CN115261408A - Screening method and application of small molecule compound for targeted inhibition of RNA polymerase I - Google Patents
Screening method and application of small molecule compound for targeted inhibition of RNA polymerase I Download PDFInfo
- Publication number
- CN115261408A CN115261408A CN202210967527.6A CN202210967527A CN115261408A CN 115261408 A CN115261408 A CN 115261408A CN 202210967527 A CN202210967527 A CN 202210967527A CN 115261408 A CN115261408 A CN 115261408A
- Authority
- CN
- China
- Prior art keywords
- rna polymerase
- fbl
- screening
- small molecule
- egfp
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 102000017143 RNA Polymerase I Human genes 0.000 title claims abstract description 44
- 108010013845 RNA Polymerase I Proteins 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000012216 screening Methods 0.000 title claims abstract description 29
- -1 small molecule compound Chemical class 0.000 title claims abstract description 19
- 230000005764 inhibitory process Effects 0.000 title claims abstract description 13
- 150000003384 small molecules Chemical class 0.000 claims abstract description 25
- 239000003112 inhibitor Substances 0.000 claims abstract description 19
- 239000013612 plasmid Substances 0.000 claims abstract description 17
- 238000013518 transcription Methods 0.000 claims abstract description 14
- 230000035897 transcription Effects 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 11
- 229940044683 chemotherapy drug Drugs 0.000 claims abstract description 9
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 7
- 238000003753 real-time PCR Methods 0.000 claims abstract description 7
- 102000007999 Nuclear Proteins Human genes 0.000 claims abstract description 6
- 108010089610 Nuclear Proteins Proteins 0.000 claims abstract description 6
- 230000001093 anti-cancer Effects 0.000 claims abstract description 6
- 101150040063 orf gene Proteins 0.000 claims abstract description 6
- 238000001890 transfection Methods 0.000 claims abstract description 6
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims abstract description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims abstract description 4
- 108020004418 ribosomal RNA Proteins 0.000 claims abstract description 4
- 238000012163 sequencing technique Methods 0.000 claims abstract description 4
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000010367 cloning Methods 0.000 claims abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 17
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 6
- 238000001638 lipofection Methods 0.000 claims description 5
- 239000012097 Lipofectamine 2000 Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 101150112014 Gapdh gene Proteins 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 3
- 239000001569 carbon dioxide Substances 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 238000009826 distribution Methods 0.000 claims description 3
- BXYDVWIAGDJBEC-UHFFFAOYSA-N n-[2-(dimethylamino)ethyl]-12-oxo-12h-benzo[g]pyrido[2,1-b]quinazoline-4-carboxamide Chemical compound C1=CC=C2C=C(C(N3C=CC=C(C3=N3)C(=O)NCCN(C)C)=O)C3=CC2=C1 BXYDVWIAGDJBEC-UHFFFAOYSA-N 0.000 claims description 3
- 239000013641 positive control Substances 0.000 claims description 3
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 2
- 230000001464 adherent effect Effects 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 239000013642 negative control Substances 0.000 claims description 2
- 239000012096 transfection reagent Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 6
- 230000008685 targeting Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 230000004907 flux Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 31
- 206010028980 Neoplasm Diseases 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- 102100029526 rRNA 2'-O-methyltransferase fibrillarin Human genes 0.000 description 5
- 230000008439 repair process Effects 0.000 description 5
- 239000000835 fiber Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 230000005782 double-strand break Effects 0.000 description 3
- 238000013537 high throughput screening Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000005971 DNA damage repair Effects 0.000 description 2
- 101150001802 FBL gene Proteins 0.000 description 2
- 102000009572 RNA Polymerase II Human genes 0.000 description 2
- 108010009460 RNA Polymerase II Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100034533 Histone H2AX Human genes 0.000 description 1
- 101001067891 Homo sapiens Histone H2AX Proteins 0.000 description 1
- 101000581326 Homo sapiens Mediator of DNA damage checkpoint protein 1 Proteins 0.000 description 1
- 102100027643 Mediator of DNA damage checkpoint protein 1 Human genes 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000012361 double-strand break repair Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 101710139639 rRNA methyltransferase Proteins 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000004708 ribosome subunit Anatomy 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000003041 virtual screening Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a screening method of a small molecular compound for targeted inhibition of RNA polymerase I, which sequentially comprises the following steps: a) Cloning FBL full-length ORF gene; b) Connecting the FBL full-length ORF gene with a pEGFP-C1 vector to obtain a recombinant plasmid; c) Sequencing and verifying the recombinant plasmid to obtain a correct plasmid; d) Right plasmid transfection HeLa cells; e) Screening a HeLa cell capable of stably expressing EGFP-FBL protein; f) Judging whether the EGFP-FBL is aggregated, if not, switching to the step g, and if so, switching to the step h; g) Prompting that the small molecules do not cause nucleolar stress, and are not considered, and then ending; h) Verifying whether the 45S pre-rRNA level is reduced and GAPDH mRNA transcription is not influenced, if so, transferring to the step i, otherwise, ending; i) Small molecule compounds that effectively inhibit RNA polymerase I, denoted Pol Ii, were selected and then terminated. The invention has simple operation, high flux screening, high detection sensitivity and strong specificity of the fluorescence quantitative PCR method, and the screened RNA polymerase I small molecular inhibitor is expected to be further developed into a novel anti-cancer chemotherapeutic drug targeting RNA polymerase I.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to a screening method and application of a small molecular compound for targeted inhibition of RNA polymerase I, and belongs to the technical field of molecular cell biology and biochemistry.
