WO2020182077A1 - Gli2抑制剂的用途以及抑制gli2的化合物的筛选方法 - Google Patents

Gli2抑制剂的用途以及抑制gli2的化合物的筛选方法 Download PDF

Info

Publication number
WO2020182077A1
WO2020182077A1 PCT/CN2020/078224 CN2020078224W WO2020182077A1 WO 2020182077 A1 WO2020182077 A1 WO 2020182077A1 CN 2020078224 W CN2020078224 W CN 2020078224W WO 2020182077 A1 WO2020182077 A1 WO 2020182077A1
Authority
WO
WIPO (PCT)
Prior art keywords
gli2
cells
alkyl
optionally substituted
smo
Prior art date
Application number
PCT/CN2020/078224
Other languages
English (en)
French (fr)
Inventor
王宇
吴付佳
Original Assignee
中国科学院动物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院动物研究所 filed Critical 中国科学院动物研究所
Publication of WO2020182077A1 publication Critical patent/WO2020182077A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C405/00—Compounds containing a five-membered ring having two side-chains in ortho position to each other, and having oxygen atoms directly attached to the ring in ortho position to one of the side-chains, one side-chain containing, not directly attached to the ring, a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, and the other side-chain having oxygen atoms attached in gamma-position to the ring, e.g. prostaglandins ; Analogues or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding

Definitions

  • the present invention relates to the prostaglandin of formula I and its analogues and pharmaceutical compositions containing these compounds for use as medicines, in particular for use as a protein GLI2 containing a zinc finger domain (ie, glioma-related oncogene-2 ) Inhibitors, used to treat, prevent or alleviate diseases caused by imbalance of the signal transduction pathway involved in GLI2 in subjects, such as tumors (especially tumor resistance), neurodegenerative diseases or developmental syndromes, and deformities.
  • a protein GLI2 containing a zinc finger domain ie, glioma-related oncogene-2
  • Inhibitors used to treat, prevent or alleviate diseases caused by imbalance of the signal transduction pathway involved in GLI2 in subjects, such as tumors (especially tumor resistance), neurodegenerative diseases or developmental syndromes, and deformities.
  • the present invention also relates to a screening method for compounds that inhibit GLI2, especially a high-content screening method (high-content screen; HCS), which is directly based on the interaction between the compound and GLI2, by visualizing GLI2 and tracking it in cells expressing GLI2 To screen candidate drugs that inhibit GLI2.
  • HCS high-content screening method
  • the GLI family is a group of transcription factor proteins with zinc finger domains. They bind to DNA through zinc finger motifs. They are named after their abnormally high expression in Glioblastoma (Kinzler KW et al., Identification). of an amplified, highly expressed gene in a human glioma, Science,1987,236(4797):70-73). It is known that the GLI family contains members called GLI1, GLI2, and GLI3, which are a platform for the integration and interweaving of several signaling pathways in the cell, such as Hedgehog (HH) signaling pathway, RAS signaling pathway, Transforming growth factor ⁇ signaling pathway (Lauth M.
  • HH Hedgehog
  • RAS Transforming growth factor ⁇ signaling pathway
  • the GLI2 gene is located at 2q14.2 and has a total of 4758 bases (https://www.ncbi.nlm.nih.gov/gene/2736).
  • the activity of the GLI2 protein encoded by this gene is regulated by many different mechanisms. Compartmentalization is one of the mechanisms regulating GLI2 protein.
  • GLI2 protein is mainly retained in the cytosol and degraded.
  • the HH signaling pathway is activated, the GLI2 protein is transported into the primary cilium (PC), where it is separated from SuFu, and then the GLI2 protein is transported to the nucleus.
  • PC primary cilium
  • GLI2 binds to specific DNA sequences in the promoter elements of target genes, and activates the transcription and expression of these target genes (for example, Gli1, Ptch1, wnt, FGF, Cyclins), thereby regulating cell growth, proliferation and differentiation .
  • GLI2 protein is found in the pathological phenotypes caused by abnormal regulation of cellular processes involved in the HH signaling pathway, RAS signaling pathway, transforming growth factor ⁇ signaling pathway, wnt signaling pathway, notch signaling pathway, etc. Activation and/or high expression. Therefore, there is an urgent need in the art for compounds that have a selective inhibitory effect on the GLI2 protein located at the confluence point of these signal transduction pathways and methods for efficiently screening for compounds that inhibit GLI2.
  • n is each independently an integer of 0-12, for example, n is each independently an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;
  • R 4 is each independently selected from the group consisting of H, -CH 3 , -COCH 3 , -OH, the salt of -OH, -COOH or the salt of -COOH, or alkyl esters with 1 to 6 carbon atoms, -CONH 2. -CONH(C 1-5 alkyl)OH;
  • R 5 is selected from H, OH, optionally substituted C 1-6 alkyl, halogen, O (optionally substituted C 1-6 alkyl);
  • R 6 is independently selected from H, OH, optionally substituted C 1-6 alkyl, halogen, O (optionally substituted C 1-6 alkyl);
  • n is each independently an integer of 0-10, for example, m is each independently an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • any two adjacent bonds At least one of is a single bond
  • R 2 is selected from H, OH or O (optionally substituted C 1-6 alkyl);
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • n is each independently an integer of 2, 3, 4, 5 or 6;
  • R 4 is each independently selected from the group consisting of H, -COOH, or the salt of -COOH or alkyl ester with carbon number of 1-3, -OH, the salt of -OH, -CONH 2 , -CONH (C 1- 3 alkyl) OH;
  • R 5 is selected from H or OH
  • R 6 is independently selected from H, C 1-3 alkyl, halogen
  • n is each independently an integer of 0, 1, 2, 3, 4;
  • any two adjacent bonds At least one of is a single bond
  • R 1 is selected from H or OH
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • n is each independently an integer of 2, 3, 4, 5 or 6;
  • R 4 is each independently selected from the group consisting of H, -COOH, or the salt of -COOH or alkyl ester with carbon number of 1-3, -OH, the salt of -OH, -CONH 2 , -CONH (C 1- 3 alkyl) OH;
  • R 5 is selected from H or OH
  • R 6 is independently selected from H, C 1-3 alkyl, halogen
  • n is each independently an integer of 0, 1, 2, 3, 4;
  • any two adjacent bonds At least one of is a single bond
  • R 1 is selected from H, optionally substituted C 1-6 alkyl, halogen
  • R 2 is selected from OH, O (optionally substituted C 1-6 alkyl)
  • R 3 and the ring carbon atom connecting R 1 are connected to each other to form a ring group of the following formula II or formula III:
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • n is each independently an integer of 2, 3, 4, 5 or 6;
  • R 4 is each independently selected from the group consisting of H, -COOH, or the salt of -COOH or alkyl ester with carbon number of 1-3, -OH, the salt of -OH, -CONH 2 , -CONH (C 1- 3 alkyl) OH;
  • R 5 is selected from H or OH
  • n is each independently an integer of 1, 2, 3, 4;
  • any two adjacent bonds At least one of is a single bond
  • the prostaglandin of formula I and its analogs can selectively inhibit the abnormal activation of the signaling pathway involved in GLI2, including but not limited to the classical HH signaling pathway (Hh ligand-dependent HH signaling pathway ) And abnormal activation of non-classical HH signaling pathway (Hh ligand-independent HH signaling pathway), RAS signaling pathway, transforming growth factor ⁇ signaling pathway, wnt signaling pathway, and notch signaling pathway, thereby
  • cancer cells e.g., cancer stem cells
  • nerve disorders for example, aseptic forebrain malformation, Greg head-polydactyl-combined digit syndrome, Pallister-Hall syndrome, Rubenstein-Teybi syndrome, basal cell nevus complex Signs,
  • the prostaglandin of Formula I and its analogues are combined with additional one or more therapeutic agents or therapies to treat, prevent, or alleviate the disease in the subject, the additional one or Multiple therapeutic agents or therapies are selected from: chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapies, cytokines, surgical treatment, and irradiation.
  • the prostaglandin of formula I and its analogues are prostaglandin A1 (PGA1), 8-iso PGA1, PGA2, prostaglandin D1 alcohol (PGD1 alcohol), prostaglandin D1, prostaglandin D2, 5- Trans prostaglandin D2 (5-trans PGD2), prostaglandin E1 (PGE1), PGE1 alcohol, 8-iso PGE1, PGE1 ethyl ester, 6-keto PGE1, PGE1 ethanolamine, PGE2, 15(R)-PGE2, 8-iso PGE2, PGE2 ethanolamine, the PGI2 (sodium salt), prostaglandin J2 (PGJ2), 15- deoxy - ⁇ 12,14 - prostaglandin J2 (15- deoxy-- ⁇ 12,14 -PGJ2), iloprost (Iloprost), Carbaprostacyclin, Ciprostene (for example, Ciprostene calcium salt), and Rivenprost.
  • PGA1 prostaglandin A1
  • the present invention provides a high-content screening method for compounds that inhibit GLI2, the method allows visualization of whether the compound inhibits GLI2 protein transport into and/or accumulation in primary cilia, the method comprising:
  • the construct expressing the primary ciliary marker of the first marker and the construct expressing the GLI2 of the second marker of step i are introduced into mammalian cells (such as NIH/3T3 cells, DAOY cells) simultaneously or sequentially in different orders , Or introduce the dual expression construct of the primary cilia marker expressing the first marker and GLI2 expressing the second marker of step i into mammalian cells (such as NIH/3T3 cells, DAOY cells) to obtain the first marker simultaneously
  • mammalian cells such as NIH/3T3 cells, DAOY cells
  • the test compound that reduces the accumulation of GLI2 protein in the primary cilia is determined as the compound that inhibits GLI2.
  • the high content screening method of compounds that inhibit GLI2 includes the following steps:
  • the inducer for example, SHH-N or SAG
  • the inducer that induces GLI2 protein accumulation in primary cilia and the test compound are contacted with the second cell line of step iii, and the inducer that induces GLI2 protein accumulation in primary cilia only Compare with the second cell line contact situation in step iii;
  • the first label and the second label in the high-content screening method for compounds that inhibit GLI2 are selected from, but not limited to, green fluorescent protein (GFP), yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, Orange fluorescent protein, preferably, enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein, enhanced blue fluorescent protein, enhanced cyan fluorescent protein, enhanced orange fluorescent protein, for example, TagRFP, tdTomato, DsRed, HcRed , AsRed, AmCyan, ZsGreen, AcGFP and ZsYellow; the primary cilia markers are selected from but not limited to ARL13B, INVERSIN, tubulin.
  • the present invention also provides a screening method for compounds that inhibit GLI2 overexpression, the method comprising:
  • GLI2 overexpressing construct i. Introducing the GLI2 overexpressing construct into mammalian cells (for example, NIH/3T3 or DAOY cells) to obtain cells overexpressing GLI2;
  • test compound preferably, the test compound identified in the second aspect above
  • iii Determine the activity level of the GLI2, for example, determine the endogenous target gene expression of GLI2 (for example, Gli1, Ptch1, Wnt2, Axin2, EGFR and cyclin D1 mRNA levels) and/or endogenous GLI1 protein The activity level and/or the level of other molecules related to GLI2 activation; and/or the detection of the activity level of the GLI-luciferase reporter gene (eg, measured by testing the activity of the luciferase reporter gene);
  • the present invention provides a screening method for compounds that inhibit GLI2 related to Hedgehog signaling pathway activation resistant to Smoothened (SMO) antagonists, the method comprising:
  • test compound preferably, the test compound identified in the second aspect above
  • cells that have abnormally activated SMO of i eg, overexpress SMO-WT or SMO-D473H or SMO-W535L
  • iii Determine the activity level of GLI2, for example, determine the endogenous target gene expression of GLI2 (for example, Gli1, Ptch1, Wnt2, Axin2, EGFR and cyclin D1 mRNA level) and/or the endogenous GLI1 protein activity level And/or the level of other molecules related to the activation of GLI2; and/or detecting the activity level of the GLI-luciferase reporter gene (eg, measured by testing the activity of the luciferase reporter gene);
  • Figure 1 Shows the generation and characterization of cell lines for high content screening (HCS) and luciferase assays.
  • Figure 1A- Figure 1B Shows the use of SHH-N, 100nM SAG, DMSO, Vimodedi (1 ⁇ M), cyclopamine (1 ⁇ M), cyclopamine+SHH-N, or Vimodedi+SHH- Representative images ( Figure 1A) and quantitative results (Figure 1B) of the localization of GLI2 cilia in N-treated 3T3/ARL13B::tagRFPT/EGFP::GLI2 cells; scale bar, 10 ⁇ m (dilute the collected SHH-N 16 times to use, Achieve about 100-fold activation as shown in Figure 3B relative to control cells without SHH-N);
  • Figure 1C shows 3T3/ARL13B::tagRFPT/EGFP compared with 3T3/ARL13B::tagRFPT cells ::GLI-luciferase measurement of the responsiveness of GLI2 cells to
  • Figure 2 Illustrates six prostaglandins and their analogs that inhibit GLI2 accumulation in primary cilia.
  • Figure 2A shows the chemical structures of these six exemplified prostaglandins and their analogs;
  • Figure 2B and Figure 2C in 3T3/ARL13B::tagRFPT/EGFP::GLI2 cells, these six exemplified prostaglandins inhibit Representative images of SHH-N-induced GLI2 accumulation in cilia ( Figure 2B) and quantitative results (Figure 2C), where 1 ⁇ M Vimodedi was used as a positive control, prostaglandin was used at 10 ⁇ M, and the data was expressed as four replicates Average and standard deviation, scale bar: 20 ⁇ m.
  • Figure 4A In 3T3/GLI-luc/GLI2 cells, exemplary dose-dependent effects of the 6 prostaglandins identified by HCS and vilmodecine on the activity of the HH pathway mediated by overexpression of GLI2, measured in quadruplicate , The data are shown as the mean and standard deviation;
  • Figure 4B qRT-PCR analysis of PGE1 (30 ⁇ M), Vimodji (10 ⁇ M), cyclopamine (10 ⁇ M) in 3T3/GLI-luc/GLI2 cells endogenous Gli1 and The effect of Ptch1 expression, data are shown as the average and standard deviation from three biological replicates, **p ⁇ 0.01; NS, not significant, student's t test.
  • Figure 5 Illustrates the results of using 3T3/GLI-luc/GLI2 cells to detect other prostaglandins and their analogs in the GLI-luciferase reporter gene assay.
  • the data represents the average and standard deviation of quadruplicate samples.
  • the figure shows the IC50 and chemical structure of each prostaglandin and its analogs.
  • Figure 6 Illustrates the dose-dependent effects of HCS-identified prostaglandins on overexpression of wild-type SMO (also denoted as SMO-WT), SMO-D473H and SMO-W535L-mediated GLI-luciferase reporter gene activity . All assays were performed in quadruplicate, and the data represents the average and standard deviation.
  • Figure 7 Shows the effect of PGE1 on the HH pathway activity produced by introducing SMO-D473H, SMO-W535L and SMO-WT, respectively, using Vimodedi as a control for comparison.
  • Figure 7A and Figure 7B respectively show the effect of Vimodji and PGE1 on 3T3/GLI-luc cells overexpressing wild-type SMO (red square), SMO-D473H (blue triangle) or SMO-W535L (green circle) The effect of GLI-luciferase reporter gene activity;
  • Figure 7C- Figure 7E qRT-PCR analysis of Vimodji (10 ⁇ M) and PGE1 (30 ⁇ M) on the overexpression of wild-type SMO ( Figure 7C), SMO-D473H, respectively ( Figure 7D) or SMO-W535L ( Figure 7E) 3T3/GLI-luc cell endogenous Gli1 and Ptch1 expression;
  • Figure 7F shows the analysis of Vimodji (10 ⁇ M) and PGE1
  • FIG. 7G-7H shows Vimodji (Fig. 7G) and PGE1 (Fig. 7H) Inhibition of GLI-luciferase reporter gene activity in 3T3/GLI-luc cells stimulated with 10nM (blue circle), 50nM (red square) or 250nM (green triangle) SAG. The data is expressed as four replicates The mean and standard deviation of, **p ⁇ 0.01; ***p ⁇ 0.001; NS means "not significant"; student's t test.
  • Figure 8 Shows the activity of prostaglandins against SHH-N or SAG-induced HH pathway.
  • Figure 8A Shows the dose-dependent effects of the 6 prostaglandins illustrated in Figure 2A on SHH-N-induced GLI-luciferase reporter gene activity in 3T3/GLI-luc cells.
  • FIG. 8B shows the qRT-PCR results of PGE1 (30 ⁇ M) acting on the target genes (Gli1, Ptch1) of GLI2 in 3T3/GLI-luc cells treated with SHH-N or SAG
  • FIG. 8C Shows the treatment of 3T3/GLI-luc cells with only HH pathway agonists (SHH-N or SAG) or their combination with PGE1 (30 ⁇ M), Vimodedi (1 ⁇ M) or cyclopamine (1 ⁇ M) , Western blot analysis results of endogenous GLI1 protein levels in cell lysates, using ⁇ -actin as a loading control
  • Figure 8D shows the effect of Raven Prostaglandin (10 ⁇ M) on SHH in 3T3/GLI-luc cells -N-induced GLI-luciferase reporter gene activity
  • Figure 8E shows that in 3T3/GLI-luc cells treated with SHH-N, Raven Prostaglandin (10 ⁇ M) acts on the target gene of GLI
  • Figure 9 shows the effect of PGE1 on the cell viability and HH pathway activity of human DAOY medulloblastoma cells and its two derivative cell lines resistant to SMO inhibitors.
  • Figure 9A-9C shows the wild-type DAOY cells ( Figure 9A), over-expressing SMO-D473H ( Figure 9B) or SMO-D473H treated with PGE1 (100 ⁇ M), Vimodedi (10 ⁇ M) and cyclopamine (10 ⁇ M) The results of cell viability determination in cells overexpressing GLI2 ( Figure 9C).
  • Figure 9D-9F Shows PGE1 (100 ⁇ M) and Vimod for qRT-PCR analysis GLI1 and PTCH1 mRNA levels in wild-type DAOY cells (Figure 9D), overexpressing SMO-D473H ( Figure 9E), or overexpressing GLI2 (Figure 9F) treated with Gee (10 ⁇ M) and cyclopamine (10 ⁇ M) ,
  • the data is shown as the mean and standard deviation from three independent experiments. *p ⁇ 0.05, **p ⁇ 0.01; ***p ⁇ 0.001; NS means "not significant"; student’s t test.
  • Figure 10A shows the results of RT-PCR detection of the Gli2 gene in the tumor cells shown
  • Figure 10B shows the effect of PGE1 (67 ⁇ M) on the cell viability of HCC38, MCF7, DLD-1 and HepG2 cells that do not express the Gli2 gene Influence, where the ordinate 1.0 represents the cell viability when PGE1 is not added.
  • Figure 12 Shows the primer sequences used for RT-PCR and qRT-PCR.
  • Figure 15 Constructed vector containing SHH-N for stably transfected cell line.
  • Figure 16 8 ⁇ GLIBS-driven firefly luciferase plasmid.
  • Figure 18 Mouse GLI2 expression plasmid driven by CMV promoter.
  • Figure 19 SMO-WT vector inserted into the pBABE retroviral plasmid.
  • Figure 20 The vector with SMO-D473H inserted into the pBABE retroviral plasmid.
  • Figure 21 The vector with SMO-W535L inserted into the pBABE retroviral plasmid.
  • Figure 22 The vector with SMO-D473H inserted into the pRRL lentiviral plasmid.
  • the activation and/or high expression of GLI2 protein is present in some pathological phenotypes. Since it is necessary for GLI2 protein to transport and accumulate in cilia in the activation and/or high expression of GLI2 protein, this provides new opportunities for the discovery of compounds that target GLI2.
  • the present invention provides a high-content screening method for compounds that inhibit the activity of GLI2 protein by inhibiting the transport of GLI2 protein to cilia and accumulation in cilia. It also provides a method for inhibiting the transport of GLI2 protein into primary cilia and / Or compounds that inhibit the accumulation of GLI2 protein in primary cilia, which are prostaglandins of formula I and their analogs.
  • alkyl refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group, which may be linear or branched. Therefore, the “alkyl” group does not contain any carbon-carbon double bonds or any carbon-carbon triple bonds.
  • C 1-6 alkyl means an alkyl group having 1 to 6 carbon atoms.
  • Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g. n-propyl or isopropyl) or butyl (e.g. n-butyl, isobutyl, sec-butyl or tert-butyl).
  • alkyl preferably refers to C1-4 alkyl, more preferably methyl or ethyl, and even more preferably methyl.
  • halogen refers to fluorine, chlorine, bromine or iodine, preferably bromine or chlorine or fluorine.
  • optionally substituted groups mean that these groups may or may not have one or more substituents, for example, 1, 2, 3, or 4 substituents. It should be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless otherwise defined, the "optionally substituted” group mentioned in this specification preferably has no more than two substituents, and in particular, may have only one substituent. Preferably, the group has no optional substituents, that is, the corresponding group is unsubstituted.
  • alkyl ester having 1 to 6 carbon atoms means an ester of C 1 -C 6 -alkyl.
  • salt refers to a salt of a compound prepared with a relatively non-toxic acid or base, depending on the substituents on the compound described herein.
  • the base addition salt can be obtained by contacting the compound in a neutral form with a sufficient amount of the desired base, purely or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts.
  • the acid addition salt can be obtained by contacting the compound in a neutral form with a sufficient amount of the desired acid, purely or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogen carbonate, phosphoric acid, monohydrophosphoric acid, dihydrophosphoric acid, sulfuric acid, monohydrogen sulfuric acid, hydroiodic acid Or phosphorous acid, etc., and those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid , Phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and other salts.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogen carbonate, phosphoric acid, monohydrophosphoric acid, di
  • solvate refers to a compound that is complexed with a solvent.
  • Solvents capable of forming solvates with the compounds in the embodiments include common organic solvents such as alcohols (methanol, ethanol, etc.), ether, acetone, ethyl acetate, halogenated solvents (dichloromethane, chloroform, etc.), hexane, and pentane.
  • alcohols methanol, ethanol, etc.
  • ether acetone
  • ethyl acetate halogenated solvents
  • hexane hexane
  • pentane hexane
  • stereoisomer refers to the isomers produced by the different arrangements of atoms in the molecule in space, which can be divided into enantiomers (isomers) and diastereoisomers (isomers). ) There are two categories.
  • High-content analysis systems usually use 384-well plates for multi-sample parallel data acquisition and analysis, which is hundreds of times faster than flow cytometers that require single sample processing one by one; at the same time, image-based analysis enables more research under natural conditions of living cells.
  • test compounds such as the distribution and transport of proteins in cells, expression regulation and signal transduction, etc.
  • primary cilia and “cilia” are used interchangeably herein, and are a special organelle present on the surface of most mammalian cells, anchored to the cell surface by centrioles.
  • primary cilia was first used in the epithelial cells of the respiratory tract. Most of the cells in the body have only one cilia, similar to the single cilia in the respiratory tract. This single cilia is called the primary cilia. Markers present on primary cilia include but are not limited to ARL13B, INVERSIN, tubulin.
  • GLI2 protein or “GLI2” are used interchangeably herein, and include naturally occurring or recombinant forms of GLI2 protein.
  • Several vertebrate GLI2 proteins have been characterized, for example, human GLI2 (GenBank accession number NM_030381.1; NM_030380.1; NM_030379.1, DQ086814.1), mouse GLI2 (GenBank accession number NM_001081125.1; XM_006529141.3; XM_011247924.2; XM_922107.1).
  • GLI2 protein activity refers to the ability to conduct GLI2 signal transduction, and includes, for example, activation of downstream genes by GLI2 transcription.
  • Hedgehog can be used interchangeably with the terms “Hh” and "HH”.
  • the Hh ligand can initiate the classic Hh signaling pathway after binding to the Hh receptor.
  • Several vertebrate Hh ligands are known in the art, for example, human SHH, murine SHH, human IHH, and murine DHH.
  • the inhibitory effect of SMO is alleviated, allowing SMO cilia to accumulate and activate (Corbit et al. (2005).
  • signal transduction or “signaling activity” refers to the biochemical causality usually triggered by protein-protein interactions such as the binding of growth factors to receptors, which result in a signal from a cell Part is transferred to another part of the cell.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom.
  • Host cells include cultured cells as well as cells inside animal tissues.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and large animals). mouse). In particular, individuals are humans.
  • treatment refers to a clinical intervention intended to alter the natural course of disease in the individual being treated.
  • the desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.
  • the prostaglandins of Formula I and their analogs are used to delay the progression of diseases caused by the activation of GLI2 protein.
  • GLI2 inhibitors are provided, which are prostaglandins and their analogs represented by the following formula I:
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • R 4 is each independently selected from the group consisting of H, -CH 3 , -COCH 3 , -OH, the salt of -OH, -COOH or the salt of -COOH, or alkyl esters with 1 to 6 carbon atoms, -CONH 2. -CONH(C 1-5 alkyl)OH;
  • R 5 is selected from H, OH, optionally substituted C 1-6 alkyl, halogen, O (optionally substituted C 1-6 alkyl);
  • R 6 is independently selected from H, OH, optionally substituted C 1-6 alkyl, halogen, O (optionally substituted C 1-6 alkyl);
  • n is each independently an integer of 0-10, for example, m is each independently an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • any two adjacent bonds At least one of is a single bond
  • R 2 is selected from H, OH or O (optionally substituted C 1-6 alkyl);
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • n is each independently an integer of 2, 3, 4, 5 or 6;
  • R 4 is each independently selected from the group consisting of H, -COOH, or the salt of -COOH or alkyl ester with carbon number of 1-3, -OH, the salt of -OH, -CONH 2 , -CONH (C 1- 3 alkyl) OH;
  • R 5 is selected from H or OH
  • R 6 is independently selected from H, C 1-3 alkyl, halogen
  • n is each independently an integer of 0, 1, 2, 3, 4;
  • any two adjacent bonds At least one of is a single bond
  • the prostaglandin of formula I and its analogs are compounds having the following structural formula:
  • the prostaglandin of formula I and its analogues are: wherein R 1 is selected from H or OH;
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • n is each independently an integer of 2, 3, 4, 5 or 6;
  • R 4 is each independently selected from the group consisting of H, -COOH, or the salt of -COOH or alkyl ester with carbon number of 1-3, -OH, the salt of -OH, -CONH 2 , -CONH (C 1- 3 alkyl) OH;
  • R 5 is selected from H or OH
  • R 6 is independently selected from H, C 1-3 alkyl, halogen
  • n is each independently an integer of 0, 1, 2, 3, 4;
  • any two adjacent bonds At least one of is a single bond
