CN116445573A - Antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as target point - Google Patents
Antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as target point Download PDFInfo
- Publication number
- CN116445573A CN116445573A CN202211060667.1A CN202211060667A CN116445573A CN 116445573 A CN116445573 A CN 116445573A CN 202211060667 A CN202211060667 A CN 202211060667A CN 116445573 A CN116445573 A CN 116445573A
- Authority
- CN
- China
- Prior art keywords
- trim54
- stat2
- plasmid
- ade
- yfp
- 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
- 108010081691 STAT2 Transcription Factor Proteins 0.000 title claims abstract description 64
- 102000004265 STAT2 Transcription Factor Human genes 0.000 title claims abstract description 64
- 101000795328 Homo sapiens Tripartite motif-containing protein 54 Proteins 0.000 title claims abstract description 57
- 102100029709 Tripartite motif-containing protein 54 Human genes 0.000 title claims abstract description 57
- 230000005764 inhibitory process Effects 0.000 title claims abstract description 29
- 230000000840 anti-viral effect Effects 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000015556 catabolic process Effects 0.000 title claims abstract description 14
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 14
- 238000007877 drug screening Methods 0.000 title claims abstract description 14
- 230000034512 ubiquitination Effects 0.000 title claims abstract description 8
- 238000010798 ubiquitination Methods 0.000 title claims abstract description 8
- 230000000694 effects Effects 0.000 claims abstract description 42
- 239000003814 drug Substances 0.000 claims abstract description 25
- 229940079593 drug Drugs 0.000 claims abstract description 19
- 238000012216 screening Methods 0.000 claims abstract description 16
- 241000700605 Viruses Species 0.000 claims abstract description 15
- KGFYHTZWPPHNLQ-AWEZNQCLSA-N rivaroxaban Chemical compound S1C(Cl)=CC=C1C(=O)NC[C@@H]1OC(=O)N(C=2C=CC(=CC=2)N2C(COCC2)=O)C1 KGFYHTZWPPHNLQ-AWEZNQCLSA-N 0.000 claims abstract description 7
- 229960001148 rivaroxaban Drugs 0.000 claims abstract description 7
- XUZMWHLSFXCVMG-UHFFFAOYSA-N baricitinib Chemical compound C1N(S(=O)(=O)CC)CC1(CC#N)N1N=CC(C=2C=3C=CNC=3N=CN=2)=C1 XUZMWHLSFXCVMG-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229950000971 baricitinib Drugs 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims description 57
- 239000013612 plasmid Substances 0.000 claims description 46
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims description 34
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 108091006047 fluorescent proteins Proteins 0.000 claims description 32
- 102000034287 fluorescent proteins Human genes 0.000 claims description 32
- 230000003993 interaction Effects 0.000 claims description 32
- 230000002401 inhibitory effect Effects 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 238000000684 flow cytometry Methods 0.000 claims description 8
- 239000013641 positive control Substances 0.000 claims description 8
- 239000013642 negative control Substances 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 73
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 34
- 102100038567 Properdin Human genes 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- 230000014509 gene expression Effects 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 9
- 102000011408 Tripartite Motif Proteins Human genes 0.000 description 8
- -1 but in resting cells Proteins 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 108010023649 Tripartite Motif Proteins Proteins 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 210000001616 monocyte Anatomy 0.000 description 6
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 238000000749 co-immunoprecipitation Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- 206010012310 Dengue fever Diseases 0.000 description 3
- 241000725619 Dengue virus Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 208000025729 dengue disease Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 208000001490 Dengue Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101150037314 STAT2 gene Proteins 0.000 description 2
- 102000001712 STAT5 Transcription Factor Human genes 0.000 description 2
- 108010029477 STAT5 Transcription Factor Proteins 0.000 description 2
- 108091053398 TRIM/RBCC family Proteins 0.000 description 2
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 2
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 2
- 241000907316 Zika virus Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 229940126586 small molecule drug Drugs 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 241001678559 COVID-19 virus Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 108010021472 Fc gamma receptor IIB Proteins 0.000 description 1
- 208000004576 Flaviviridae Infections Diseases 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 102100029205 Low affinity immunoglobulin gamma Fc region receptor II-b Human genes 0.000 description 1
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 description 1
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 108010044012 STAT1 Transcription Factor Proteins 0.000 description 1
- 102000006381 STAT1 Transcription Factor Human genes 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 108010019992 STAT4 Transcription Factor Proteins 0.000 description 1
- 102000005886 STAT4 Transcription Factor Human genes 0.000 description 1
- 108010011005 STAT6 Transcription Factor Proteins 0.000 description 1
- 102000013968 STAT6 Transcription Factor Human genes 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- 102000042631 TRIM/RBCC family Human genes 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000000887 Transcription factor STAT Human genes 0.000 description 1
- 108050007918 Transcription factor STAT Proteins 0.000 description 1
- 101150043993 Trim54 gene Proteins 0.000 description 1
- 208000020329 Zika virus infectious disease Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 108091006088 activator proteins Proteins 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 108010082025 cyan fluorescent protein Proteins 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to an antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as a target spot, and a series of antiviral ADE drugs are screened out based on the screening system: baricitinib, rivaroxaban, and the like, expands a new application field for the medicaments, provides candidate medicaments for the blank of no ADE treatment scheme at present, and has important biological significance for preventing and treating viruses with ADE effect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to an antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as a target spot.