[ background of the invention ]
Cancer has been a major problem in the life and health field, and seriously threatens human health. For patients with advanced cancer, chemotherapy is relied upon to maintain survival because most distal organs are inoperable due to tumor invasion. To date, over 250 anticancer drugs have been used in clinical cancer chemotherapy. However, the most common first-line anticancer chemotherapeutic drugs inhibit genomic DNA metabolism or mitosis of the cells, but do not specifically eliminate tumor cells. Therefore, long-term use of these chemotherapeutic drugs can kill normal cells, causing serious side effects, as well as causing drug resistance of tumor cells, resulting in death of patients with advanced cancer. The method for developing and screening the novel anti-cancer drug targeting the tumor has important research and development values.
RNA polymerase I is a potential tumor therapy target. On the one hand, tumor cells can undergo unregulated expansion due to cell cycle disorders, and the massive proliferation of tumor cells must rely on abundant ribosomes to synthesize proteins necessary for proliferation. The synthesis and assembly of ribosomes is dependent on rRNA transcribed by RNA polymerase I, and in almost all tumors, the expression level of RNA polymerase I is much higher than in normal tissues. Therefore, the function of RNA polymerase I is inhibited, the rRNA transcription level is reduced, and the cell nucleus carbohydrate assembly can be blocked, so that proteins required for mass proliferation of tumor cells cannot be provided to play a role in tumor inhibition. On the other hand, since many cancer cells are deficient in DNA damage repair, these cancer cells are abnormally sensitive to the blockade of specific repair pathways. Blocking these pathways can cause death of cancer cells that are deficient in DNA damage repair. It has been proved by research that, in the process of repairing DNA double-strand break, precursor rRNA participates in the recruitment of damage repair factors such as MDC1, γ H2AX, BRCA1, etc., and inhibition of rRNA transcription can block foci formation in the process of DNA double-strand break, so that the repair factors cannot be loaded to the damaged site, thereby blocking repair, and finally leading to unstable cell genome and apoptosis. Screening small molecule inhibitor of RNA polymerase I, inhibiting rDNA transcription, thereby blocking repair of double-strand break of tumor cells and finally inducing death of tumor cells. Therefore, the small-molecule chemotherapeutic drug is used for inhibiting the transcription activity of RNA polymerase I, on one hand, the ribosome biosynthesis can be quickly inhibited, on the other hand, the double-strand break repair pathway of cells is blocked, and the small-molecule chemotherapeutic drug is a novel target with great potential in the aspect of tumor treatment. Therefore, the development of a sensitive and high-flux screening method for the RNA polymerase I inhibitor has important research value.