  • the prostaglandin of formula I and its analogs are compounds having the following structural formula:
  • the prostaglandin of formula I and its analogs are: wherein R 1 is selected from H, optionally substituted C 1-6 alkyl, halogen;
  • R 2 is selected from OH, O (optionally substituted C 1-6 alkyl)
  • R 3 and the ring carbon atom connecting R 1 are connected to each other to form a ring group of the following formula II or formula III:
  • X 1 , X 2 , X 3 are CH, CH 2 , O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine.
  • X 1 is O or S or direct bond
  • X 2 Is O or S or direct bond or carbon-carbon triple bond or optionally substituted phenyl or pyridine
  • X 3 is CH or CH 2 ;
  • n is each independently an integer of 2, 3, 4, 5 or 6;
  • R 5 is selected from H or OH
  • n is each independently an integer of 1, 2, 3, 4;
  • any two adjacent bonds At least one of is a single bond
  • the prostaglandin of formula I and its analogs are compounds having the following structural formula:
  • the prostaglandins represented by formula I and their analogs can inhibit the transport of GLI2 protein into primary cilia, and/or inhibit the accumulation of GLI2 protein in primary cilia, and/or inhibit the activation of target genes in the nucleus by GLI2 protein.
  • GLI2 protein occurs in diseases caused by abnormal regulation of cellular processes involved in HH signaling pathway, RAS signaling pathway, transforming growth factor ⁇ signaling pathway, wnt signaling pathway, notch signaling pathway, etc. Physical phenotype.
  • Both the Hh ligand-dependent mechanism and the Hh ligand-independent mechanism of the HH pathway activation caused by the Hh ligand-independent mechanism can lead to abnormal activation of the GLI2 protein, wherein the Hh ligand-dependent mechanism is the Hh ligand (for example, human SHH, murine SHH, After human IHH and mouse DHH) bind to the Hh receptor PTCH1, the inhibitory effect of PTCH1 on SMO is alleviated, and SMO is activated and subsequently causes the activation of GLI2 protein; wherein the Hh ligand-independent mechanism is independent of Hh ligand It binds to the Hh receptor, but the mechanism of GLI2 protein activation, such as the inability to inhibit SMO due to mutations in the loss of PTCH1 function, resulting in activation of SMO and subsequent activation of GLI2 protein; it can also be due to mutations in which SMO gains function that cause SMO activation and subsequently GLI2 Protein activation; GLI
  • SMO inhibitors that inhibit the activation of the HH pathway have been identified in the prior art.
  • the first SMO inhibitor identified in the field that directly binds to SMO is cyclopamine (Chen et al. (2002). Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes&development 16,2743-2748), The cyclopamine is a natural compound found in wild California Veratrum (Veratrum californicum).
  • the inventors developed a method for high-content screening of compounds that inhibit GLI2.
  • the method can compare a relatively large number of compounds inhibiting the in vitro activity of GLI2 under different mechanisms of abnormal GLI2 protein activation.
  • this type of high-content screening is performed in a multi-well microtiter plate (for example, in a 96-well plate or a 384-well plate or a plate with 1536 wells or 3456 wells).
  • the high-content screening method of the present invention can be used to screen compounds selected from small molecules, proteins, peptides and nucleic acids and/or candidate drugs for inhibiting the activity of GLI2 protein, and the method allows visualization of the compounds and /Or whether the candidate drug inhibits the transport of GLI2 protein into and/or accumulation in primary cilia, the method includes:
  • the construct expressing the primary ciliary marker of the first marker and the construct expressing the GLI2 of the second marker of step i are introduced into mammalian cells simultaneously or sequentially in different orders, or the first marker of step i is expressed
  • the dual expression construct of the primary cilia marker and the second marker GLI2 is introduced into mammalian cells to obtain a cell line that simultaneously expresses the first marker primary cilia marker and the second marker GLI2;
  • the test compound that reduces the accumulation of GLI2 protein in the primary cilia is determined as the compound that inhibits GLI2.
  • the "primary cilia marker” can be used to determine the primary cilia.
  • Primary cilia markers are known in the art and include but are not limited to ARL13B, INVERSIN, tubulin.
  • mammalian cells are mammalian host cell lines suitable for the production of foreign proteins, including but not limited to human embryonic kidney lines (293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1) , African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), CHO cells, COS Cells, NIH/3T3 cells, DAOY cells.
  • human embryonic kidney lines (293 or 293F cells
  • BHK baby hamster kidney cells
  • CV1 African green monkey kidney cells
  • HELA human cervical cancer cells
  • MDCK canine kidney cells
  • BBL 3A Buffalo rat liver cells
  • W138 human lung cells
  • COS Cells COS Cells
  • NIH/3T3 cells NIH/3T3 cells
  • the SHH-N treatment group is compared with the DMSO treatment group, There is no significant difference in the number of EGFP::GLI2 localizations on primary cilia)
  • GLI2 protein is expressed, HH pathway agonists are not required.
  • tagRFPT is used to label the primary cilia marker and EGFP is used to label the GLI2 protein.
  • the compound that inhibits GLI2 screened by the high-content screening method of the present invention is a prostaglandin represented by Formula I and its analogs.
  • GLI2 overexpressing construct i. Introducing the GLI2 overexpressing construct into mammalian cells (for example, NIH/3T3 or DAOY cells) to obtain cells overexpressing GLI2;
  • test compound preferably, the compound that inhibits GLI2 screened by the high-content screening method of the present invention
  • iii Determine the activity level of the GLI2, for example, determine the endogenous target gene expression of GLI2 (for example, Gli1, Ptch1, Wnt2, Axin2, EGFR and cyclin D1 mRNA levels) and/or endogenous GLI1 protein The activity level and/or the level of other molecules related to GLI2 activation; and/or the detection of the activity level of the GLI-luciferase reporter gene (eg, measured by testing the activity of the luciferase reporter gene);
  • the method is a screening method for a compound that inhibits GLI2 related to Hedgehog signaling pathway activation that is resistant to a Smoothened (SMO) antagonist, the method comprising:
  • test compound preferably, the compound that inhibits GLI2 screened by the high-content screening method of the present invention
  • SMO of i are abnormally activated (eg, overexpression of SMO-WT or SMO-D473H or SMO-W535L) Cell contact
  • iii Determine the activity level of GLI2, for example, determine the endogenous target gene expression of GLI2 (for example, Gli1, Ptch1, Wnt2, Axin2, EGFR and cyclin D1 mRNA levels) and/or the endogenous GLI1 protein activity level And/or the level of other molecules related to the activation of GLI2; and/or detecting the activity level of the GLI-luciferase reporter gene (eg, measured by testing the activity of the luciferase reporter gene);
  • the compound that inhibits GLI2 obtained by the screening method disclosed herein is the prostaglandin represented by formula I and its analogs.
  • the compound can inhibit cancer cells (for example, cancer stem cells), thereby being used to treat, prevent or alleviate tumors in subjects (especially drug-resistant tumors that exhibit tumor resistance).
  • cancer refers to solid mammalian tumors and hematological malignancies in which GLI2 is activated/overexpressed.
  • GLI2 activated/overexpressed solid mammalian tumors include head and neck, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, colorectum, rectum, anus, kidney, urethra, Bladder, prostate, urethra, penis, testis, gynecological organs, ovary, breast, endocrine system, skin, and cancers with GLI2 activation/overexpression in the central nervous system including the brain; GLI2 activation/overexpression sarcoma of soft tissue and bone; and Melanoma with activated/overexpressed GLI2 from skin and eye.
  • GLI2 activated/overexpressed hematological malignancies includes GLI2 activated/overexpressed childhood leukemias and lymphomas, Hodgkin's disease, lymphomas of lymphocyte and skin origin, acute and chronic leukemias, plasma cell tumors.
  • GLI2 activated/overexpressed cancers at any stage of development can be treated, such as early stage, metastatic, and recurrent cancers.
  • GLI2 activated/overexpressed cancers at any stage of development can be treated, such as early stage, metastatic, and recurrent cancers.
  • cancers that can be treated by the prostaglandins represented by formula I and their analogs include, but are not limited to, GLI2 activated/overexpressed glioma, medulloblastoma (e.g., cerebellar medulloblastoma), pericytes Tumor, primitive neuroectodermal tumor (PNETS), basal cell carcinoma (BCC), small cell lung cancer, large cell lung cancer, gastrointestinal tumors, rhabdomyosarcoma, breast cancer, soft tissue sarcoma, pancreatic tumor, bladder tumor and prostate tumor.
  • GLI2 activated/overexpressed glioma e.g., cerebellar medulloblastoma
  • pericytes Tumor e.g., pericytes Tumor
  • PNETS primitive neuroectodermal tumor
  • BCC basal cell carcinoma
  • small cell lung cancer large cell lung cancer
  • gastrointestinal tumors rhabdomyosarcoma
  • breast cancer soft tissue sarcoma
  • “Sensitive tumor” as used herein means a tumor (eg, medulloblastoma) that responds to treatment with SMO inhibitor anticancer regimens.
  • resistant tumor or "refractory tumor” means a previously sensitive tumor that reappears after shrinking due to treatment or temporarily eliminated due to treatment in the continuous presence of SMO inhibitor ( For example, medulloblastoma).
  • SMO inhibitor For example, medulloblastoma
  • Drug-resistant tumors showed reduced sensitivity or did not respond to smoothened inhibition.
  • Successful treatment of drug-resistant tumors can cause, for example, increased sensitivity of tumor cells to new or previously tried anti-cancer regimens and/or chemotherapeutics, and can lead to, for example, subsequent tumor cell death and prevention of metastasis.
  • the prostaglandins represented by Formula I and their analogs are used to treat, prevent, or alleviate neurodegenerative diseases or developmental syndromes and malformations (e.g., Acephalic forebrain malformation, Greg head-polydactyl-complex digit syndrome, Pallister-Hall syndrome, Rubenstein-Teybi syndrome, basal cell nevus syndrome, posterior polydactyly).
  • neurodegenerative diseases or developmental syndromes and malformations e.g., Acephalic forebrain malformation, Greg head-polydactyl-complex digit syndrome, Pallister-Hall syndrome, Rubenstein-Teybi syndrome, basal cell nevus syndrome, posterior polydactyly.
  • the present invention also provides a combination of the prostaglandin represented by formula I and its analogues with one or more additional therapeutic agents or therapies for the treatment, prevention or alleviation of the signal transduction pathways involved in GLI2 in a subject.
  • the additional one or more therapeutic agents or therapies are selected from: chemotherapy, targeted anti-cancer therapy, oncolytic drugs, cytotoxic agents, immune-based therapy, cytokines, surgical treatment, and irradiation.
  • the implementation of the present invention will utilize cell biology, cell culture, molecular biology (including recombinant technology), microbiology, biochemistry, zoology, virology, and immunology known and available to those skilled in the art.
  • Conventional technology This type of technology is described in the following documents: Molecular Cloning: A Laboratory Manual, 3rd Edition (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, 2004); Animal Cell Culture (RIFreshney, eds, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FMAusubel et al.
  • Cell line NIH/3T3 cells were cultured in DMEM supplemented with 10% (v/v) calf serum. Cos7 cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, and DAOY cells were cultured in MEM supplemented with 10% (v/v) fetal bovine serum. Keep all cell lines in a humid environment of 37°C, 95% air and 5% CO 2 .
  • Chemical libraries used in high-content screening including Prestwick Chemical Library (Prestwick Chemical), Spectrum Collection (Microsource Discovery Systems), pharmacologically active compound library (LOPAC, Sigma), FDA approved drug library (Topscience), and customized in-house Compound library.
  • Imaging analysis The cells expressing the first labeled primary ciliary marker and the second labeled GLI2 were placed at 1 ⁇ 10 4 cells/well in 50 ⁇ l of a 384-well imaging plate pre-coated with 1% gelatin (Sigma) Medium. After the cells reach confluence (1-2 days), change the medium to DMEM medium with 0.5% calf serum, add the test compound to each well in the presence or absence of SHH-N, 24 hours later Cells were fixed with 4% paraformaldehyde, stained with Hoechst (Thermo Fisher) and imaged. Cells were imaged with Operetta High Content Screening System (Perkin Elmer) using a 40x high numerical aperture objective. Harmony 4.1 software (PerkinElmer) is used for high-content screening data management and image quantification. Make the same microscope settings and input parameters throughout the imaging measurement.
  • RT-PCR Reverse transcription PCR
  • TRIzol reagent 15596018, Thermo Fisher Scientific
  • DNase I A1907, Thermo Fisher Scientific
  • GoScript cDNA Synthesis Kit GoScript cDNA Synthesis Kit (Promega) according to the manufacturer’s instructions cDNA.
  • High-fidelity KOD-Plus-New KOD-401, TOYOBO was used for PCR amplification to determine gene expression.
  • Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal reference.
  • the primer sequences used to quantify the target gene expression of GLI2 are listed in FIG. 12.
  • Cells are lysed in RIPA buffer (25mM Tris ⁇ HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), in which every 10ml RIPA is supplemented with 1 protease and phosphatase inhibitor (A32961, Thermo Fisher Scientific). Separate equal amounts of cell lysates by SDS-PAGE, and then transfer to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked in 5% skim milk at room temperature for 2 hours, and then the membrane was incubated with the primary antibody prepared in the blocking solution at 4°C overnight.
  • RIPA buffer 25mM Tris ⁇ HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS
  • A32961 protease and phosphatase inhibitor
  • PVDF polyvinylidene fluoride
  • HRP horseradish peroxidase
  • mice were fixed in 4% paraformaldehyde for 30 minutes, permeabilized in 0.3% triton-X 100 for 15 minutes, blocked in a blocking solution containing 2% BSA and 0.3% Triton-X for 1 hour, and then used for mice
  • the monoclonal anti-EP4 antibody (sc-55596, Santa Cruz, 1:200 dilution) was incubated overnight at 4°C. After incubating overnight, the cells were washed 3 times for 10 minutes each, and then incubated with secondary antibody Alexa Fluor 488 goat anti-mouse IgG (H+L) (A-11001, Thermo Fisher Scientific, 1:500 dilution) at room temperature1 Hours, then wash 3 times, 10 minutes each time.
  • the cells were stained with Hoechst, washed with PBS, covered with a cover glass and the sample was imaged.
  • a Zeiss LSM 780 confocal microscope was used to collect images with a 63x oil objective lens, and the images were processed with ZEN software (Zeiss).
  • CCK-8 reagent B34302, Biomake
  • the collected fresh tumor samples were fixed with formalin and then embedded in paraffin. Before staining, the sections were dewaxed with xylene and a series of reduced concentrations of ethanol to water, and then rinsed with deionized water. For H&E staining, the sections were incubated with hematoxylin and eosin dyes separately according to standard procedures.
  • NPG NOD-Prkdc scid Il2rg null mice
  • the tumor volume was measured with a vernier caliper every 3 days, and the tumor volume calculation formula was as follows: length ⁇ width ⁇ width ⁇ 0.5.
  • each tumor sample was harvested and divided into several parts for qRT-PCR, H&E staining and immunohistochemical analysis.
  • This example developed a method for high content screening (HCS) for compounds capable of inhibiting the transport of GLI2 to cilia and/or accumulation in cilia and constructed the vectors and cell lines used in the method.
  • HCS high content screening
  • the high content screening method of compounds that inhibit GLI2 includes the following steps:
  • NIH/3T3 cells i. NIH/3T3 cells (CRL-1658, ATCC) were cultured in DMEM supplemented with 10% (v/v) bovine calf serum (BCS), and passaged according to conventional methods.
  • the ARL13B construct with the red fluorescent tagRFPT marker was introduced into HH-reactive NIH/3T3 cells to obtain the parental 3T3/ARL13B::tagRFPT cell line (the plasmid backbone is pRRL lentiviral plasmid Addgene:#31484, ARL13B::tagRFPT plasmid
  • the map is shown in Figure 13, and the sequence of the inserted Arl13b-tagRFPT is shown in SEQ ID NO: 2)
  • iii Introduce a lentiviral plasmid expressing EGFP::GLI2 into the 3T3/ARL13B::tagRFPT cell line obtained in step ii, wherein the mouse Gli2 gene sequence is used to construct the EGFP::GLI2 fusion (the plasmid backbone is pRRL slow
  • the virus plasmid Addgene:#31484, EGFP::GLI2 plasmid map is shown in Figure 14, and the inserted EGFP-Gli2-3 ⁇ Flag-3 ⁇ Myc sequence is shown in SEQ ID NO: 3).
  • the expression will be regulated by the HH signal transduction pathway.
  • the monoclonal cells with low EGFP::GLI2 expression were cultured, and the obtained cell line was named 3T3/ARL13B::tagRFPT/EGFP::GLI2 cell line.
  • SHH-N (SHH N-terminal signal peptide, its sequence is shown in SEQ ID NO:1, and the plasmid shown in Figure 15 is used to construct Cos7 stably expressing SHH-N After expanding the cell to overgrown, collect the supernatant expressing SHH-N.
  • SHH-N is: 1:16 dilution with DMEM) or SAG (a small molecule SMO agonist, Millipore: #566660; use concentration: 100 nM) respectively induced the accumulation of GLI2 protein in primary cilia; Vimodedi or cyclopamine attenuated SHH-N-induced GLI2 protein cilia accumulation ( Figure 1A and B).
  • the 3T3/ARL13B::tagRFPT/EGFP::GLI2 cell line and its parent 3T3/ARL13B were tested using HH pathway agonists (SHH-N or SAG) and/or antagonists (cyclopamine or vimmodec):
  • the :tagRFPT cell line responds to the GLI-luciferase activity of the HH pathway agonist and/or antagonist.
  • Figure 1C it can be seen from the measurement of the relative GLI-luciferase activity that the GLI-dependent transcriptional activity is responsive to the treatment of the HH pathway agonist and/or antagonist. This is in contrast to the transfer of endogenous GLI2 to cilia reported in the prior art (Haycraft et al. (2005).
  • Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.
  • 3T3/ARL13B::tagRFPT/EGFP::GLI2 cell line can be used for high-content screening of compounds that affect the localization of GLI2 cilia.
  • the cells were imaged with the Operetta High Content Screening System (Perkin Elmer) using a 40x high numerical aperture objective lens. Harmony 4.1 software (PerkinElmer) is used for high-content screening data management and image quantification. Make the same microscope settings and input parameters throughout the imaging measurement.
  • the test compound is a compound that inhibits GLI2.
  • the chemical libraries used include Prestwick Chemical Library (Prestwick Chemical), Spectrum Collection (Microsource Discovery Systems), and pharmacologically active compound library (LOPAC, Sigma), FDA approved drug library (Topscience), and customized enterprise compound library.
  • Cyclopamine was purchased from Sigma, and SAG was purchased from Millipore. Vimodji and Forskolin were purchased from Selleck. Purchase prostaglandins and their analogs from Cayman.
  • the SHH-N conditioned medium was collected as previously described (Wang et al. (2012). Smoothened transforms Hedgehog signal by forming a complex with Evc/Evc2. Cell research 22, 1593-1604).
  • the control conditioned medium was collected from wild-type Cos7 cells (CRL-1651, ATCC).
  • prostaglandins with the structure shown in formula I and their analogs, including prostaglandin A1 (PGA1), prostaglandin D1 alcohol (PGD1 alcohol), and 5-trans prostaglandin Su D2 (5- trans PGD2), prostaglandin E1 (PGE1), prostaglandin J2 (PGJ2), 15- deoxy - ⁇ 12,14 - prostaglandin J2 (15- deoxy-- ⁇ 12,14 -PGJ2), the The structure of the compound is shown in Figure 2A, and the results of inhibition of GLI2 cilia accumulation are shown in Figure 2B and Figure 2C.
  • an HH signal activity reporter cell line with high expression of GLI2 was constructed, and the inhibitory effect of prostaglandins and their analogs on GLI2 expression was tested.
  • the 3T3/GLI-luc reporter cell line was generated. Specifically, the wild-type NIH/3T3 cells were first introduced into a firefly luciferase plasmid driven by 8 ⁇ GLIBS with a lentivirus (the plasmid backbone is pRRL lentiviral plasmid Addgene: #31484, and the plasmid map is shown in attached Figure 16. The sequence of firefly luciferase is shown in SEQ ID NO: 4).
  • lentivirus is used to re-introduce the Renilla luciferase plasmid driven by the CMV promoter (plasmid backbone is pRRL).
  • 3T3/GLI-luc/GLI2 reporter cell line a cell line with high expression of GLI2 was generated, named 3T3/GLI-luc/GLI2 reporter cell line.
  • the lentiviral plasmid containing the mouse GLI2 expression cassette driven by the CMV promoter ( Figure 3D) was delivered to the 3T3/GLI-luc reporter cell line (plasmid backbone is pRRL lentiviral plasmid Addgene: #31484, the plasmid map is shown in the attachment Figure 18, the inserted mouse GLI2 sequence is shown in SEQ ID NO: 6), and the 3T3/GLI-luc/GLI2 reporter cell line was obtained, which overexpressed mouse GLI2. Therefore, the foreign source can be easily detected by Western blotting Expression of GLI2 ( Figure 3F).
  • SMO point mutations can lead to drug resistance to Vimodji.
  • This example detects the HH pathway activity mediated by two drug-refractory SMO mutants SMO-D473H and SMO-W535L (also known as SMO-A1 or SMO-M2) (Xie et al. (1998). Activating Smoothened mutations) in sporadic basal-cell carcinoma. Nature 391, 90-92; Yauch et al. (2009). Smoothened Mutation Confers Resistance to a Hedgehog Pathway Inhibitor in Medulloblastoma Science 326, 572-574), the SMO mutant is treated in Vimodji (Atwood et al. (2015).
  • Plasmid backbone is pBABE retroviral plasmid Addgene: #1764, the plasmid map is shown in Figure 19, Figure 20 and Figure 21, respectively, inserted SMO WT-EGFP sequence, SMO D473H-EGFP sequence, SMO W535L-EGFP see SEQ ID NO: 7, 8, 9 respectively)
  • prostaglandins and their analogs can effectively inhibit the GLI2 protein activity caused by the two SMO mutants, and have the same efficacy against wild-type SMO. All six prostaglandins identified in Example 1 inhibit these two SMO mutants.
  • Figures 6A-E and Figure 7B illustrate the relative GLI luciferase activity of several specific prostaglandins.
  • prostaglandins and their analogs show pan-inhibition of multiple drug resistance causes (including GLI2 overexpression and SMO mutations) caused by SMO-targeted cancer treatments, and can act at the downstream level of SMO .
  • This example explores the potential application of prostaglandins and their analogs in the treatment of refractory tumors, where the refractory tumors are related to GLI2.
  • DAOY cell line (#HTB-186, ATCC) was used in this example.
  • the DAOY cell line is a human SHH subtype MB cell line with known PTCH1 mutations (Singh et al. (2015).
  • the inventors produced two DAOY cell lines that stably overexpress human SMO-D473H and indirectly cause abnormal GLI2 or stably overexpress human GLI2 directly cause abnormal GLI2 through lentiviral delivery.
  • the plasmid backbone is the pRRL lentiviral plasmid Addgene: #31484.
  • the plasmid map is shown in Figure 22 and Figure 23 respectively.
  • SMO D473H-P2A-firefly luciferase sequence and human Gli2-P2A-firefly luciferase sequence See SEQ ID NO: 10 and SEQ ID NO: 11), which are hereinafter referred to as DAOY/SMO-D473H cells and DAOY/GLI2 cells.
  • PGE1 was used to treat wild-type DAOY cells, DAOY/SMO-D473H cells and DAOY/GLI2 cells, respectively, to detect cell viability and the expression of GLI2 target genes (GLI1 and PTCH1). It can be seen from Figure 9A-C that the cell viability of these three types of cells was significantly reduced after PGE1 treatment, while DAOY/SMO-D473H cells and DAOY/GLI2 cells showed the expected resistance to Vimodedi and cyclopamine; It can be seen from Fig.
  • PGE1 As a control of wild-type DAOY cells, PGE1 was also used to treat cells HCC38 (#CRL-2314,ATCC), MCF7 (#HTB-22,ATCC), DLD-1 (#CCL-221,ATCC) that do not express Gli2 gene. , HepG2 (#HB-8065, ATCC), as can be seen from Figure 10A and Figure 10B, the cell viability of these four cells did not change significantly after PGE1 treatment, indicating that for the cells that do not express Gli2 gene, PGE1 treatment Will not cause a significant decrease in cell viability.
  • DAOY cells overexpressing GLI2 were subjected to tumor xenotransplantation in mice, and treated with PGE1 or Vimodji, respectively.
  • the results showed that daily administration of 15 mg/kg PGE1 significantly inhibited tumor growth ( Figure 11A and B).
  • the daily saturation treatment with 30 mg/kg Vimodjib did not inhibit tumor growth ( Figure 11A and B).
  • the in vivo administration of PGE1 significantly reduced the expression of GLI2 target genes (including GLI1 and PTCH1) in these tumors ( Figure 11C).
  • hematoxylin and eosin (H&E) staining on tumor tissues are shown in Figure 11D.
  • Tumor tissues treated with PGE1 showed signs of necrosis, such as basophilic tumor cells losing regular cell morphology and nuclear pyknosis (black arrows), while tumor tissues treated with vehicle control or Vimodedi showed good tumor cell morphology ( Red Arrow).
  • PGE1 treatment significantly reduced Ki67-positive proliferating cells compared with vehicle control and Vimodedi ( Figure 11E and F).