Background
Antibody-dependent enhancement (ADE-Dependent Enhancement) refers to the phenomenon that antibodies with weak or no neutralizing ability after vaccination or infection with viruses form complexes with viral particles that not only do not block but rather promote viral infection of cells and increase viral replication by disrupting interferon signaling pathways. Halstead et al scientist in 1973 first described the ADE phenomenon in dengue infection, and later found that ADE phenomenon was widely present in many flaviviridae infections, e.g., encephalitis b virus, zika virus and dengue virus all had the potential to cause ADE. ADE effects are also an important challenge to be faced in the control of SARS-CoV-2. At present, no specific drug for treating ADE is marketed, and ADE has become a major disturbance in the prevention and treatment of viral infection. Therefore, the mechanism of ADE was explored to find that targets that inhibit ADE are of great importance for the control of viral infectious diseases.
Signal transduction and transcription activator protein (Signal Transducers and Activators of Transcription, STAT) is a unique family of proteins capable of binding DNA, and the STAT family includes 7 structurally and functionally related proteins STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, which generally respond to various extracellular cytokines and growth factor signals and are a class of SH2 signaling molecules that contain proteins capable of binding phosphorylated tyrosine. STAT is an intranuclear transcription factor, but in resting cells, STAT is present in the cytoplasm. Wherein, STAT2 molecules are transferred into the nucleus to combine with specific DNA after being activated, thereby influencing gene transcription and participating in the growth, differentiation, survival and apoptosis of cells. At present, a plurality of researches show that STAT2 gene deletion or overexpression has important influence on tumorigenesis and development, and is closely related to tumor angiogenesis, tumor proliferation and tumor apoptosis.
The name of the Tripartite motif (TRIM) family is due to its 3 conserved domains. The three domains are in order from N-terminus to C-terminus, RING domain, 1 or 2B-box domains, a Coiled-coil domain, and the family has a variable C-terminus. Currently, the human genome has found and identified nearly 70 TRIM protein members, and the functions of its family members are becoming more and more important. Research shows that members of the TRIM family are involved in many important biological processes such as cell differentiation, proliferation, development, apoptosis, etc. Most TRIM has E3 ubiquitin ligase activity and is involved in a variety of physiological processes including cell proliferation, DNA repair, signal transduction and transcription, and in recent years extensive research has found that TRIM is associated with the development of cancer.
However, there is no report on the interaction of STAT2 with TRIM54 as a target for blocking ADE effect.
Disclosure of Invention
In order to solve the technical problems, the invention provides an antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as a target point, which comprises the steps of respectively fusing and expressing TRIM54 and STAT2 with a first fluorescent protein (FP 1) and a second fluorescent protein (FP 2) to form FP1-TRIM54 and FP2-STAT2, detecting the intensity of fluorescence resonance transfer (FRET) by a flow cytometer or an enzyme-labeled instrument after transfecting cells to evaluate the interaction condition of the TRIM54 and STAT2, and screening out compounds for specifically inhibiting ADE
The first object of the invention is to provide an antiviral ADE drug screening method with TRIM54 ubiquitination degradation STAT2 inhibition as a target, comprising the following steps:
s1, respectively constructing a first plasmid and a second plasmid, introducing the first plasmid and the second plasmid into cells, and detecting FRET signals; the first plasmid and the second plasmid respectively contain a gene for encoding TRIM54 and a gene for encoding STAT2, the first plasmid and the second plasmid respectively contain a gene for encoding a first fluorescent protein and a gene for encoding a second fluorescent protein, and the first fluorescent protein and the second fluorescent protein can generate fluorescence resonance energy transfer;
s2, incubating the compound to be detected with the cells in the step S1, and screening out the drug for inhibiting the ADE effect of the virus according to the change of the FRET signal.