rRNA is transcribed by rDNA as a template under the action of RNA polymerase I, and the whole process occurs in nucleolus. The nucleolus is located in the nucleus and is usually a single or multiple homogeneous spherical bodies with a round or oval granular structure, without an envelope. The kernel has a three-layer structure and comprises a fiber center, a compact fiber component and a particle component from inside to outside. The fiber center is surrounded by dense fiber components, the main component being rDNA. The dense fiber component consists of dense fibers, which are the site for newly synthesized rRNA and its binding proteins. The particle component consists of ribonucleoprotein particles, which are precursor particles of the mature ribosomal subunits being processed. Processing and maturation of rRNA is dependent on FBL action by rRNA methyltransferase, which is normally distributed evenly in the nucleolus. When RNA polymerase I is abnormal, rDNA transcription is inhibited, and nucleolus is stressed, so that FBL forms 1-3 aggregates with different numbers in nucleolus and transfers to nucleolus edge. According to the invention, heLa cells which can stably express FBL protein with EGFP fluorescent signals are screened out by utilizing the change of the location of FBL in nucleolus when nucleolus is stressed. When the cells are treated by the small molecule inhibitor, the positioning of EGFP fluorescent signals is observed under a fluorescent microscope, so that whether the small molecule can specifically inhibit the activity of RNA polymerase I or not is judged, and whether the rRNA transcription level is inhibited or not is verified by a real-time fluorescent quantitative PCR method.
The traditional screening method of RNA polymerase I small molecule inhibitors is to use recombinant RNA polymerase I in vitro, generate rDNA from P32 labeled ribonucleotide and then perform agarose gel electrophoresis, and evaluate the activity of RNA polymerase I by detecting the radioactive intensity of P32. The method has the following difficulties in the actual operation process: 1. it is difficult to obtain RNA polymerase I protein with good purity and activity; p32 has radioactivity, cannot be operated under general experimental conditions, and has high requirements on laboratory level and operators; 3. the method can not meet the requirement of high-throughput screening, and has complex operation and longer test period.
[ contents of the invention ]
The invention aims to solve the problems in the prior art and provides a method for screening a small molecular compound for targeted inhibition of RNA polymerase I, which does not need to prepare RNA polymerase I protein, does not need to use a P32 radioactive material, and has low operation requirements, low requirements on laboratory conditions and low screening cost.
In order to achieve the purpose, the invention provides a screening method of a small molecule compound for targeted inhibition of RNA polymerase I, which sequentially comprises the following steps: a) Cloning FBL full-length ORF gene; b) Connecting the FBL full-length ORF gene with a pEGFP-C1 vector to obtain a recombinant plasmid; c) Sequencing and verifying the recombinant plasmid to obtain a correct plasmid; d) Right plasmid transfection HeLa cells; e) Screening a HeLa cell capable of stably expressing EGFP-FBL protein; f) Judging whether the EGFP-FBL is aggregated, if not, switching to the step g, and if so, switching to the step h; g) Prompting that the small molecules do not cause nucleolar stress, and are not considered, and then ending; h) Verifying whether the 45S pre-rRNA level is reduced and GAPDH mRNA transcription is not influenced, if so, transferring to the step i, otherwise, ending; i) Small molecule compounds that effectively inhibit RNA polymerase I, denoted Pol Ii, were selected and then terminated.
Preferably, the right plasmid is transfected into the HeLa cell by using a liposome transfection reagent in the step d).
Preferably, lipofectamine 2000 is used as the lipofection reagent in the step d).
Preferably, the step e) is performed 24 hours after transfection, and then the HeLa cells capable of stably expressing the EGFP-FBL protein are obtained by using 0.5mg/ml G418 for screening.
Preferably, the HeLa cells stably expressing the EGFP-FBL protein are prepared into 10 in the step f) 4 Adding 100 mu L/ml of cell suspension into a flat-bottom transparent 96-well cell culture plate, placing the culture plate in a 37 ℃ and 5% carbon dioxide incubator for overnight culture until cells grow completely adherent, preparing 160 small-molecule compound solutions by using DMSO (dimethyl sulfoxide), adding a small-molecule compound into each well of cells, treating the cells for 2 hours, and then placing the cells under a fluorescence microscope to observe the distribution of EGFP-FBL fluorescence signals, wherein the EGFP-FBL fluorescence signals are dispersed and uniformly distributed in HeLa nucleolus cells by using 0.1% DMSO as a negative control; with 1 μ M BMH21 treatment as a positive control, EGFP-FBL fluorescence signals aggregated into 1 or more high bright spot signals, distributed at the nucleolar border.
Preferably, the step h) adopts real-time fluorescent quantitative PCR analysis to verify whether the level of 45Spre-rRNA of the cells is remarkably reduced, and the mRNA transcription level of the GAPDH gene does not influence.
In order to achieve the purpose, the invention also provides the application of the RNA polymerase I small molecule inhibitor, and the RNA polymerase I small molecule inhibitor obtained by the method is used for anti-cancer chemotherapeutic drugs.