Abstract

用作药物的式I的前列腺素和其类似物以及包含这些化合物的药物组合物，特别是用作含锌指结构域的蛋白GLI2(即神经胶质瘤相关的癌基因-2)的抑制剂，以及用于治疗、预防或减轻受试者中的肿瘤(尤其是肿瘤抗性)或GLI2参与的信号传导通路失调导致的神经变性疾病或发育综合征和畸形。还提供了抑制GLI2的化合物的筛选方法。

Description

GLI2抑制剂的用途以及抑制GLI2的化合物的筛选方法 技术领域
本发明涉及用作药物的式I的前列腺素和其类似物以及包含这些化合物的药物组合物，特别是用作含有锌指结构域的蛋白质GLI2(即，神经胶质瘤相关的癌基因-2)的抑制剂，用于治疗、预防或减轻受试者中的GLI2参与的信号传导通路失调导致的疾病，例如肿瘤(尤其是肿瘤抗性)、神经变性疾病或发育综合征、畸形。本发明还涉及抑制GLI2的化合物的筛选方法，特别是高内涵筛选方法(high-content screen；HCS)，所述方法直接基于化合物与GLI2的相互作用，通过可视化GLI2并追踪其在表达GLI2的细胞中的细胞定位来筛选抑制GLI2的候选药物。
背景技术
GLI家族是一组具有锌指结构域的转录因子蛋白，它们通过锌指基序结合DNA，因首次在恶性胶质瘤(Glioblastoma)中发现其异常高表达而得名(Kinzler K.W.等人，Identification of an amplified,highly expressed gene in a human glioma，Science,1987,236(4797):70-73)。已知GLI家族包含称为GLI1、GLI2、GLI3的成员，它们是细胞内的几种信号传导途径共同整合交织的平台，所述信号传导途径例如Hedgehog(HH)信号传导途径、RAS信号传导途径、转化生长因子β信号传导途径(Lauth M.等人，Non-canonical activation of GLI transcription factors:implication for targeted anti-cancer therapy,Cell Cycle,2007,6(20):2458-2463)、wnt信号传导途径、notch信号传导途径(Beiqin Yu等人，The role of Gli2-ABCG2 signaling axis for 5Fu resistance in gastric cancer,Journal of Genetics and Genomics,2017，44(8):375-383)等，其中GLI2在这三种GLI家族成员中处于核心地位(Kim Y.等人，Selective down-regulation of glioma-associated oncogene 2 inhibits the proliferation of hepatocellular carcinoma cells,Cancer Res.,2007,67(8):3583-3593)。
GLI2基因定位于2q14.2，共有4758个碱基(https://www.ncbi.nlm.nih.gov/gene/2736)，由该基因编码的GLI2蛋白的活性受到多个不同机制的调节，区域化(compartmentalization)是调节GLI2蛋白的机制之一。通常，GLI2蛋白主要保留在胞质溶胶中并降解。当HH信号传导途径活化时，GLI2蛋白转运入初级纤毛(primary cilium；PC)，在那里它与SuFu分离，然后GLI2蛋白再转运至细胞核。在细胞核中GLI2与靶基因的启动子元件中的特定DNA序列结合，并激活这些靶基因(例如，Gli1、Ptch1、wnt、FGF、Cyclins)的转录和表达，进而调控细胞的生长、增殖与分化。
在HH信号传导途径、RAS信号传导途径、转化生长因子β信号传导途径、wnt信号传导途径、notch信号传导途径等参与的细胞过程的异常调节所导致的病理学表型中，发现均存在GLI2蛋白的活化和/或高表达。因此，本领域迫切需要对位于这些信号传导途径的汇合点处的GLI2蛋白具有选择性抑制作用的化合物和高效筛选抑制GLI2的化合物的方法。
发明概述
本发明人通过锐意研究，开发了一种选择性抑制GLI2的化合物的高内涵筛选方法，所述方法直接分析化合物库中的多种化合物对GLI2的初级纤毛转运和/或积聚的影响，其中所述GLI2的初级纤毛转运和/或积聚是GLI2活化的关键事件，并使用所述方法发现了式I的前列腺素和其类似物能够选择性抑制GLI2蛋白转运入初级纤毛、和/或抑制GLI2蛋白在初级纤毛中的积聚、和/或抑制GLI2蛋白对细胞核中靶基因的激活。
Figure PCTCN2020078224-appb-000001
其中
R 1选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、或者R 1与其连接的环碳原子一起形成C(＝O)；
R 2选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、或者R 2与其连接的环碳原子一起形成C(＝O)；
R 3选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、＝O、或者R 3与连接R 1的环碳原子一起相互连接形成5元环基团；
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是0－12的整数，例如，n各自独立地是0、1、2、3、4、5、6、7、8、9、10、11、12的整数；
R 4各自独立地选自H、-CH 3、-COCH 3、-OH、所述-OH的盐、-COOH或所述-COOH的盐或碳原子数1－6的烷基酯、-CONH 2、-CONH(C 1-5烷基)OH；
R 5选自H、OH、任选取代的C 1-6烷基、卤素、O(任选取代的C 1-6烷基)；
R 6独立地选自H、OH、任选取代的C 1-6烷基、卤素、O(任选取代的C 1-6烷基)；
m各自独立地是0－10的整数，例如，m各自独立地是0、1、2、3、4、5、6、7、8、9、10的整数；
每个
Figure PCTCN2020078224-appb-000002
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000003
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；
优选地，
其中R 1与其连接的环碳原子一起形成C(＝O)；
R 2选自H、OH或者O(任选取代的C 1-6烷基)；
R 3选自H或者＝O；
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是2、3、4、5或6的整数；
R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
R 5选自H或OH；
R 6独立地选自H、C 1-3烷基、卤素；
m各自独立地是0、1、2、3、4的整数；
每个
Figure PCTCN2020078224-appb-000004
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000005
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；或者
优选地，
其中R 1选自H或者OH；
R 2与其连接的环碳原子一起形成C(＝O)；
R 3是H；
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是2、3、4、5或6的整数；
R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
R 5选自H或OH；
R 6独立地选自H、C 1-3烷基、卤素；
m各自独立地是0、1、2、3、4的整数；
每个
Figure PCTCN2020078224-appb-000006
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000007
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；或者
优选地，
其中R 1选自H、任选取代的C 1-6烷基、卤素；
R 2选自OH、O(任选取代的C 1-6烷基)
R 3与连接R 1的环碳原子一起相互连接，形成下式II或式III的环基团：
Figure PCTCN2020078224-appb-000008
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是2、3、4、5或6的整数；
R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
R 5选自H或OH；
R 6独立地选自H、C 1-3烷基、卤素；
m各自独立地是1、2、3、4的整数；
每个
Figure PCTCN2020078224-appb-000009
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000010
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物。
在一个实施方案中，式I的前列腺素和其类似物能够选择性抑制GLI2参与的信号传导途径的异常活化，包括但不限于经典的HH信号传导途径(Hh配体依赖性的HH信号传导途径)和非经典的HH信号传导途径(Hh配体非依赖性的HH信号传导途径)、RAS信号传导途径、转化生长因子β信号传导途径、wnt信号传导途径、notch信号传导途径的异常活化，从而通过抑制癌细胞(例如，癌症干细胞)的增殖来治疗、预防或减轻受试者中的肿瘤(尤其是肿瘤抗性)；或者用来治疗、预防或减轻受试者中的GLI2参与的信号传导通路失调导致的神经变性疾病或发育综合征和畸形(例如，前脑无裂畸形、格雷格头-多指-并指综合征、Pallister-Hall综合征、Rubenstein-Teybi综合征、基底细胞痣综合征、轴后性多指症)。
又在一个实施方案中，将式I的前列腺素和其类似物与额外的一种或多种治疗剂或疗法组合来治疗、预防或减轻受试者中的疾病，所述额外的一种或多种治疗剂或疗法选自：化疗、靶向抗癌疗法、溶瘤药物、细胞毒性剂、基于免疫的疗法、细胞因子、外科处置、照射术。
在进一步的实施方案中，式I的前列腺素和其类似物是前列腺素A1(PGA1)、8-异PGA1、PGA2、前列腺素D1醇(PGD1醇)、前列腺素D1、前列腺素D2、5-反式前列腺素D2(5-反式PGD2)、前列腺素E1(PGE1)、PGE1醇、8-异PGE1、PGE1乙基酯、6-酮PGE1、PGE1乙醇胺、PGE2、15(R)-PGE2、8-异PGE2、PGE2乙醇胺、PGI2(钠盐)、前列腺素J2(PGJ2)、15-脱氧-Δ 12,14-前列腺素J2(15-脱氧-Δ 12,14-PGJ2)、伊洛前列素(Iloprost)、Carbaprostacyclin、西前列烯(Ciprostene)(例如，西前列烯钙盐)和瑞文前列腺素(Rivenprost)。
在第二方面，本发明提供了一种抑制GLI2的化合物的高内涵筛选方法，所述方法允许可视化化合物是否抑制GLI2蛋白转运入初级纤毛和/或在初级纤毛中积聚，所述方法包括：
i.分别构建表达第一标记的初级纤毛标志物的构建体和表达第二标记的GLI2的构建体；或者构建表达第一标记的初级纤毛标志物和表达第二标记的GLI2的双表达构建体，其中第一标记不同于第二标记；
ii.将步骤i的表达第一标记的初级纤毛标志物的构建体和表达第二标记的GLI2的构建体同时或以不同顺序依次地引入哺乳动物细胞(如NIH/3T3细胞、DAOY细胞)中，或者将步骤i的表达第一标记的初级纤毛标志物和表达第二标记的GLI2的双表达构建体引入哺乳动物细胞(如NIH/3T3细胞、DAOY细胞)中，获得同时表达第一标记的初级纤毛标志物和第二标记的GLI2的细胞系；
iii.将受试化合物与步骤ii的GLI2蛋白活化或者过量表达的细胞系接触；
iv.通过基于图像的高内涵分析，与未接触受试化合物的步骤ii的GLI2蛋白活化或者过量表达的细胞系相比较，确定减少GLI2蛋白积聚于初级纤毛的受试化合物作为抑制GLI2的化合物。
在一个实施方案中，抑制GLI2的化合物的高内涵筛选方法包括如下步骤：
i.将表达第一标记的初级纤毛标志物的构建体引入哺乳动物细胞(例如，NIH/3T3细胞)中，获得表达第一标记的初级纤毛标志物的第一细胞系；
ii.向步骤i获得的细胞系中引入表达第二标记的GLI2的构建体；
iii.筛选具有低第二标记的GLI2表达的单克隆细胞并培养，获得同时表达第一标记的初级纤毛标志物和低表达第二标记的GLI2的第二细胞系，其中第一标记不同于第二标记；
iv.将诱导GLI2蛋白积聚于初级纤毛的诱导剂(例如，SHH-N或SAG)和受试化合物与步骤iii的第二细胞系接触，并与仅将诱导GLI2蛋白积聚于初级纤毛的诱导剂与步骤iii的第二细胞系接触的情形比较；
v.选择减弱GLI2蛋白积聚于初级纤毛的受试化合物作为抑制GLI2的化合物。
在一些实施方案中，抑制GLI2的化合物的高内涵筛选方法中的第一标记和第二标记分别选自但不限于绿色荧光蛋白(GFP)、黄色荧光蛋白、蓝色荧光蛋白、青色荧光蛋白、橙色荧光蛋白，优选地，增强型绿色荧光蛋白(EGFP)、增强型黄色荧光蛋白、增强型蓝色荧光蛋白、增强型青色荧光蛋白、增强型橙色荧光蛋白，例如，TagRFP、tdTomato、DsRed、HcRed、AsRed、AmCyan、ZsGreen、AcGFP和ZsYellow；初级纤毛标志物选自但不限于ARL13B、INVERSIN、微管蛋白。
在第三方面，本发明还提供了抑制过量表达GLI2的化合物的筛选方法，所述方法包括：
i.将过量表达GLI2的构建体引入哺乳动物细胞(例如，NIH/3T3或DAOY细胞)中，获得过量表达GLI2的细胞；
ii.使受试化合物(优选地，上述第二方面鉴定的受试化合物)与i的过量表达GLI2的细胞接触；
iii.测定所述GLI2的活性水平，例如，测定GLI2的内源性靶基因表达(例如，Gli1、Ptch1、Wnt2、Axin2、EGFR和细胞周期蛋白D1 mRNA水平)和/或内源性GLI1蛋白的活性水平和/或与GLI2活化有关的其他分子的水平；和/或检测GLI-萤光素酶报告基因活性水平(如，通过测试萤光素酶报告基因活性来测量)；
iv.将所述活性水平与没有接触受试化合物的细胞中GLI2的活性水平进行比较，并选择使GLI2的活性水平降低的受试化合物。
在第四方面，本发明提供了抑制耐受Smoothened(SMO)拮抗剂的Hedgehog信号传导途径活化相关的GLI2的化合物的筛选方法，所述方法包括：
i.培养SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞；
ii.使受试化合物(优选地，上述第二方面鉴定的受试化合物)与i的SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞接触；
iii.测定GLI2的活性水平，例如，测定GLI2的内源性靶基因表达(例如，Gli1、Ptch1、Wnt2、Axin2、EGFR和细胞周期蛋白D1 mRNA水平)和/或内源性GLI1蛋白的活性水平和/或与GLI2活化有关的其他分子的水平；和/或检测GLI-萤光素酶报告基因活性水平(如，通过测试萤光素酶报告基因活性来测量)；
iv.与没有接触受试化合物的i的SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞的GLI2的活性水平进行比较，并选择使GLI2活性水平降低的受试化合物。
附图简述：
图1：显示了用于高内涵筛选(HCS)和萤光素酶测定法的细胞系的产生和表征。图1A－图1B：显示了在用SHH-N、100nM SAG、DMSO、维莫德吉(1μM)、环巴胺(1μM)、环巴胺+SHH-N、或者维莫德吉+SHH-N处理的3T3/ARL13B::tagRFPT/EGFP::GLI2细胞中GLI2纤毛定位的代表性图像(图1A)和定量结果(图1B)；比例尺,10μm(将收集的SHH-N稀释16倍使用，达到如图3B中所示相对于未加入SHH-N的对照细胞而言100倍左右的激活)；图1C：显示了与3T3/ARL13B::tagRFPT细胞相比较，3T3/ARL13B::tagRFPT/EGFP::GLI2细胞对HH途径激动剂和拮抗剂的响应性的GLI-萤光素酶测量，其中分别以100nM、1μM、1μM使用SAG、环巴胺和维莫德吉，数据表示为四个重复的平均值和标准偏差。
图2：例示了六种抑制GLI2在初级纤毛中积聚的前列腺素和其类似物。图2A：给出了这六种例示的前列腺素和其类似物的化学结构；图2B和图2C：在3T3/ARL13B::tagRFPT/EGFP::GLI2细胞中，这六种例示的前列腺素抑制SHH-N诱导的GLI2在纤毛积聚的代表性图像(图2B)和定量结果(图2C)，其中使用1μM维莫德吉用作阳性对照，前列腺素以10μM使用，数据表示为四个重复的平均值和标准偏差，比例尺：20μm。
图3A－图3C：显示了稳定表达的3T3/GLI-luc报告细胞系的萤光素酶测定法结果；图3D－图3F：显示了过量表达GLI2的GLI-萤光素酶报告系统的构建(图3D)、萤光素酶测定法 鉴定结果(图3E)、和通过蛋白质印迹分析的鉴定结果(图3F)，图中的GLIBS：GLI结合位点；fLuc：萤火虫萤光素酶；minP：最小启动子；P(A),poly(A)；LTR：长末端重复序列。
图4A：在3T3/GLI-luc/GLI2细胞中，例示的HCS鉴定的6种前列腺素和维莫德吉对GLI2过量表达介导的HH途径活性的剂量依赖性效应，测量以一式四份进行，数据显示为平均值和标准偏差；图4B：qRT-PCR分析PGE1(30μM)、维莫德吉(10μM)、环巴胺(10μM)对3T3/GLI-luc/GLI2细胞中内源Gli1和Ptch1表达的影响，数据显示为来自三个生物学重复的平均值和标准偏差，**p<0.01；NS，不显著，student’t检验。
图5：例示了使用3T3/GLI-luc/GLI2细胞在GLI-萤光素酶报告基因测定法中检测其它的前列腺素和其类似物的结果。数据代表一式四份样品的平均值和标准偏差。图中给出了每种前列腺素和其类似物的IC50和化学结构。