Further, the nucleotide sequence of the gene encoding TRIM54 is shown as SEQ ID NO. 1.
Further, the nucleotide sequence of the gene for encoding STAT2 is shown as SEQ ID NO. 2.
Further, test compounds with reduced FRET signals are screened as candidate antiviral ADE effect drugs.
Further, in one embodiment of the present invention, the first fluorescent protein is a cyan fluorescent protein CFP, and the second fluorescent protein is a yellow fluorescent protein YFP.
Further, when drug screening is performed, cells into which a third plasmid containing a gene encoding the first fluorescent protein and a gene encoding the second fluorescent protein are introduced are used as positive controls.
Further, when drug screening is performed, cells into which a fourth plasmid and a fifth plasmid, each of which contains a gene encoding a first fluorescent protein and a gene encoding a second fluorescent protein, are introduced are used as negative controls.
Further, the FRET signal is detected by flow cytometry or fluoroenzyme labeling.
Further, in one embodiment of the invention, the cell is a 293T cell.
The invention provides a new target for inhibiting virus ADE effect, wherein the target is TRIM54 and STAT2:
human monocytes-macrophages mainly express three classes of activated fcγrs: fcγria, fcγriia and fcγriiia. Although there is evidence that fcyriiia can be involved in mediating dengue virus ADE, there is more evidence that fcyriia, but not fcyria and fcyriiia, are key receptors for ADE. For example, studies performed in Vietnam, guba, pakistan, and Mexico, respectively, indicate that the FcgammaRIIa polymorphism is closely related to the prognosis of dengue virus infection. Recent evidence from multiple laboratories further suggests that fcγriia is involved in mediating ADE of dengue, zika and ebola virus. Antibodies exist in forms that may affect the ADE effect, such as fucose modification of an antibody affecting its affinity for FcR, antibodies in dimeric form induce the ADE effect more readily than monomers, etc. In addition, the ITAM motif in the cytoplasmic domain of fcγriia is a key domain that mediates the ADE effect of the virus, while the ITIM motif in fcγriib plays a protective role.
The applicant found in the study that acting of the immune complex on human monocytes through fcyriia on the membrane surface enhances their susceptibility to viruses during which time TRIM54 expression is significantly increased and which can inhibit IFN-I signaling by degrading STAT2, resulting in increased susceptibility of monocytes to viruses. Interference with TRIM54 expression can significantly reverse ADE effects, so it is thought that the interaction of TRIM54 and STAT2 may be a link to immune complex-induced enhancement of monocyte sensitivity to viruses suggesting that it may act as a target for blocking ADE effects. Because TRIM54 is non-constitutively expressed, and only expressed in mononuclear macrophages acted by immune complexes, the interaction of TRIM54 and STAT2 is specifically interfered without causing the wide influence of cells on IFN-I related signal paths, and the specificity is higher. Therefore, small molecule compounds which target TRIM54/STAT2 interaction and inhibit ADE effect are screened, and the method has important biological significance for preventing and treating viruses with the ADE effect.
The second object of the invention is to provide an application of a substance inhibiting the interaction between TRIM54 and STAT2 in preparing antiviral ADE effect medicines.
A third object of the invention is to provide the use of a substance characterising the interaction of TRIM54 and STAT2 in antiviral ADE effect drug screening.
The fourth object of the invention is to provide the application of Baricitinib or Rivaroxaban in preparing the drug for inhibiting the virus ADE effect.
Further, the Baricitinib or Rivaroxaban is used to block TRIM54 and STAT2 interactions.
It is a fifth object of the present invention to provide an antiviral ADE effect drug, including bacitracinib or Rivaroxaban.