The invention has the beneficial effects that: compared with the traditional screening method, the invention screens RNA polymerase I small molecule inhibitors by using HeLa cells stably expressing EGFP-FBL protein, screens out small molecules which specifically inhibit the activity of RNA polymerase I, and has no influence on the activity of RNA polymerase II. The method has the advantages of simple operation, high throughput screening, high detection sensitivity and strong specificity of the fluorescent quantitative PCR method. And screening is carried out at the cellular level, so that the activity of the small molecules can be better reflected, meanwhile, the cost of high-throughput screening can be greatly reduced, and the screened RNA polymerase I small molecule inhibitor is expected to be further developed into a novel anti-cancer chemotherapeutic drug targeting RNA polymerase I.
The features and advantages of the present invention will be described in detail by the embodiments with reference to the accompanying drawings.
[ description of the drawings ]
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is a diagram of the EGFP-FBL nuclear localization unchanged;
FIG. 3 is a diagram of EGFP-FBL signal aggregation;
FIG. 4 is a graph of the results of two screening Pol I small molecule inhibitors of the present invention on the EGFP-FBL localization;
FIG. 5 is a graphical representation of the results of testing the effect of two small molecule inhibitors of Pol I on transcription of 45S rRNA, screened in the examples of the invention.
[ detailed description ] embodiments
Referring to FIG. 1, the full-length ORF sequence of the human FBL gene (the full-length ORF sequence of the human FBL gene is referred to NM-001436.4 sequence in NCBI database) was cloned by PCR technique and pEGFP-C1 vector was ligated and the correct plasmid was obtained by sequencing verification. HeLa cells were transfected with the correct plasmid using Lipofectamine 2000 as a lipofection reagent, and 24 hours after transfection, selection was performed using 0.5mg/ml G418, and HeLa cells stably expressing EGFP-FBL protein were obtained. Preparing 10 HeLa cells capable of stably expressing EGFP-FBL protein 4 Cell suspension per ml, 100. Mu.L/well suspension was added to flat-bottom, clear 96-well cell culture plates. The culture plate is placed in an incubator at 37 ℃ and 5% carbon dioxide for culture overnight until the cells are fully attached to the wall and grow. 160 small molecule compounds (160 RNA polymerase I small molecule compounds obtained after virtual screening of ChemDiv compound library) solutions are prepared by DMSO, one small molecule compound is added into each well, the compound concentration is 20 mu M, and the DMSO concentration is not more than 0.1%. Cells were treated for 2 hours and then placed under a fluorescence microscope to observe the distribution of EGFP-FBL fluorescence signals. 0.1% DMSO treatment as a negative pairAccording to the formula, EGFP-FBL fluorescent signals are dispersed and uniformly distributed in HeLa nucleolus, as shown in figure 2; with 1 μ M BMH21 treatment as a positive control, EGFP-FBL fluorescence signals aggregated into 1 or more highlight dots, distributed at the nucleolar border as shown in fig. 3.
After treatment and observation, 2 small molecules capable of obviously changing the EGFP-FBL fluorescence signal positioning are obtained by co-screening: pol Ii-1 and Pol Ii-2 are shown in FIG. 4, which are the results of testing the influence of 2 screened Pol I small molecule inhibitors on the EGFP-FBL localization, and the right structural formula represents the molecular structural formulas of the 2 Pol I small molecule inhibitors respectively. After pol Ii-1 and pol Ii-2 treatment, the EGFP-FBL fluorescence signal is gathered into 1 or more high-light spot signals and distributed at the edge of nucleolus, which indicates that pol Ii-1 and pol Ii-2 treatment can induce nucleolus stress of cells, and the two compounds are possibly RNA polymerase inhibitors. Further real-time fluorescent quantitative PCR analysis shows that after Pol Ii-1 and Pol Ii-2 are treated, the level of 45Spre-rRNA of the cell is obviously reduced, the mRNA transcription level of GAPDH gene is not influenced, as shown in figure 5, 2 Pol I small molecule inhibitors can obviously inhibit the transcription of 45S rRNA and have no influence on the transcription of GAPDH mRNA, which indicates that 2 small molecule inhibitors obtained by screening can inhibit the activity of RNA polymerase I and have no influence on the activity of RNA polymerase II. The pol Ii-1 and pol Ii-2 obtained by screening by the method are suggested to be specific small molecule inhibitors of RNA polymerase I, and are expected to be further developed into novel anti-cancer chemotherapeutic drugs targeting RNA polymerase I.