图6：例示了HCS鉴定的前列腺素分别对过量表达野生型SMO(也表示为SMO-WT)、SMO-D473H和SMO-W535L介导的GLI-萤光素酶报告基因活性的剂量依赖性效应。所有测定法均一式四份进行，数据代表平均值和标准偏差。
图7：显示了PGE1对抗通过分别引入SMO-D473H、SMO-W535L和SMO-WT而产生的HH途径活性的作用，使用维莫德吉作为比较用对照。图7A和图7B：分别表示维莫德吉和PGE1对过量表达野生型SMO(红色方块)、SMO-D473H(蓝色三角形)或SMO-W535L(绿色圆圈)的3T3/GLI-luc细胞中的GLI-萤光素酶报告基因活性的影响；图7C－图7E：分别显示qRT-PCR分析维莫德吉(10μM)和PGE1(30μM)对过量表达野生型SMO(图7C)、SMO-D473H(图7D)或SMO-W535L(图7E)的3T3/GLI-luc细胞中内源Gli1和Ptch1表达的影响；图7F：显示了通过蛋白质印迹分析维莫德吉(10μM)和PGE1(30μM)对过量表达野生型SMO、SMO-D473H或SMO-W535L的3T3/GLI-luc细胞中内源GLI1蛋白表达的影响；图7G－图7H：显示了维莫德吉(图7G)和PGE1(图7H)对用10nM(蓝色圆圈)、50nM(红色方块)或250nM(绿色三角形)SAG刺激的3T3/GLI-luc细胞中GLI-萤光素酶报告基因活性的抑制，数据表示为四个重复的平均值和标准偏差，**p<0.01；***p<0.001；NS表示“不显著”；student’t检验。
图8：显示了前列腺素对抗SHH-N或SAG诱导的HH途径活性。图8A：显示了在3T3/GLI-luc细胞中，图2A例示的6种前列腺素对SHH-N诱导的GLI-萤光素酶报告基因活性的剂量依赖性作用，数据表示为四个重复的平均值和标准偏差；图8B：显示了在用SHH-N或SAG处理的3T3/GLI-luc细胞中，PGE1(30μM)作用于GLI2的靶基因(Gli1,Ptch1)的qRT-PCR结果；图8C：显示了仅用HH途径激动剂(SHH-N或SAG)或用它们与PGE1(30μM)、维莫德吉(1μM)或环巴胺(1μM)的组合处理3T3/GLI-luc细胞后，细胞裂解物中内源GLI1蛋白水平的蛋白质印迹分析结果，使用β-肌动蛋白作为上样对照；图8D：显示了在3T3/GLI-luc细胞中，瑞文前列腺素(10μM)对SHH-N诱导的GLI-萤光素酶报告基因活性的影响；图8E：显示了在用SHH-N处理的3T3/GLI-luc细胞中，瑞文前列腺素(10μM)作用于GLI2的靶基因(Gli1,Ptch1)的qRT-PCR结果。
图9：显示了PGE1对人DAOY髓母细胞瘤细胞和它的2种抗SMO抑制剂的衍生细胞系的细胞活力和HH途径活性的影响。图9A－图9C：显示了在用PGE1(100μM)、维莫德吉(10μM)和环巴胺(10μM)处理的野生型DAOY细胞(图9A)、过量表达SMO-D473H(图9B)或过量表达GLI2(图9C)的细胞中的细胞活力测定结果，数据表示为四个重复的平均值和标准偏差；图9D－图9F：显示了qRT-PCR分析用PGE1(100μM)、维莫德吉(10μM)和环巴胺(10μM)处理的野生型DAOY细胞(图9D)、过量表达SMO-D473H(图9E)或过量表达GLI2(图9F)的DAOY细胞中的GLI1和PTCH1的mRNA水平，数据显示为来自三个独立实验的平均值和标准偏差。*p<0.05,**p<0.01；***p<0.001；NS表示“不显著”；student’t检验。
图10A：显示了在所示的各肿瘤细胞中Gli2基因的RT-PCR检测结果；图10B：显示了PGE1(67μM)对不表达Gli2基因的细胞HCC38、MCF7、DLD-1、HepG2细胞活力的影响，其中纵坐标1.0表示不添加PGE1时的细胞活力。
图11：显示了使用过量表达GLI2的人DAOY细胞进行异种移植测定法的结果。图11A：显示了在第51天实验结束时，代表性异种移植免疫缺陷小鼠的宏观外观；图11B：显示了用溶媒对照、维莫德吉(30mg/kg)或PGE1(15mg/kg)处理时，肿瘤体积的变化与时程的关系，数据描述为平均值和标准偏差，每组n＝8；图11C：显示了通过qRT-PCR分析分别用溶媒对照、维莫德吉(30mg/kg)或PGE1(15mg/kg)处理的肿瘤中，GLI1和PTCH1 mRNA表达情况，数据代表三个样本的平均值和标准偏差；图11D－图11E：显示了分别用溶媒对照或维莫德吉(30mg/kg)或PGE1(15mg/kg)处理的肿瘤组织的H&E和Ki67染色的代表性图像，比例尺代表20μm；图11F：显示了对图11E中肿瘤的Ki67染色的定量结果。数据表示三个样品的平均值和标准偏差，*p<0.05；**p<0.01；***p<0.001；NS表示“不显著”。
图12：显示了用于RT-PCR和qRT-PCR的引物序列。
图13：pRRL慢病毒质粒中插入了ARL13B::tagRFPT的载体。
图14：pRRL慢病毒质粒中插入了EGFP::GLI2的载体。
图15：构建的包含SHH-N的稳定转染细胞系用载体。
图16：8×GLIBS驱动的萤火虫萤光素酶质粒。
图17：CMV启动子驱动的海肾萤光素酶质粒。
图18：CMV启动子驱动的小鼠GLI2表达质粒。
图19：pBABE逆转录病毒质粒中插入了SMO-WT的载体。
图20：pBABE逆转录病毒质粒中插入了SMO-D473H的载体。
图21：pBABE逆转录病毒质粒中插入了SMO-W535L的载体。
图22：pRRL慢病毒质粒中插入了SMO-D473H的载体。
图23：pRRL慢病毒质粒中插入了人GLI2的载体。
发明详述：
GLI2蛋白的活化和/或高表达存在于一些病理学表型中。由于在GLI2蛋白的活化和/或高表达中，GLI2蛋白向纤毛转运和在纤毛中积聚是必要的，这为靶向GLI2的化合物的发现提供了新机会。本发明提供了一种通过抑制GLI2蛋白向纤毛转运和在纤毛中积聚来抑制GLI2蛋白活性的化合物的高内涵筛选方法，还提供了通过所述方法获得的、能够抑制GLI2蛋白转运入初级纤毛和/或抑制GLI2蛋白在初级纤毛中积聚的化合物，它们是式I的前列腺素和其类似物。
进一步地，本发明建立了模拟抗药机制的细胞模型，以及使用所述细胞模型来筛选抑制GLI2蛋白活性的化合物。通过所述体外方法证实了式I的前列腺素和其类似物还能够在具有抗药机制的细胞中抑制GLI2蛋白活性。动物的体内实验也获得了与此一致的结果。
除非下文中另外定义，否则本说明书中的术语如本领域通常所用那样使用。
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
本文中使用的术语“烷基”是指单价饱和无环(即非环状)烃基，其可以是直链或支链的。因此，“烷基”基团不包含任何碳-碳双键或任何碳-碳三键。“C 1-6烷基”表示具有1-6个碳原子的烷基。优选的示例性烷基是甲基、乙基、丙基(例如正丙基或异丙基)或丁基(例如正丁基、异丁基、仲丁基或叔丁基)。除非另有说明，术语“烷基”优选指C 1-4烷基，更优选指甲基或乙基，甚至更优选指甲基。
术语“卤素”是指氟、氯、溴或碘，优选为溴或氯或氟。
在本说明书中，“任选取代的”基团是指这些基团可以具有或者不具有一个或多个取代基，例如，1、2、3或4个取代基。应当理解，取代基的最大数量受被取代部分上可用的连接位点数量的限制。除非另外定义，否则本说明书中提到的“任选取代的”基团优选具有不超过两个取代基，特别地，可以仅具有一个取代基。优选地，基团不存在任选的取代基，即相应的基团是未取代的。
“氧代”例如代表连同与之结合的碳原子一起形成酮基(C＝O)的残基。
“碳原子数1－6的烷基酯”是指C 1-C 6-烷基的酯。作为非限制性实例，可以提及的是甲基、乙基、正丙基或异丙基、正丁基、仲丁基或叔丁基的酯。
术语“盐”是指取决于本文所述的化合物上的取代基，用相对无毒的酸或碱制备的化合物的盐。当实施方案中的化合物含有相对酸性的官能度时，可通过纯净地或在适合的惰性溶剂中使中性形式的所述化合物与足量的所需碱相接触来获得碱加成盐。药学上可接受的碱加成盐的实例包括钠、钾、钙、铵、有机氨基或镁盐，或类似盐。当实施方案的化合物含有相对碱性的官能度时，可通过纯净地或在适合的惰性溶剂中使中性形式的所述化合物与足量的所需酸相接触来获得酸加成盐。药学上可接受的酸加成盐的实例包括衍生自无机酸如盐酸、氢 溴酸、硝酸、碳酸、一氢碳酸、磷酸、一氢磷酸、二氢磷酸、硫酸、一氢硫酸、氢碘酸或亚磷酸等的那些，以及衍生自相对无毒的有机酸如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、富马酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸、甲磺酸等的盐。还包括氨基酸如精氨酸等的盐等，以及有机酸如葡萄糖醛酸或半乳糖醛酸等的盐(参见，例如，Berge,S.M.等人,“Pharmaceutical Salts”,Journal of Pharmaceutical Science,1977,66,1-19)。
如本文所用，术语“溶剂化物”是指与溶剂复合的化合物。能与实施方案中的化合物形成溶剂化物的溶剂包括常见有机溶剂如醇(甲醇、乙醇等)、醚、丙酮、乙酸乙酯、卤化溶剂(二氯甲烷、氯仿等)、己烷和戊烷。另外，当水是络合溶剂时，与水复合的化合物称为“水合物”。
如本文所用，术语“立体异构体(stereoisomer)”是指由分子中原子在空间上排列方式不同所产生的异构体,可分为对映(异构)体和非对映(异构)体两大类。
术语“高内涵筛选(high content screening，HCS)”或“高内涵分析(high content analysis，HCA)”在本文中可互换使用，是指在保持细胞结构和功能完整性前提下，同时检测多种受试化合物对细胞形态、生长、分化、迁移、凋亡、代谢途径及信号传导各个环节的影响，确定受试化合物的生物活性和/或潜在毒性等。
高内涵分析系统通常采用的是384孔板多样品平行数据采集和分析，这比需要单个样品逐个处理的流式细胞仪快数百倍；同时基于图像的分析使得在活细胞自然条件下研究多种受试化合物对蛋白质的影响(如蛋白质在细胞内的分布转运、表达调控和信号传导等)成为可能。
术语“初级纤毛”和“纤毛”在本文中可互换地使用，是一种存在于大多数哺乳动物细胞表面的特殊细胞器，通过中心粒锚定于细胞表面。术语“初级纤毛”首次被用在呼吸道的上皮细胞中。机体大部分的细胞只有一根纤毛，与呼吸道的单根纤毛类似，这种单个的纤毛称为初级纤毛。在初级纤毛上存在的标志物包括但不限于ARL13B、INVERSIN、微管蛋白。
本文所用的“干扰”、“抑制”或“阻断”信号传导途径可以互换使用，是指抑制GLI2蛋白转运入初级纤毛、和/或抑制GLI2蛋白在初级纤毛中的积聚、和/或抑制GLI2蛋白对细胞核中靶基因的激活。由式I的前列腺素和其类似物导致的“干扰”、“抑制”或“阻断”信号传导途径不需要是完全的干扰、抑制或阻断。
“GLI2蛋白”或“GLI2”在本文中可互换地使用，包括天然存在的或重组形式的GLI2蛋白。已经表征了几种脊椎动物GLI2蛋白，例如，人GLI2(GenBank登录号NM_030381.1；NM_030380.1；NM_030379.1、DQ086814.1)、小鼠GLI2(GenBank登录号NM_001081125.1；XM_006529141.3；XM_011247924.2；XM_922107.1)。
术语“GLI2蛋白活性”是指能够进行GLI2信号传导，并且包括例如通过GLI2转录活化下游基因。
术语“Gli”是指编码GLI蛋白的基因。因此，Gli2是编码GLI2蛋白的基因。
术语“Hedgehog”可与术语“Hh”、“HH”互换使用。Hh配体在结合Hh受体后可启动经 典的Hh信号传导途径。在哺乳动物中存在三种Hh配体，分别是音猬(Sonic hedgehog；Shh)、印度刺猬(Indian hedgehog；Ihh)和沙漠刺猬(Desert hedgehog；Dhh)。几种脊椎动物Hh配体是本领域中已知的，例如，人SHH、鼠SHH、人IHH和鼠DHH。在经典的Hh信号传导途径中，Hh配体与Hh受体PTCH1结合后，SMO的抑制作用得以缓解，使SMO纤毛积累和活化(Corbit等人(2005).Vertebrate Smoothened functions at the primary cilium.Nature 437,1018-1021；Rohatgi等人(2007).Patched1regulates hedgehog signaling at the primary cilium.Science 317,372-376)，随后GLI2以活化的全长形式从纤毛转运至细胞核(Kim等人(2009).Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus.Proceedings of the National Academy of Sciences of the United States of America 106,21666-21671)，在细胞核中GLI2诱导靶基因(包括GLI1和PTCH1)的协调表达。
如本文所用，短语“信号转导”或“信号传导活性”指通常由蛋白质-蛋白质相互作用如生长因子与受体结合引发的生物化学因果关系，所述生物化学因果关系导致信号从细胞的一个部分传输至细胞的另一个部分。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代。宿主细胞包括培养的细胞，也包括动物组织内部的细胞。
术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体是人。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，式I的前列腺素和其类似物用来延缓GLI2蛋白活化所致的疾病进展。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
II.抑制GLI2蛋白的前列腺素和其类似物
通过本发明的高内涵筛选方法，提供了有效的GLI2抑制剂，它们是如下式I所示的前列腺素和其类似物：
Figure PCTCN2020078224-appb-000011
其中
R 1选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、或者R 1与其连接的环碳原子一起形成C(＝O)；
R 2选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、或者R 2与其连接的环碳原子一起形成C(＝O)；
R 3选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、＝O、或者R 3与连接R 1的环碳原子一起相互连接形成5元环基团；
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是0－12的整数，例如，n各自独立地是0、1、2、3、4、5、6、7、8、9、10、11、12的整数；
R 4各自独立地选自H、-CH 3、-COCH 3、-OH、所述-OH的盐、-COOH或所述-COOH的盐或碳原子数1－6的烷基酯、-CONH 2、-CONH(C 1-5烷基)OH；
R 5选自H、OH、任选取代的C 1-6烷基、卤素、O(任选取代的C 1-6烷基)；
R 6独立地选自H、OH、任选取代的C 1-6烷基、卤素、O(任选取代的C 1-6烷基)；
m各自独立地是0－10的整数，例如，m各自独立地是0、1、2、3、4、5、6、7、8、9、10的整数；
每个
Figure PCTCN2020078224-appb-000012
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000013
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物。
在一些实施方案中，式I所示的前列腺素和其类似物是：其中R 1与其连接的环碳原子一起形成C(＝O)；
R 2选自H、OH或者O(任选取代的C 1-6烷基)；
R 3选自H或者＝O；
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是2、3、4、5或6的整数；
R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
R 5选自H或OH；
R 6独立地选自H、C 1-3烷基、卤素；
m各自独立地是0、1、2、3、4的整数；
每个
Figure PCTCN2020078224-appb-000014
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000015
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；或者
在具体的实施方案中，式I所示的前列腺素和其类似物是具有如下结构式的化合物：
Figure PCTCN2020078224-appb-000016
Figure PCTCN2020078224-appb-000017
在一些实施方案中，式I所示的前列腺素和其类似物是：其中R 1选自H或者OH；
R 2与其连接的环碳原子一起形成C(＝O)；
R 3是H；
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是2、3、4、5或6的整数；
R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
R 5选自H或OH；
R 6独立地选自H、C 1-3烷基、卤素；
m各自独立地是0、1、2、3、4的整数；
每个
Figure PCTCN2020078224-appb-000018
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000019
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物。
在具体的实施方案中，式I所示的前列腺素和其类似物是具有如下结构式的化合物：
Figure PCTCN2020078224-appb-000020
在一些实施方案中，式I所示的前列腺素和其类似物是：其中R 1选自H、任选取代的C 1-6烷基、卤素；
