导剂会诱导所述检测细胞发生凋亡；
    并且，当所述测试物导致所述检测细胞中的所述目标信号通路激活，并且表 达所述的保护蛋白元件，且所述的测试物不导致所述保护蛋白元件降解或减少时，则所述第二诱导剂不诱导所述检测细胞发生凋亡；
    当所述测试物导致所述检测细胞中的所述目标信号通路激活，并且表达所述的保护蛋白元件，且所述的测试物不导致所述保护蛋白元件降解或减少时，则所述的测试物被认为是候选激活剂。
  3. 如如权利要求1或2所述的筛选体系，其特征在于，所述目标信号通路选自Wnt通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路或p53通路。
  4. 一种筛选目标信号通路抑制剂的方法，其特征在于，包括步骤：
    (a)在激活培养条件下，将加入待筛选的测试物的培养体系作为实验组；将不加入测试化合物的培养体系作为空白对照组，其中，所述的培养体系中含有培养的活的检测细胞，所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，其中，当所述检测细胞在激活培养条件下，细胞中的所述目标信号通路激活时，所述检测细胞表达所述自杀蛋白元件和保护蛋白元件，所述自杀蛋白元件在第一诱导剂存在下会诱导所述检测细胞发生凋亡；和
    (b)向所述实验组和空白对照组中加入第一诱导剂，观测实验组和空白对照组中检测细胞的存活情况；
    其中，当实验组中检测细胞的存活数量显著高于对照组时，则表明该测试物为候选抑制剂。
  5. 一种筛选目标信号通路激活剂的方法，其特征在于，包括步骤：
    (a)在常规培养条件下，将加入待筛选的测试物的培养体系作为实验组；将不加入测试化合物的培养体系作为空白对照组，其中，所述的培养体系中含有培养的活的检测细胞；所述检测细胞的基因组中整合有表达盒，所述表达盒中，目标信号通路的报告元件、自杀蛋白元件和保护蛋白元件可操作性地连接，
    其中，当所述检测细胞在常规培养条件下，细胞中的所述目标信号通路未激活时，所述检测细胞不表达所述的自杀蛋白元件和保护蛋白元件；所述检测细胞在第二诱导剂存在下发生凋亡；和
    (b)向所述实验组和空白对照组中加入第二诱导剂，观测实验组和空白对照组中检测细胞的存活情况；
    其中，当实验组中检测细胞的存活数量显著高于对照组时，则表明该测试物为候选激活剂。
  6. 一种如权利要求1或2所述的筛选体系的用途，其特征在于，用于筛选目标信号通路激活剂和/或抑制剂。
  7. 一种用于筛选针对目标信号通路的抑制剂或激活剂的筛选装置，其特征在于，所述装置包括：
    (d1)凋亡筛选模块，所述的凋亡筛选模块包括一个或多个培养单元，所述培养单元中设有n个用于培养活的检测细胞的培养室(或孔)(compartment)，其中在所述培养室中含有权利要求1或2所述的用于筛选针对目标信号通路的抑制剂或激活剂的筛选体系；n为≥2的正整数；
    (d2)数据采集模块，所述数据采集模块被配置为对凋亡筛选模块中各个培养室中的所述检测细胞的凋亡情况进行数据采集；
    (d3)筛选分析模块，所述筛选分析模块被配置为对来自所述数据采集模块的细胞凋亡情况进行分析，获得待筛选的测试物是否是针对目标信号通路的抑制剂或激活剂的分析结果；和
    (d4)输出模块，所述输出模块输出筛选分析模块的分析结果。
  8. 如权利要求7所述的筛选装置，其特征在于，所述培养单元的数量为1-200个，较佳地4-100个，更佳地8-50个，最佳地10-20个。
  9. 一种表达盒，其特征在于，所述表达盒从5'-3'具有如式I所示的结构：
    Z1-Z2-Z3-Z4  式I
    式中，各“-”独立地为键或核苷酸连接序列；
    Z1为报告元件；
    Z2为自杀蛋白元件的编码序列；和
    Z3为无或自剪切序列；
    Z4为保护蛋白元件的编码序列；
    其中，所述报告元件包含目标信号通路中最下游转录因子的结合位点Y1、下游启动子Y2，其中Y1与Y2操作性地连接，从而当目标信号通路的最下游转录因子的结合位点Y1结合目标信号通路最下游的转录因子时，下游启动子Y2驱动自杀蛋白元件和保护蛋白元件的表达。
  10. 如权利要求9所述的表达盒，其特征在于，所述目标信号通路选自Wnt 通路、TGFβ通路、Hippo通路、Keap1-Nrf2通路、VHL-HIF1α通路、JAK-STAT1/2通路、MAP/ERK通路、cAMP/PKA通路、NFκB通路或p53通路。
  11. 一种载体，其特征在于，所述载体中含有如权利要求9所述的表达盒。
  12. 一种宿主细胞，其特征在于，所述宿主细胞的基因组的一个或多个位点整合有权利要求9所述的表达盒，或者所述宿主细胞中含有权利要求11所述的载体。
  13. 如如权利要求12所述的宿主细胞，其特征在于，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
PCT/CN2022/085516 2021-04-09 2022-04-07 基于细胞死活表型的信号通路激活剂和抑制剂的新型筛选系统 WO2022214022A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010064142A1 (en) * 2008-12-05 2010-06-10 Christoph Merten Screening method for the identification of inhibitors of viral entry
CN103881979A (zh) * 2014-03-20 2014-06-25 北京大学 抗丙肝病毒药物高通量筛选模型及其应用
US20200190509A1 (en) * 2017-06-06 2020-06-18 Katholieke Universiteit Leuven Methods for identifying activators and suppressors
WO2021051016A1 (en) * 2019-09-13 2021-03-18 Massachusetts Institute Of Technology Systems and assays for identifying pu.1 inhibitors
CN112553286A (zh) * 2020-11-05 2021-03-26 北京大学深圳医院 自杀基因/前药系统疗效的评价方法和药物筛选方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010064142A1 (en) * 2008-12-05 2010-06-10 Christoph Merten Screening method for the identification of inhibitors of viral entry
CN103881979A (zh) * 2014-03-20 2014-06-25 北京大学 抗丙肝病毒药物高通量筛选模型及其应用
US20200190509A1 (en) * 2017-06-06 2020-06-18 Katholieke Universiteit Leuven Methods for identifying activators and suppressors
WO2021051016A1 (en) * 2019-09-13 2021-03-18 Massachusetts Institute Of Technology Systems and assays for identifying pu.1 inhibitors
CN112553286A (zh) * 2020-11-05 2021-03-26 北京大学深圳医院 自杀基因/前药系统疗效的评价方法和药物筛选方法

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