The present embodiment relates to the instrument list:
this example relates to a list of cells, reagents:
| name (R) | Manufacturer of the product |
| Human cervical cancer cell line HeLa | American ATCC |
| DMEM cell culture medium | Gibco Inc. of USA |
| Opti-MEM cell culture medium | Gibco Inc. of USA |
| Fetal bovine serum | Gibco Inc. of USA |
| pEGFP-C1 plasmid | Addgene |
| Lipofectamine 2000 | Thermo Fisher Scientific |
| G418 | Sigma |
The present example relates to real-time fluorescent quantitative PCR primers:
| GAPDH-S | GTCTCCTCTGACTTCAACAGCG | |
| GAPDH- | ACCACCCTGTTGCTGTAGCCAA | |
| 45S rRNA- | AAGGAAGGAGGTGGGTGGA | |
| 45S rRNA-AS | CAGATCGCTAGAGAAGGCT |
the above embodiments are illustrative of the invention, and are not intended to limit the invention, and any simple modifications of the invention fall within the scope of the invention.
Claims (7)
1. A method for screening a small molecule compound for targeted inhibition of RNA polymerase I, which is characterized by comprising the following steps: the method sequentially comprises the following steps: a) Cloning FBL full-length ORF gene; b) Connecting the FBL full-length ORF gene with a pEGFP-C1 vector to obtain a recombinant plasmid; c) Sequencing and verifying the recombinant plasmid to obtain a correct plasmid; d) Right plasmid transfection HeLa cells; e) Screening a HeLa cell capable of stably expressing EGFP-FBL protein; f) Judging whether the EGFP-FBL is aggregated, if not, switching to the step g, and if so, switching to the step h; g) Prompting that the small molecules do not cause nucleolar stress, and are not considered, and then ending; h) Verifying whether the 45S pre-rRNA level is reduced and GAPDH mRNA transcription is not influenced, if so, transferring to the step i, otherwise, ending; i) Small molecule compounds that effectively inhibit RNA polymerase I, denoted Pol Ii, were selected and then terminated.
2. The method for screening a small molecule compound for targeted inhibition of RNA polymerase I as claimed in claim 1, wherein: in the step d), a proper plasmid is transfected into the HeLa cell by using a liposome transfection reagent.
3. The method for screening a small molecule compound for targeted inhibition of RNA polymerase I as claimed in claim 2, wherein: the lipofection reagent in the step d) adopts Lipofectamine 2000.
4. The method for screening a small molecule compound for targeted inhibition of RNA polymerase I as claimed in claim 1, wherein: and e) after 24 hours of transfection, screening by using 0.5mg/ml G418 to obtain HeLa cells capable of stably expressing EGFP-FBL protein.
5. The method for screening a small molecule compound for targeted inhibition of RNA polymerase I as claimed in claim 1, wherein: in the step f), heLa cells capable of stably expressing EGFP-FBL protein are prepared into 10 4 Adding 100 mu L/ml of cell suspension into a flat-bottom transparent 96-well cell culture plate, placing the culture plate in a 37 ℃ and 5% carbon dioxide incubator for overnight culture until cells grow completely adherent, preparing 160 small-molecule compound solutions by using DMSO (dimethyl sulfoxide), adding a small-molecule compound into each well of cells, treating the cells for 2 hours, and then placing the cells under a fluorescence microscope to observe the distribution of EGFP-FBL fluorescence signals, wherein the EGFP-FBL fluorescence signals are dispersed and uniformly distributed in HeLa nucleolus cells by using 0.1% DMSO as a negative control; with 1 μ M BMH21 treatment as a positive control, EGFP-FBL fluorescence signals aggregated into 1 or more high bright spot signals, distributed at the nucleolar border.
6. The method for screening a small molecule compound which can be targeted to inhibit RNA polymerase I according to any one of claims 1 to 5, wherein: and h) adopting real-time fluorescent quantitative PCR analysis in the step to verify whether the level of 45S pre-rRNA of the cell is obviously reduced or not, and the mRNA transcription level of the GAPDH gene is not influenced.