R 2选自OH、O(任选取代的C 1-6烷基)
R 3与连接R 1的环碳原子一起相互连接，形成下式II或式III的环基团：
Figure PCTCN2020078224-appb-000021
X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
n各自独立地是2、3、4、5或6的整数；
R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所 述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
R 5选自H或OH；
R 6独立地选自H、C 1-3烷基、卤素；
m各自独立地是1、2、3、4的整数；
每个
Figure PCTCN2020078224-appb-000022
独立为单键或双键,其中任何两个相邻键
Figure PCTCN2020078224-appb-000023
中的至少一个为单键；
和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物。
在具体的实施方案中，式I所示的前列腺素和其类似物是具有如下结构式的化合物：
Figure PCTCN2020078224-appb-000024
式I所示的前列腺素和其类似物能够抑制GLI2蛋白转运入初级纤毛、和/或抑制GLI2蛋白在初级纤毛中的积聚、和/或抑制GLI2蛋白对细胞核中靶基因的激活。
III.抑制GLI2的化合物的高内涵筛选方法
GLI2蛋白的活化和/或高表达存在于HH信号传导途径、RAS信号传导途径、转化生长因子β信号传导途径、wnt信号传导途径、notch信号传导途径等参与的细胞过程的异常调节所导致的病理学表型中。Hh配体依赖性机制和Hh配体非依赖性机制引起的HH途经活化均可导致GLI2蛋白的异常活化，其中所述Hh配体依赖性机制是Hh配体(例如，人SHH、鼠SHH、人IHH和鼠DHH)与Hh受体PTCH1结合后，使得PTCH1对SMO的抑制作用得以缓解，SMO活化并随后引起GLI2蛋白活化；其中所述Hh配体非依赖性机制是不依赖于Hh配体与Hh受体结合，但GLI2蛋白活化的机制，例如由于PTCH1功能丧失的突变引起无法抑制SMO，导致SMO活化并随后引起GLI2蛋白活化；也可由于SMO获得功能的突变导致SMO活化并随后引起GLI2蛋白活化；也可由于SUFU中的失活突变引起GLI2蛋白活化；也可由于Gli2基因组扩增引起GLI2蛋白的过量表达。
现有技术中已鉴定出了多种抑制HH途径活化的SMO抑制剂。本领域鉴定出的直接结合SMO的第一个SMO抑制剂是环巴胺(cyclopamine)(Chen等人(2002).Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened.Genes&development 16,2743-2748)，所述环巴胺是在野生加利福利亚藜芦(Veratrum californicum)中发现的一种天然化合物。此后，已开发出了更多的SMO抑制剂，并且其中几种(包括维莫德吉、索尼得吉、LY2940680、PF04449913和BMS-833923)在HH途径活化依赖性癌症的临床前和临床研究中显示了有前景的功效(Wu等人(2017).Hedgehog Signaling:From Basic Biology to Cancer Therapy.Cell chemical biology 24,252-280)。美国食品和药品管理局(FDA)已批准维莫德吉和索尼得吉用于治疗晚期基底细胞癌(BCC)(这是白种人群体中最常见的癌症)(Burness,C.B.(2015).Sonidegib:First Global Approval.Drugs 75,1559-1566；Dlugosz等人(2012).Vismodegib.Nat Rev Drug Discov 11,437-438)。但是，已观察到对这些药物的获得性抗性，这限制了这些药物在临床中的长期功效。为了解决抗药性这一难题，可以通过靶向SMO下游的HH信号传导组分GLI2蛋白，抑制GLI2蛋白的活化和/或高表达来实现。
本发明人通过锐意研究，开发了一种高内涵筛选抑制GLI2的化合物的方法，所述方法能够比较在GLI2蛋白异常活化的不同机制下，数目相对巨大的化合物抑制GLI2的体外活性。通常，这类高内涵筛选在多孔微量滴定板中(例如，在96孔平板或384孔平板或具有1536孔或3456孔的平板中)进行。
在一个实施方案中，本发明的高内涵筛选方法能够用于筛选选自小分子、蛋白质、肽和核酸的化合物和/或候选药物对GLI2蛋白活性的抑制，所述方法允许可视化所述化合物和/或候选药物是否抑制GLI2蛋白转运入初级纤毛和/或在初级纤毛中积聚，所述方法包括：
i.分别构建表达第一标记的初级纤毛标志物的构建体和表达第二标记的GLI2的构建体；或者构建表达第一标记的初级纤毛标志物和表达第二标记的GLI2的双表达构建体，其中第一标记不同于第二标记；
ii.将步骤i的表达第一标记的初级纤毛标志物的构建体和表达第二标记的GLI2的构建体同时或以不同顺序依次地引入哺乳动物细胞中，或者将步骤i的表达第一标记的初级纤毛标志物和表达第二标记的GLI2的双表达构建体引入哺乳动物细胞中，获得同时表达第一标记的初级纤毛标志物和第二标记的GLI2的细胞系；
iii.将受试化合物与步骤ii的GLI2蛋白活化或者过量表达的细胞系接触；
iv.通过基于图像的高内涵分析，与未接触受试化合物的步骤ii的GLI2蛋白活化或者过量表达的细胞系相比较，确定减少GLI2蛋白积聚于初级纤毛的受试化合物作为抑制GLI2的化合物。
所述“初级纤毛标志物”可以用来确定初级纤毛。初级纤毛标志物是本领域已知的并且包括但不限于ARL13B、INVERSIN、微管蛋白。
所述“哺乳动物细胞”是适于产生外源蛋白质的哺乳动物宿主细胞系，包括但不限于人胚肾系(293或293F细胞)、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、 人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、CHO细胞、COS细胞、NIH/3T3细胞、DAOY细胞。
在一个实施方案中，当步骤ii.中的获得的细胞系以低水平(DMSO处理组观测不到EGFP::GLI2在初级纤毛上的定位，SHH-N处理组与DMSO处理组相比，EGFP::GLI2在初级纤毛上的定位数差异极显著)表达GLI2蛋白时，使用HH途径激动剂活化GLI2蛋白。所述HH途径激动剂为经典的Hedgehog信号传导途径激动剂，例如SHH-N(SHH N末端信号肽，其序列见SEQ ID NO:1)或SAG(一种小分子SMO激动剂，Millipore：#566660)等。
在一个实施方案中，当步骤ii.中的获得的细胞系以高水平(DMSO处理组可以明显观测到EGFP::GLI2在初级纤毛上的定位，SHH-N处理组与DMSO处理组相比，EGFP::GLI2在初级纤毛上的定位数差异不显著)表达GLI2蛋白时，无需使用HH途径激动剂。
在一个实施方案中，使用tagRFPT标记初级纤毛标志物，并使用EGFP标记GLI2蛋白。
通过本发明的高内涵筛选方法，能够高效地筛选到抑制GLI2的化合物。
在一些实施方案中，通过本发明的高内涵筛选方法筛选到的抑制GLI2的化合物是式I所示的前列腺素和其类似物。
IV.抑制Hh配体非依赖性机制中GLI2活化的化合物的筛选方法
本发明还公开了抑制Hh配体非依赖性机制中GLI2活化的化合物的筛选方法。
在一个实施方案中，所述方法是抑制过量表达GLI2的化合物的筛选方法，所述方法包括：
i.将过量表达GLI2的构建体引入哺乳动物细胞(例如，NIH/3T3或DAOY细胞)中，获得过量表达GLI2的细胞；
ii.使受试化合物(优选地，通过本发明的高内涵筛选方法筛选到的抑制GLI2的化合物)与i的过量表达GLI2的细胞接触；
iii.测定所述GLI2的活性水平，例如，测定GLI2的内源性靶基因表达(例如，Gli1、Ptch1、Wnt2、Axin2、EGFR和细胞周期蛋白D1 mRNA水平)和/或内源性GLI1蛋白的活性水平和/或与GLI2活化有关的其他分子的水平；和/或检测GLI-萤光素酶报告基因活性水平(如，通过测试萤光素酶报告基因活性来测量)；
iv.将所述活性水平与没有接触受试化合物的细胞中GLI2的活性水平进行比较，并选择使GLI2的活性水平降低的受试化合物。
在另一个实施方案中，所述方法是抑制耐受Smoothened(SMO)拮抗剂的Hedgehog信号传导途径活化相关的GLI2的化合物的筛选方法，所述方法包括：
i.培养SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞；
ii.使受试化合物(优选地，通过本发明的高内涵筛选方法筛选到的抑制GLI2的化合物)与i的SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞接触；
iii.测定GLI2的活性水平，例如，测定GLI2的内源性靶基因表达(例如，Gli1、Ptch1、 Wnt2、Axin2、EGFR和细胞周期蛋白D1 mRNA水平)和/或内源性GLI1蛋白的活性水平和/或与GLI2活化有关的其他分子的水平；和/或检测GLI-萤光素酶报告基因活性水平(如，通过测试萤光素酶报告基因活性来测量)；
iv.与没有接触受试化合物的i的SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞的GLI2的活性水平进行比较，并选择使GLI2活性水平降低的受试化合物。
V.抑制GLI2活化的化合物的用途
通过本文公开的筛选方法得到的抑制GLI2的化合物是式I所示的前列腺素和其类似物。所述化合物能够抑制癌细胞(例如，癌症干细胞)，从而用于治疗、预防或减轻受试者中的肿瘤(尤其是表现出肿瘤抗性的耐药肿瘤)。
本文所用的术语“癌症”是指GLI2活化/过量表达的实体哺乳动物肿瘤以及血液性恶性肿瘤。“GLI2活化/过量表达的实体哺乳动物肿瘤”包括头和颈、肺、间皮瘤、纵膈、食管、胃、胰腺、肝胆系统、小肠、结肠、结直肠、直肠、肛门、肾、尿道、膀胱、前列腺、尿道、阴茎、睾丸、妇科器官、卵巢、乳腺、内分泌系统、皮肤、包括脑的中枢神经系统的GLI2活化/过量表达的癌症；软组织和骨的GLI2活化/过量表达的肉瘤；以及皮肤和眼内来源的GLI2活化/过量表达的黑素瘤。术语“GLI2活化/过量表达的血液性恶性肿瘤”包括GLI2活化/过量表达的儿童白血病和淋巴瘤、霍奇金病、淋巴细胞和皮肤来源的淋巴瘤、急性和慢性白血病、浆细胞肿瘤。此外，可以治疗在任何发展阶段的GLI2活化/过量表达的癌症，诸如初期、转移的和复发的癌症。关于众多癌症类型的信息可参见例如美国癌症协会(American Cancer Society)或例如Wilson等(1991)Harrison’s Principles of Internal Medicine，第12版，McGraw-Hill,Inc。人和兽用的用途都考虑在内。
尤其可通过式I所示的前列腺素和其类似物治疗的癌症包括但不限于GLI2活化/过量表达的神经胶质瘤、成神经管细胞瘤(例如小脑成神经管细胞瘤)、周皮细胞瘤、原始神经外胚瘤(PNETS)、基底细胞癌(BCC)、小细胞肺癌、大细胞肺癌、胃肠道的肿瘤、横纹肌肉瘤、乳腺癌、软组织肉瘤、胰腺肿瘤、膀胱肿瘤和前列腺肿瘤。
本文所用的“敏感肿瘤”意指对SMO抑制剂抗癌方案的治疗响应的肿瘤(例如成神经管细胞瘤)。
本文所用的“耐药肿瘤”或“难治性肿瘤”意指在SMO抑制剂的持续存在下，由于治疗引起收缩后再生长的或由于治疗被暂时性消除后再出现的以前敏感的肿瘤(例如成神经管细胞瘤)。耐药肿瘤表现出敏感性降低或对smoothened抑制没有反应。对耐药肿瘤的成功治疗可以引起例如肿瘤细胞对新型或以前尝试的抗癌方案和/或化学治疗剂的敏感性增加，并可以导致例如随后的肿瘤细胞死亡和防止转移。
在一些实施方案中，式I所示的前列腺素和其类似物用于治疗、预防或减轻受试者中的GLI2参与的信号传导通路失调导致的神经变性疾病或发育综合征和畸形(例如，前脑无裂畸形、格雷格头-多指-并指综合征、Pallister-Hall综合征、Rubenstein-Teybi综合征、基底细胞 痣综合征、轴后性多指症)。
本发明还提供了式I所示的前列腺素和其类似物与额外的一种或多种治疗剂或疗法组合，用于治疗、预防或减轻受试者中的GLI2参与的信号传导通路失调导致的疾病，所述额外的一种或多种治疗剂或疗法选自：化疗、靶向抗癌疗法、溶瘤药物、细胞毒性剂、基于免疫的疗法、细胞因子、外科处置、照射术。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例：
一般方法：
除非另外说明，本发明的实施将利用本领域技术人员已知并可获得的细胞生物学、细胞培养、分子生物学(包括重组技术)、微生物学、生物化学、动物学、病毒学和免疫学的常规技术。这类技术在如下文献中描述：Molecular Cloning:A laboratory Manual,第3版(Sambrook等人,2001)Cold Spring Harbor Press；Oligonucleotide Synthesis(P.Herdewijn编著,2004)；Animal Cell Culture(R.I.Freshney编著,1987)；Methods in Enzymology(Academic Press,Inc.)；Current Protocols in Molecular Biology(F.M.Ausubel等人编著,1987)；PCR:The Polymerase Chain Reaction(Mullis等人编著,1994)；Current Protocols in Immunology(J.E.Coligan等人编著,1991)；和Short Protols in Molecular Biology(Wiley and Sons,1999)。除非另外规定，本文中所用的全部术语及科学术语具有与本发明所属领域的技术人员通常理解的相同含义。
细胞系：NIH/3T3细胞培养于补充有10％(v/v)小牛血清的DMEM中。Cos7细胞培养于补充有10％(v/v)胎牛血清的DMEM中,DAOY细胞培养于补充有10％(v/v)胎牛血清的MEM中。将所有细胞系保持在37℃，95％空气和5％CO 2的潮湿环境中。
高内涵筛选中使用的化学库：包括Prestwick化学库(Prestwick Chemical)、Spectrum Collection(Microsource Discovery Systems)、药理活性化合物库(LOPAC,Sigma)、FDA批准的药物库(Topscience)、以及定制的企业内化合物库。
成像分析：将表达第一标记的初级纤毛标志物和第二标记的GLI2的细胞以1×10 4个细胞/孔置于预先包被有1％明胶(Sigma)的384孔成像板中的50μl培养基中。在细胞达到汇合后(1-2天)，将培养基更换为0.5％小牛血清的DMEM培养基，在SHH-N存在下或者不存在下，向各孔中加入受试化合物，24小时后用4％多聚甲醛固定细胞，并用Hoechst(Thermo Fisher)进行染色后成像，用Operetta High Content Screening System(Perkin Elmer)使用40倍高数值孔径物镜对细胞进行成像。Harmony 4.1软件(PerkinElmer)用于高内涵筛选数据管理和图像定量。在整个成像测定中进行相同的显微镜设置和输入参数。
逆转录PCR(RT-PCR)
使用TRIzol试剂(15596018,Thermo Fisher Scientific)从培养的细胞或快速冷冻的肿瘤中 分离总RNA。将每一RNA样品用DNase I(AM1907，Thermo Fisher Scientific)在37℃孵育30分钟以除去任何污染的基因组DNA，然后作为cDNA合成的模板，根据制造商的说明书使用GoScript cDNA Synthesis Kit(Promega)合成cDNA。使用高保真KOD-Plus-New(KOD-401，TOYOBO)进行PCR扩增以确定基因表达。
实时荧光定量PCR(qRT-PCR)
取1/10稀释的cDNA作为模板，使用SYBR Green-based PCR Master Mix(11201ES08，YEASEN)在Roche@480Real-Time PCR System上进行PCR扩增。使用甘油醛-3-磷酸脱氢酶(Gapdh)作为内部参考。用于定量GLI2的靶基因表达的引物序列列于图12中。
蛋白质印迹分析
在RIPA缓冲液(25mM Tris·HCl pH 7.6,150mM NaCl,1％NP-40,1％脱氧胆酸钠，0.1％SDS)中裂解细胞，其中每10ml RIPA补充有1片蛋白酶和磷酸酶抑制剂(A32961,Thermo Fisher Scientific)。通过SDS-PAGE分离等量的细胞裂解物，然后转移到聚偏二氟乙烯(PVDF)膜上。将膜在5％的脱脂牛奶中室温封闭2小时，随后将膜用封闭液配置的一级抗体在4℃孵育过夜。孵育过夜后，用TBST缓冲液洗3次，每次10分钟，然后用1:5000稀释的辣根过氧化物酶(HRP)标记的二级抗体(中国北京，ZSBIO)在室温孵育1小时，用TBST缓冲液洗3次，每次15分钟，然后用化学发光试剂(32106，Thermo Fisher Scientific)进行蛋白条带检测。分别使用了如下一级抗体：小鼠抗GLI1(2643,Cell signaling)、兔抗GLI2(AF3635，R&D)、小鼠抗β-肌动蛋白(ab8226,Abcam)。
免疫荧光成像分析：
将3T3/ARL13B::tagRFPT细胞按20000个细胞/孔接种在放有盖玻片的24孔板中，用含有10％(v/v)小牛血清的DMEM培养基继续培养细胞至完全汇合，此时将培养基变换为含有0.5％小牛血清的DMEM培养基继续培养24h。然后将细胞在4％多聚甲醛中固定30分钟，在0.3％triton-X 100中通透15分钟，在含2％BSA和0.3％Triton-X的封闭液中封闭1小时，然后用小鼠单克隆抗EP4抗体(sc-55596,Santa Cruz,1:200稀释)在4℃孵育过夜。孵育过夜后，将细胞洗3次，每次10分钟，然后用二级抗体Alexa Fluor 488山羊抗小鼠IgG(H+L)(A-11001,Thermo Fisher Scientific,1:500稀释)室温孵育1小时，然后再洗3次，每次10分钟。细胞最后用Hoechst进行染色，用PBS洗涤后盖上盖玻片并对样品进行成像。使用Zeiss LSM 780共聚焦显微镜用63倍油物镜收集图像，并使用ZEN软件(Zeiss)处理图像。
细胞活力测定法
以每孔1000个细胞接种至96孔板中，用受试化合物处理48小时。将CCK-8试剂(B34302，Biomake)以10μl/孔直接加入板中，并在37℃孵育4小时。使用Perkin Elmer板读数仪评估细胞活力。
H&E染色和免疫组织化学
将收集的新鲜肿瘤样本用福尔马林固定，然后用石蜡进行包埋。染色前，切片用二甲苯和一系列下降浓度的乙醇脱腊至水，然后用去离子水漂洗。对于H&E染色，按照标准程序将 切片分别与苏木精和伊红染料进行孵育。对于免疫组织化学染色，用柠檬酸钠缓冲液处理切片以进行热诱导的抗原修复，然后用1:600稀释的兔抗Ki67抗体(ab15580，Abcam)4℃孵育过夜，用PBS洗3次，每次10分钟，然后用抗兔HRP(DAB)(ZLI-9001，ZSBIO，北京，中国)进行DAB染色。用去离子水冲洗载玻片，用一系列上升浓度的乙醇和二甲苯脱水，然后盖上盖玻片。得到的载玻片在Aperio VESA8系统(Leica Biosystems)上进行40倍数字扫描。
异种移植实验
将含有5×10 6个GLI2过量表达的DAOY细胞的总体积为100μl PBS:Matrigel的1:1混合物皮下注射到每一只4-6周龄的NOD-Prkdc scid Il2rg null(NPG)小鼠(VITALSTAR，北京，中国)侧腹。当肿瘤生长到100mm 3的中位数大小时，将小鼠随机分成三组(每组n＝8)并分别用仅溶媒、维莫德吉(30mg/kg，每日一次，灌胃给药)、PGE1(15mg/kg，每日一次腹，腔注射给药)处理51天。用游标卡尺每3天一次测量肿瘤体积，肿瘤体积计算公式如下：长×宽×宽×0.5。处理结束时收获每个肿瘤样本并分为几份分别用于qRT-PCR、H&E染色和免疫组织化学分析。
实施例1抑制GLI2的化合物的高内涵筛选方法
本实施例开发了一种用于高内涵筛选(HCS)能够抑制GLI2转运至纤毛和/或在纤毛中积聚的化合物的方法并构建了在所述方法中使用的载体和细胞系。
抑制GLI2的化合物的高内涵筛选方法包括以下步骤：
i.将NIH/3T3细胞(CRL-1658，ATCC)培养于补充有10％(v/v)小牛血清(bovine calf serum；BCS)的DMEM中，按照常规方法进行传代。
ii.将具有红色荧光tagRFPT标记的ARL13B构建体引入HH反应性NIH/3T3细胞中，获得亲本3T3/ARL13B::tagRFPT细胞系(质粒骨架为pRRL慢病毒质粒Addgene:#31484，ARL13B::tagRFPT质粒图谱见图13，插入的Arl13b-tagRFPT的序列见SEQ ID NO:2)
iii.向步骤ii获得的3T3/ARL13B::tagRFPT细胞系中再引入表达EGFP::GLI2的慢病毒质粒，其中使用鼠源Gli2基因序列用于构建EGFP::GLI2融合体(质粒骨架为pRRL慢病毒质粒Addgene:#31484,EGFP::GLI2质粒图谱见图14，插入的EGFP-Gli2-3×Flag-3×Myc的序列见SEQ ID NO:3)。
iv.当外源导入的GLI2以低水平表达时，所述表达会受到HH信号传导途径的调节。对具有低EGFP::GLI2表达的单克隆细胞进行培养，将获得的细胞系命名为3T3/ARL13B::tagRFPT/EGFP::GLI2细胞系。
在3T3/ARL13B::tagRFPT/EGFP::GLI2细胞系中，SHH-N(SHH N末端信号肽，其序列见SEQ ID NO:1，用图15所示的质粒构建稳定表达SHH-N的Cos7细胞，扩增该细胞至长满后收集表达有SHH-N的上清液，SHH-N使用浓度为：用DMEM进行1:16稀释后的浓度)或SAG(一种小分子SMO激动剂，Millipore：#566660；使用浓度：100nM)分别诱导GLI2蛋白积聚于初级纤毛；维莫德吉或环巴胺减弱SHH-N诱导的GLI2蛋白纤毛积聚(图1A和 B)。
使用HH途径激动剂(SHH-N或SAG)和/或拮抗剂(环巴胺或维莫德吉)，测定了3T3/ARL13B::tagRFPT/EGFP::GLI2细胞系及其亲本3T3/ARL13B::tagRFPT细胞系响应于所述HH途径激动剂和/或拮抗剂的GLI-萤光素酶活性。如图1C所示，通过相对GLI-萤光素酶活性的测定可见，GLI依赖性转录活性响应于所述HH途径激动剂和/或拮抗剂的处理。这与现有技术中报道的内源性GLI2转运至纤毛(Haycraft等人(2005).Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.PLoS genetics 1,e53；Kiprilov等人(2008).Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery.The Journal of cell biology 180,897-904)等的行为一致。
这些数据表明3T3/ARL13B::tagRFPT/EGFP::GLI2细胞系能够用于影响GLI2纤毛定位的化合物的高内涵筛选。
v.将3T3/ARL13B::tagRFPT/EGFP::GLI2细胞系与受试化合物接触，通过成像分析测定纤毛尖端具有GLI2的纤毛百分数。
如下实施成像分析。将3T3/ARL13B::tagRFPT/EGFP::GLI2细胞以1×10 4个细胞/孔置于预先包被有1％明胶(Sigma)的384孔成像板中的50μl培养基中。在细胞达到汇合后(1-2天)，在SHH-N存在下，将各受试化合物(各受试化合物使用浓度为10μM，维莫德吉的使用浓度为1μM)加入0.5％小牛血清培养基中24小时，然后用4％多聚甲醛固定细胞，并用Hoechst(Thermo Fisher)染色成像，用Operetta High Content Screening System(Perkin Elmer)使用40倍高数值孔径物镜对细胞进行成像。Harmony 4.1软件(PerkinElmer)用于高内涵筛选数据管理和图像定量。在整个成像测定中进行相同的显微镜设置和输入参数。
当纤毛尖端具有GLI2的纤毛百分数时，表明受试化合物是抑制GLI2的化合物。
在对抑制SHH-N诱导的EGFP::GLI2纤毛积聚的受试化合物进行高内涵筛选时，使用的化学库包括Prestwick化学库(Prestwick Chemical)、Spectrum Collection(Microsource Discovery Systems)、药理活性化合物库(LOPAC,Sigma)、FDA批准的药物库(Topscience)、以及定制的企业内化合物库。环巴胺购自Sigma，SAG购自Millipore。维莫德吉和佛司可林购自Selleck。自Cayman购买前列腺素和其类似物。如之前所述(Wang等人(2012).Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2.Cell research 22,1593-1604)收集SHH-N条件培养基。从野生型Cos7细胞(CRL-1651,ATCC)收集对照条件培养基。
获得了多种抑制GLI2在纤毛积聚的化合物，它们是具有式I所示结构的前列腺素和其类似物，包括前列腺素A1(PGA1)、前列腺素D1醇(PGD1醇)、5-反式前列腺素D2(5-反式PGD2)、前列腺素E1(PGE1)、前列腺素J2(PGJ2)、15-脱氧-Δ 12,14-前列腺素J2(15-脱氧-Δ 12,14-PGJ2)，所述化合物的结构见图2A，对GLI2纤毛积聚的抑制结果见图2B和图2C。
实施例2前列腺素及其类似物对GLI2表达的抑制作用
本实施例构建了具有GLI2高表达的HH信号活性报道细胞系，并测试了前列腺素及其类 似物对GLI2表达的抑制作用。
首先，产生了3T3/GLI-luc报告细胞系。具体而言，将野生型NIH/3T3细胞先用慢病毒导入受8×GLIBS驱动的萤火虫萤光素酶质粒(质粒骨架为pRRL慢病毒质粒Addgene:#31484,质粒图谱见附图16，插入的萤火虫萤光素酶的序列见SEQ ID NO:4)，在稳定表达萤火虫萤光素酶的亚克隆中用慢病毒再次导入受CMV启动子驱动的海肾萤光素酶质粒(质粒骨架为pRRL慢病毒质粒Addgene:#31484,质粒图谱见附图17，插入的海肾萤光素酶序列见SEQ ID NO:5)，获得稳定的3T3/GLI-luc报告细胞系。在所述8×GLIBS萤火虫萤光素酶质粒中，存在8个串联重复的GLI结合位点(GLIBS)，能够通过GLI依赖性转录活性来驱动萤火虫萤光素酶的表达。使用组成型海肾萤光素酶报告基因用作内部对照。
如下实施GLI-萤光素酶报告基因测定法。将3T3/GLI-luc细胞系或其衍生细胞系分别培养于96孔测定板，进行扩增培养。在细胞汇合后，将培养基更换为补充有0.5％(v/v)小牛血清的DMEM培养基，并用受试化合物(将受试化合物从100μM或30μM倍比稀释)处理36h。然后用酶标仪(PerkinElmer)依次读取萤火虫萤光素酶活性和海肾萤光素酶活性。使用海肾萤光素酶信号归一化萤火虫萤光素酶信号。
分别将SAG、SHH-N或维莫德吉与稳定的3T3/GLI-luc报告细胞系一起孵育36小时，测定相对GLI萤光素酶活性，结果如图3A-C所示。
然后，产生了具有GLI2高表达的细胞系，命名为3T3/GLI-luc/GLI2报告细胞系。具体而言，递送含有CMV启动子驱动的小鼠GLI2表达盒(图3D)的慢病毒质粒至3T3/GLI-luc报告细胞系(质粒骨架为pRRL慢病毒质粒Addgene:#31484，质粒图谱见附图18，插入的小鼠GLI2序列见SEQ ID NO:6)，获得3T3/GLI-luc/GLI2报告细胞系，其过量表达小鼠GLI2，由此，通过蛋白质印迹可以容易地检测到该外源GLI2的表达(图3F)。
与3T3/GLI-luc报告细胞系相比，3T3/GLI-luc/GLI2报告细胞系引发了强的萤光素酶报告基因活性(图3E)。
在3T3/GLI-luc/GLI2报告细胞系中，对于浓度高达10μM的维莫德吉，没有观察到对GLI2诱导的萤光素酶活性的抑制作用(图4A)。对3T3/GLI-luc/GLI2报告细胞系而言使用的维莫德吉浓度10μM远高于维莫德吉对3T3/GLI-luc报告细胞系产生抑制作用的饱和剂量(图3C)。
使用实施例1鉴定的所有6种前列腺素和其他17种前列腺素类似物，在3T3/GLI-luc/GLI2报告细胞系中通过GLI-萤光素酶报告基因测定法测定了它们对GLI2的抑制作用(图4A、图5)。在这些前列腺素中，PGE1(前列腺素E1)，也称为前列地尔(alprostadil)，是FDA批准的常用于治疗肺高血压、勃起功能障碍和外周动脉闭塞性疾病的药物(Murali等人,1992；Weiss等人,2002)。
另外，在3T3/GLI-luc/GLI2报告细胞系中，通过检测GLI2诱导的靶基因(包括Gli1和Ptch1)mRNA水平，证实了PGE1有效抑制GLI2诱导的靶基因(包括Gli1和Ptch1)的内源性表达，而维莫德吉和环巴胺没有显示这样的抑制作用(图4B)
实施例3.前列腺素及其类似物抑制SMO突变体介导的HH途径活性
在HH信号传导途径的活化中，SMO点突变会导致对维莫德吉的药物耐受性。本实施例检测了由两种药物难治性SMO突变体SMO-D473H和SMO-W535L(也称为SMO-A1或SMO-M2)介导的HH途径活性(Xie等(1998).Activating Smoothened mutations in sporadic basal-cell carcinoma.Nature 391,90-92；Yauch等(2009).Smoothened Mutation Confers Resistance to a Hedgehog Pathway Inhibitor in Medulloblastoma.Science 326,572-574)，所述SMO突变体是在维莫德吉治疗期间经历破坏性癌症复发的患者中确定的(Atwood等(2015).Smoothened variants explain the majority of drug resistance in basal cell carcinoma.Cancer cell 27,342-353；Sharpe等(2015).Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma.Cancer cell 27,327-341)，并测试了前列腺素及其类似物抑制SMO突变体介导的HH途径活性。
产生了在3T3/GLI-luc细胞中或SMO-D473H或SMO-W535L的亚克隆(质粒骨架为pBABE逆转录病毒质粒Addgene:#1764,质粒图谱分别见图19、图20和图21，插入的SMO WT-EGFP序列、SMO D473H-EGFP序列、SMO W535L-EGFP分别见SEQ ID NO:7、8、9)
与维莫德吉相反，前列腺素及其类似物可以有效地抑制所述两种SMO突变体导致的GLI2蛋白活性，具有针对野生型SMO等同的效力。实施例1中鉴定的所有6种前列腺素均抑制这两种SMO突变体。图6A－E和图7B中例举了几种具体前列腺素的相对GLI萤光素酶活性。
进一步地，分别通过内源性Gli1和Ptch1 mRNA表达(图7C-E)、和内源性GLI1蛋白水平(图7F)证实了PGE1的技术效果。另外，当使用升高的SAG浓度时，未观察到PGE1的IC50移位(图7H)，这与维莫德吉获得的结果(图7G)相反。
除了由SAG引起的HH途径活性之外，由SHH-N在HH途径的更上游水平引起的途径活性也可以被PGE1抑制，这可通过多种测定法来检测，结果如图8A-C所示。
将这些数据结合在一起可见，前列腺素及其类似物显示对SMO靶向癌症治疗引起的多种抗药原因(包括GLI2过量表达和SMO突变)的泛抑制，并且可以在SMO的下游水平发挥作用。
实施例4前列腺素及其类似物抑制药物难治性异种移植物的生长
本实施例探讨了前列腺素及其类似物在治疗难治性肿瘤中的潜在应用，其中所述难治性肿瘤与GLI2相关。
在现有技术中，癌症研究利用的往往是源自遗传修饰小鼠的肿瘤同种异体移植物。为了最小化人和小鼠的物种间变异，本实施例使用了DAOY细胞系(#HTB-186,ATCC)。所述DAOY细胞系是一种具有已知PTCH1突变的人SHH亚型MB细胞系(Singh等人(2015).The Orphan G Protein-coupled Receptor Gpr175(Tpra40)Enhances Hedgehog Signaling by Modulating cAMP Levels.Journal of Biological Chemistry 290,29663-29675；Triscott等人(2013).Personalizing the treatment of pediatric medulloblastoma:Polo-like kinase 1as a molecular target in high-risk children.Cancer research 73,6734-6744)。
为了模拟GLI2异常引起的抗药性，本发明人通过慢病毒递送产生了分别稳定地过量表达人SMO-D473H间接地引起GLI2异常或稳定地过量表达人GLI2直接地引起GLI2异常的两种DAOY细胞系(质粒骨架为pRRL慢病毒质粒Addgene:#31484，质粒图谱分别见附图22和附图23，插入的SMO D473H-P2A-萤火虫萤光素酶序列、人Gli2-P2A-萤火虫萤光素酶序列分别见SEQ ID NO:10和SEQ ID NO:11)，下文中称为DAOY/SMO-D473H细胞和DAOY/GLI2细胞。
使用PGE1分别处理野生型DAOY细胞、DAOY/SMO-D473H细胞和DAOY/GLI2细胞，检测了细胞成活力和GLI2靶基因(GLI1和PTCH1)的表达。由图9A－C可见，在PGE1处理后这三种细胞的细胞活力均显著降低，而DAOY/SMO-D473H细胞和DAOY/GLI2细胞显示了对维莫德吉和环巴胺的预期抗性；由图9D－F可见，在PGE1处理后这三种细胞的GLI2靶基因(GLI1和PTCH1)mRNA水平均显著下降，而DAOY/SMO-D473H细胞和DAOY/GLI2细胞同样显示了对维莫德吉和环巴胺的预期抗性。这些结果表明，PGE1具有体外抗药物难治性肿瘤细胞生长和抗HH途径活性的作用。
作为野生型DAOY细胞的对照，还使用PGE1分别处理不表达Gli2基因的细胞HCC38(#CRL-2314,ATCC)、MCF7(#HTB-22,ATCC)、DLD-1(#CCL-221,ATCC)、HepG2(#HB-8065,ATCC)，由图10A和图10B可见，在PGE1处理后这四种细胞的细胞成活力没有显著变化，表明对不表达Gli2基因的所述细胞而言，PGE1处理不会引起细胞活力显著下降。
进一步地，将GLI2过量表达的DAOY细胞(即，DAOY/GLI2细胞)进行小鼠体内肿瘤异种移植，并分别用PGE1或维莫德吉处理。结果表明，每天施用15mg/kg PGE1显著地抑制肿瘤的生长(图11A和B)。相反地，每天用30mg/kg维莫德吉进行的饱和处理没有抑制肿瘤的生长(图11A和B)。与PGE1体外对GLI2的抑制作用一致的是，PGE1的体内施用在这些肿瘤中显著地减弱了GLI2的靶基因(包括GLI1和PTCH1)的表达(图11C)。
对肿瘤组织的苏木精和伊红(H&E)染色结果示于图11D中。PGE1处理的肿瘤组织显示坏死的迹象，如嗜碱性肿瘤细胞失去规则的细胞形态和发生核固缩(黑色箭头)，而溶媒对照或维莫德吉处理的肿瘤组织显示良好的肿瘤细胞形态(红色箭头)。与PGE1递送所致的肿瘤生长抑制一致的是，与溶媒对照和维莫德吉相比，PGE1处理使得Ki67阳性增殖细胞显著降低(图11E和F)。
由这些结果可见，PGE1作为本研究中鉴定的许多前列腺素的代表，将为针对当前一代SMO拮抗剂难以治愈的肿瘤的进一步治疗提供潜在的机会。
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。在这个方面，本发明范围仅由以下权利要求限定。