7. The application of a small molecule inhibitor of RNA polymerase I is characterized in that: the RNA polymerase I small molecule inhibitor obtained in the claims 1-6 is used for the development of anti-cancer chemotherapeutic drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210967527.6A CN115261408A (en) | 2022-08-12 | 2022-08-12 | Screening method and application of small molecule compound for targeted inhibition of RNA polymerase I |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210967527.6A CN115261408A (en) | 2022-08-12 | 2022-08-12 | Screening method and application of small molecule compound for targeted inhibition of RNA polymerase I |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115261408A true CN115261408A (en) | 2022-11-01 |
Family
ID=83750727
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210967527.6A Pending CN115261408A (en) | 2022-08-12 | 2022-08-12 | Screening method and application of small molecule compound for targeted inhibition of RNA polymerase I |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115261408A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017191187A2 (en) * | 2016-05-03 | 2017-11-09 | Université Libre de Bruxelles | Nucleolar structure evaluation and manipulation |
| CN110128477A (en) * | 2018-02-09 | 2019-08-16 | 天津医科大学 | Based on kernel stress platinum-like compounds |
| CN114796226A (en) * | 2021-12-17 | 2022-07-29 | 新乡医学院 | Application of olaparib in induction of nucleolar stress |
-
2022
- 2022-08-12 CN CN202210967527.6A patent/CN115261408A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017191187A2 (en) * | 2016-05-03 | 2017-11-09 | Université Libre de Bruxelles | Nucleolar structure evaluation and manipulation |
| CN110128477A (en) * | 2018-02-09 | 2019-08-16 | 天津医科大学 | Based on kernel stress platinum-like compounds |
| CN114796226A (en) * | 2021-12-17 | 2022-07-29 | 新乡医学院 | Application of olaparib in induction of nucleolar stress |
Non-Patent Citations (2)
| Title |
|---|
| DANYANG CHEN等: "Nucleolar Components Involved in Ribosome Biogenesis Cycle between the Nucleolus and Nucleoplasm in Interphase Cells" * |
| KASPAR BURGER等: "Chemotherapeutic drugs inhibit ribosome biogenesis at various levels" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8809296B2 (en) | Apoptosis inducer for cancer cell | |
| CN108359692B (en) | Luciferase report system of specific targeting hDGK theta gene | |
| Sokolov et al. | Changes in gene expression as one of the key mechanisms involved in radiation‑induced bystander effect | |
| WO2020181663A1 (en) | Long-chain non-coding rna and use thereof | |
| Zhang et al. | A novel lncRNA, lnc403, involved in bovine skeletal muscle myogenesis by mediating KRAS/Myf6 | |
| Lv et al. | LncRNA PVT1 aggravates the progression of glioma via downregulating UPF1. | |
| Liu et al. | Targeting of CDKN 1B by miR‐222‐3p may contribute to the development of intervertebral disc degeneration | |
| Cui et al. | HOXA10 promotes the development of bladder cancer through regulating FOSL1. | |
| Wu et al. | mTORC1-Rps15 axis contributes to the mechanisms underlying global translation reduction during senescence of mouse embryonic fibroblasts | |
| Das et al. | The importance of RNA modifications: From cells to muscle physiology | |
| Xie et al. | Influence of UGT1A1 gene methylation level in colorectal cancer cells on the sensitivity of the chemotherapy drug CPT-11 | |
| CN108034655B (en) | Application of long non-coding RNA and composition thereof in diagnosis/treatment of colorectal cancer | |
| CN115261408A (en) | Screening method and application of small molecule compound for targeted inhibition of RNA polymerase I | |
| JP2022514573A (en) | Na + / K + ATPase inhibitor for use in the prevention or treatment of metastases | |
| CN112143790B (en) | Method for assessing risk of Down syndrome based on m6A methylation modification of NRIP1 mRNA and application thereof | |
| Aguado et al. | Exploring aging interventions in human brain organoids | |
| Fang et al. | MIIP inhibits malignant progression of hepatocellular carcinoma through regulating AKT. | |
| Vaidya et al. | Exposure to a pathological condition may be required for the cells to secrete exosomes containing mtDNA aberration | |
| CN114517204A (en) | circPOLK for tumor treatment target and diagnosis biomarker and application thereof | |
| CN114934048A (en) | circMTMR14 for tumor treatment target and diagnosis biomarker, kit and application | |
| EP3699301A1 (en) | Novel recql4 muteins and personalized treatment of glioblastoma patients | |
| Zhou et al. | RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma | |
| CN113151260B (en) | tRF022252 for inhibiting tumor metastasis and application thereof | |
| Li et al. | Mechanistic insights on non-coding RNAs in learning and memory | |
| Villaverde et al. | TMET-26. Primary oligodendroglioma cell culture viability: an in vitro study with metabolic modulators |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20221101 |
|
| RJ01 | Rejection of invention patent application after publication |