Claims (10)

  1. 神经胶质瘤相关的癌基因-2(GLI2)抑制剂的用途，用于制备治疗、预防或减轻受试者中与GLI2转录因子激活相关的疾病的药物，其中所述GLI2抑制剂是式I所示的前列腺素和其类似物：
    Figure PCTCN2020078224-appb-100001
    其中
    R 1选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、或者R 1与其连接的环碳原子一起形成C(＝O)；
    R 2选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、或者R 2与其连接的环碳原子一起形成C(＝O)；
    R 3选自H、任选取代的C 1-6烷基、OH、卤素、O(任选取代的C 1-6烷基)、＝O、或者R 3与连接R 1的环碳原子一起相互连接形成5元环基团；
    X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
    n各自独立地是0－12的整数，例如，n各自独立地是0、1、2、3、4、5、6、7、8、9、10、11、12的整数；
    R 4各自独立地选自H、-CH 3、-COCH 3、-OH、所述-OH的盐、-COOH或所述-COOH的盐或碳原子数1－6的烷基酯、-CONH 2、-CONH(C 1-5烷基)OH；
    R 5选自H、OH、任选取代的C 1-6烷基、卤素、O(任选取代的C 1-6烷基)；
    R 6独立地选自H、OH、任选取代的C 1-6烷基、卤素、O(任选取代的C 1-6烷基)；
    m各自独立地是0－10的整数，例如，m各自独立地是0、1、2、3、4、5、6、7、8、9、10的整数；
    每个
    Figure PCTCN2020078224-appb-100002
    独立为单键或双键,其中任何两个相邻键
    Figure PCTCN2020078224-appb-100003
    中的至少一个为单键；
    和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；
    优选地，
    其中R 1与其连接的环碳原子一起形成C(＝O)；
    R 2选自H、OH或者O(任选取代的C 1-6烷基)；
    R 3选自H或者＝O；
    X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
    n各自独立地是2、3、4、5或6的整数；
    R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
    R 5选自H或OH；
    R 6独立地选自H、C 1-3烷基、卤素；
    m各自独立地是0、1、2、3、4的整数；
    每个
    Figure PCTCN2020078224-appb-100004
    独立为单键或双键,其中任何两个相邻键
    Figure PCTCN2020078224-appb-100005
    中的至少一个为单键；
    和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；或者
    优选地，
    其中R 1选自H或者OH；
    R 2与其连接的环碳原子一起形成C(＝O)；
    R 3是H；
    X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
    n各自独立地是2、3、4、5或6的整数；
    R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
    R 5选自H或OH；
    R 6独立地选自H、C 1-3烷基、卤素；
    m各自独立地是0、1、2、3、4的整数；
    每个
    Figure PCTCN2020078224-appb-100006
    独立为单键或双键,其中任何两个相邻键
    Figure PCTCN2020078224-appb-100007
    中的至少一个为单键；
    和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物；或者
    优选地，
    其中R 1选自H、任选取代的C 1-6烷基、卤素；
    R 2选自OH、O(任选取代的C 1-6烷基)
    R 3与连接R 1的环碳原子一起相互连接，形成下式II或式III的环基团：
    Figure PCTCN2020078224-appb-100008
    X 1、X 2、X 3是CH、CH 2、O或S或直接键或碳碳三键或任选取代的苯基或吡啶，优选地，X 1是O或S或直接键，X 2是O或S或直接键或碳碳三键或任选取代的苯基或吡啶，X 3是CH或CH 2；
    n各自独立地是2、3、4、5或6的整数；
    R 4各自独立地选自H、-COOH或所述-COOH的盐或碳原子数1－3的烷基酯、-OH、所述-OH的盐、-CONH 2、-CONH(C 1-3烷基)OH；
    R 5选自H或OH；
    R 6独立地选自H、C 1-3烷基、卤素；
    m各自独立地是1、2、3、4的整数；
    每个
    Figure PCTCN2020078224-appb-100009
    独立为单键或双键,其中任何两个相邻键
    Figure PCTCN2020078224-appb-100010
    中的至少一个为单键；
    和其立体异构体(例如，对映体、非对映体)、其可药用盐、溶剂化物。
  2. 根据权利要求1所述的用途，其中所述GLI2抑制剂选择性抑制GLI2的表达和活性，例如，抑制GLI2转运至初级纤毛和/或抑制GLI2在初级纤毛中的积聚。
  3. 根据权利要求1或2所述的用途，其中所述GLI2抑制剂抑制GLI2参与的信号传导通路，包括但不限于经典的和非经典的Hedgehog信号传导途径、RAS信号传导途径、TGFβ信号传导途径、wnt信号传导途径、notch信号传导途径。
  4. 根据权利要求1－3中任一项所述的用途，其中所述GLI2抑制剂抑制GLI2活化/过量表达的癌细胞(例如，癌症干细胞)，从而用于制备治疗、预防或减轻受试者中的GLI2活化/过量表达的肿瘤(尤其是肿瘤抗性)的药物，所述GLI2活化/过量表达的肿瘤是GLI2活化/过量表达的实体哺乳动物肿瘤以及血液性恶性肿瘤，例如GLI2活化/过量表达的神经胶质瘤、成神经管细胞瘤(例如小脑成神经管细胞瘤)、周皮细胞瘤、原始神经外胚瘤(PNETS)、基底细胞癌(BCC)、小细胞肺癌、大细胞肺癌、胃肠道的肿瘤、横纹肌肉瘤、乳腺癌、软组织肉瘤、胰腺肿瘤、膀胱肿瘤和前列腺肿瘤；或者所述GLI2抑制剂用于制备治疗、预防或减轻受试者中的GLI2参与的信号传导通路失调导致的神经变性疾病或发育综合征和畸形(例如，前脑无裂畸形、格雷格头-多指-并指综合征、Pallister-Hall综合征、Rubenstein-Teybi综合征、基底细胞痣综合征、轴后性多指症)的药物。
  5. 根据权利要求1－4中任一项所述的用途，其中所述GLI2抑制剂与额外的一种或多种治疗剂或疗法组合，所述额外的一种或多种治疗剂或疗法选自：化疗、靶向抗癌疗法、溶瘤 药物、细胞毒性剂、基于免疫的疗法、细胞因子、外科处置、照射术。
  6. 根据权利要求1－5中任一项所述的用途，其中所述GLI2抑制剂选自：前列腺素A1(PGA1)、8-异PGA1、PGA2、前列腺素D1醇(PGD1醇)、前列腺素D1、前列腺素D2、5-反式前列腺素D2(5-反式PGD2)、前列腺素E1(PGE1)、PGE1醇、8-异PGE1、PGE1乙基酯、6-酮PGE1、PGE1乙醇胺、PGE2、15(R)-PGE2、8-异PGE2、PGE2乙醇胺、PGI2(钠盐)、前列腺素J2(PGJ2)、15-脱氧-Δ 12,14-前列腺素J2(15-脱氧-Δ 12,14-PGJ2)、伊洛前列素(Iloprost)、Carbaprostacyclin、西前列烯(钙盐)和瑞文前列腺素。
  7. 抑制GLI2的化合物的高内涵筛选方法，所述方法包括：
    i.分别构建表达第一标记的初级纤毛标志物的构建体和表达第二标记的GLI2的构建体；或者构建表达第一标记的初级纤毛标志物和表达第二标记的GLI2的双表达构建体，其中第一标记不同于第二标记；
    ii.将步骤i的表达第一标记的初级纤毛标志物的构建体和表达第二标记的GLI2的构建体同时或以不同顺序依次地引入哺乳动物细胞(如NIH/3T3细胞、DAOY细胞)中，或者将步骤i的表达第一标记的初级纤毛标志物和表达第二标记的GLI2的双表达构建体引入哺乳动物细胞(如NIH/3T3细胞、DAOY细胞)中，获得同时表达第一标记的初级纤毛标志物和第二标记的GLI2的细胞系；
    iii.将受试化合物与步骤ii的GLI2蛋白活化或者过量表达的细胞系接触；
    iv.通过基于图像的高内涵分析，与未接触受试化合物的步骤ii的GLI2蛋白活化或者过量表达的细胞系相比较，确定减少GLI2蛋白积聚于初级纤毛的受试化合物作为抑制GLI2的化合物；
    其中，例如当步骤ii中的获得的细胞系以低水平表达GLI2蛋白时，使用HH途径激动剂活化GLI2蛋白。
  8. 根据权利要求7的高内涵筛选方法，其中第一标记和第二标记分别选自绿色荧光蛋白(GFP)、黄色荧光蛋白、蓝色荧光蛋白、青色荧光蛋白、橙色荧光蛋白，优选地，增强型绿色荧光蛋白(EGFP)、增强型黄色荧光蛋白、增强型蓝色荧光蛋白、增强型青色荧光蛋白、增强型橙色荧光蛋白，例如，TagRFP、tdTomato、DsRed、HcRed、AsRed、AmCyan、ZsGreen、AcGFP和ZsYellow；初级纤毛标志物选自ARL13B、INVERSIN、微管蛋白。
  9. 抑制过量表达GLI2的化合物的筛选方法，所述方法包括：
    i.将过量表达GLI2的构建体引入哺乳动物细胞(例如，NIH/3T3或DAOY细胞)中，获得过量表达GLI2的细胞；
    ii.使受试化合物(优选地，权利要求7或8鉴定的受试化合物)与i的过量表达GLI2的细胞接触；
    iii.测定所述GLI2的活性水平，例如，测定GLI2的内源性靶基因表达(例如，Gli1、Ptch1、Wnt2、Axin2、EGFR和细胞周期蛋白D1 mRNA水平)和/或内源性GLI1蛋白的活性水平和/或与GLI2活化有关的其他分子的水平；和/或检测GLI-萤光素酶报告基因活性水平(如， 通过测试萤光素酶报告基因活性来测量)；
    iv.将所述活性水平与没有接触受试化合物的细胞中GLI2的活性水平进行比较，并选择使GLI2的活性水平降低的受试化合物。
  10. 抑制耐受Smoothened(SMO)拮抗剂的Hedgehog信号传导途径活化相关的GLI2的化合物的筛选方法，所述方法包括：
    i.培养SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞；
    ii.使受试化合物(优选地，权利要求7或8鉴定的受试化合物)与i的SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞接触；
    iii.测定GLI2的活性水平，例如，测定GLI2的内源性靶基因表达(例如，Gli1、Ptch1、Wnt2、Axin2、EGFR和细胞周期蛋白D1 mRNA水平)和/或内源性GLI1蛋白的活性水平和/或与GLI2活化有关的其他分子的水平；和/或检测GLI-萤光素酶报告基因活性水平(如，通过测试萤光素酶报告基因活性来测量)；
    iv.与没有接触受试化合物的i的SMO异常活化(如，过量表达SMO-WT或SMO-D473H或SMO-W535L)的细胞的GLI2的活性水平进行比较，并选择使GLI2活性水平降低的受试化合物。
PCT/CN2020/078224 2019-03-08 2020-03-06 Gli2抑制剂的用途以及抑制gli2的化合物的筛选方法 WO2020182077A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910174832.8 2019-03-08
CN201910174832.8A CN111658628B (zh) 2019-03-08 2019-03-08 Gli2抑制剂的用途以及抑制gli2的化合物的筛选方法

Publications (1)

Publication Number Publication Date
WO2020182077A1 true WO2020182077A1 (zh) 2020-09-17

Family

ID=72382350

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/078224 WO2020182077A1 (zh) 2019-03-08 2020-03-06 Gli2抑制剂的用途以及抑制gli2的化合物的筛选方法

Country Status (2)

Country Link
CN (1) CN111658628B (zh)
WO (1) WO2020182077A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786339A (zh) * 2022-10-24 2023-03-14 北京爱思益普生物科技股份有限公司 一种tmej检测底物、其制备方法及应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2608924B1 (fr) * 1986-12-29 1990-07-20 Pasteur Institut Compositions therapeutiques contenant des derives soufres de prostaglandines, nouveaux derives soufres et leur procede de preparation
US7714014B2 (en) * 2005-12-09 2010-05-11 The Regents Of The University Of California Targeting GLI proteins in human cancer by small molecules
WO2011008305A1 (en) * 2009-07-15 2011-01-20 Enzon Pharmaceuticals, Inc. Rna antagonists targeting gli2 for the treatment of leukemia
NO2721710T3 (zh) * 2014-08-21 2018-03-31
US10330683B2 (en) * 2015-02-04 2019-06-25 Genentech, Inc. Mutant smoothened and methods of using the same
CN106421786A (zh) * 2015-08-06 2017-02-22 复旦大学 Hedgehog通路抑制剂和纳米药物递送系统的药物组合物及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KUMP, E. ET AL.: "Gli2 upregulates cFlip and renders basal cell carcinoma ce- lls resistan", ONCOGENE, vol. 27, 11 February 2008 (2008-02-11), pages 3856 - 3864, XP055740979 *
LI, F. Y. ET AL.: "Prostaglandin E1 and Its Analog Misoprostol Inhibit Human CML Stem Cell Self-Renewal via EP4 Receptor Activation and Repression of AP-1", CELL STEM CELL, vol. 21, 24 August 2017 (2017-08-24), pages 359 - 373, XP085189929 *
LU , F. ET AL.: "Virtual screening for potential allosteric inhibitors of cyclin-dependent kinase 2 from traditional chinese medicine", MOLECULES, vol. 21, 1259, 31 December 2012 (2012-12-31), XP055740977 *
WANG, X.P. ET AL.: "Multiple roles of dihomo-g-linolenic acid against prolife- ration diseases", LIPIDS IN HEALTH AND DISEASE, vol. 11, 31 December 2012 (2012-12-31), pages 25, XP021118589 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786339A (zh) * 2022-10-24 2023-03-14 北京爱思益普生物科技股份有限公司 一种tmej检测底物、其制备方法及应用

Also Published As

Publication number Publication date
CN111658628B (zh) 2022-03-01
CN111658628A (zh) 2020-09-15

Similar Documents

Publication Publication Date Title
Challa et al. Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis
Ho et al. Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA)
Tuson et al. Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube
Jiang Hedgehog signaling mechanism and role in cancer
Shan et al. Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3
Lim et al. Systemic autophagy insufficiency compromises adaptation to metabolic stress and facilitates progression from obesity to diabetes
Lee et al. Phosphoinositide 3-kinase signaling mediates β-catenin activation in intestinal epithelial stem and progenitor cells in colitis
Yu et al. The circadian clock gene Bmal1 controls intestinal exporter MRP2 and drug disposition
Chen et al. Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved
Lüdtke et al. Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung
Berbari et al. Mutations in Traf3ip1 reveal defects in ciliogenesis, embryonic development, and altered cell size regulation
Arruda et al. Defective STIM-mediated store operated Ca2+ entry in hepatocytes leads to metabolic dysfunction in obesity
Wojciech et al. The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development
Chen et al. Induction of nuclear protein-1 by thyroid hormone enhances platelet-derived growth factor A mediated angiogenesis in liver cancer
Rojas et al. The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus
Van Bree et al. Systemic inflammation with enhanced brain activation contributes to more severe delay in postoperative ileus
Somatilaka et al. Ankmy2 prevents smoothened-independent hyperactivation of the Hedgehog pathway via cilia-regulated adenylyl cyclase signaling
Skrzypski et al. TRPV6 channel modulates proliferation of insulin secreting INS-1E beta cell line
Watanabe et al. Requirement of Gαq/Gα11 signaling in the preservation of mouse intestinal epithelial homeostasis
WO2020182077A1 (zh) Gli2抑制剂的用途以及抑制gli2的化合物的筛选方法
Jitsukawa et al. Loss of sorting nexin 5 stabilizes internalized growth factor receptors to promote thyroid cancer progression
Zhao et al. Schisandrol B promotes liver enlargement via activation of PXR and YAP pathways in mice
Vaes et al. NDRG 4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons
Sui et al. Epidermal growth factor receptor and hedgehog signaling pathways are active in esophageal cancer cells from rat reflux model
Roybal et al. Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHβ

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20770060

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20770060

Country of ref document: EP

Kind code of ref document: A1