By means of the scheme, the invention has at least the following advantages:
the invention discloses a new target for inhibiting virus ADE effect and provides an application of treating ADE by taking a substrate STAT2 for inhibiting E3 ubiquitin ligase TRIM54 ubiquitination degradation as a target. According to the invention, the research objects TRIM54 and STAT2 are respectively fused with a first fluorescent protein (FP 1) and a second fluorescent protein (FP 2) to form FP1-TRIM54 and FP2-STAT2, and after cells are transfected, the interaction condition of the TRIM54 and STAT2 is evaluated by detecting the intensity of fluorescence resonance transfer (FRET) through a flow cytometer or an enzyme-labeled analyzer. The small molecular compound which specifically inhibits TRIM54/STAT2 interaction is obtained through large-scale screening of the platform. And further verifying the inhibiting effect of small molecules on TRIM54/STAT2 interaction by using molecular biology, finally verifying the inhibiting effect of compounds on ADE effect, and finally screening a series of small molecule compounds which target TRIM54/STAT2 interaction and inhibit ADE effect, thereby having important biological significance on the virus prevention and treatment with ADE effect.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.
FIG. 1 is the construction of eukaryotic plasmids; wherein a is a vector pCMV-N-CFP schematic diagram; b is the amplified product of TRIM54 gene (left), and the result of EcoRI and XhoI digestion of plasmid pCMV-N-CFP-TRIM 54; c is the amplified product of YFP gene (left), and plasmid pCMV-N-CFP-YFP is digested with EcoRI and XhoI; d is a vector pCMV-C-YFP schematic diagram; e is the amplification product of STAT2 gene (left), and plasmid pCMV-C-YFP-STAT2 is digested with BamH I and Hind III;
FIG. 2 shows the expression of CFP-TRIM54 and YFP-STAT2 in 293T cells; wherein a is the expression condition of YFP-STAT2 and CFP-TRIM54 by immunoblotting analysis; b is the fluorescence intensity of YFP-STAT2 and CFP-TRIM54 analyzed by a flow cytometer;
FIG. 3 is a flow cytometer analysis of TRIM54 and STAT2 binding efficiency: binding rate of CFP and YFP, binding rate of CFP-YFP, and binding rate of CFP-TRIM54 and YFP-STAT 2;
FIG. 4 shows the detection analysis of fluorescence resonance energy transfer intensity by a microplate reader: binding rate of CFP and YFP, aggregation rate of CFP-YFP, and binding rate of CFP-TRIM54 and YFP-STAT 2;
FIG. 5 shows the results of preliminary screening of small molecule compounds; wherein a is an analysis result of the enzyme label instrument; b is the analysis result of the flow cytometer;
FIG. 6 shows the result of rescreening small molecule compounds; wherein a is an analysis result of the enzyme label instrument; b is the analysis result of the flow cytometer;
FIG. 7 shows the results of inhibition of TRIM54 degradation of STAT2 by compounds; transfecting a plasmid encoding Flag-TRIM54 into 293T cells, adding a compound into an experimental group after 9 hours, adding DMSO (Med) with the same dilution into a control group, collecting cells after 24 hours, and then performing immunoblotting (Western blotting) to detect the protein level of STAT2 in a cell lysate;
FIG. 8 shows the results of inhibition of TRIM54 interaction with STAT2 by compounds; the plasmid encoding Flag-TRIM54 was transfected into 293T cells, cells were collected after 24h and cell lysates were incubated with Anti-Flag Affinity gel and compound (10. Mu.M) for 12h at4℃and control groups were added with the same dilution of DMSO (Med). Detecting the expression level of STAT2 in the immunoprecipitation complex by using an immunoblotting experiment (Western blotting);
FIG. 9 is an analysis of the effect of compounds on ADE; wherein a represents the detection by a flow cytometer after incubation for 72 hours with the addition of RPMI, 4G2, DENV2 or IC (DENV 2+4G 2) to K562 cells, respectively; b represents the addition of compound (10 μm) to K562 cells treated with DENV2 or IC, respectively, control group, incubation with DMSO (Med) at the same dilution for 72h, followed by detection by flow cytometry; c represents the addition of compound (1,3,10,30 μm) to IC-treated K562 cells for incubation for 72h and detection by flow cytometry;
FIG. 10 is a schematic diagram of the principle of fluorescence resonance energy transfer; wherein a is the coexpression of fluorescent proteins CFP and YFP in 293T cells; b is expression of fusion protein CFP-YFP in 293T cells; c is the co-expression of CFP-TRIM54 and YFP-STAT2 in 293T cells.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The invention aims to provide a drug screening platform based on a fluorescence resonance energy transfer technology and targeting TRIM54/STAT2 interaction. Fluorescence Resonance Energy Transfer (FRET) refers to the transfer of energy to an adjacent acceptor molecule by dipole interaction when two fluorescent chromophores are sufficiently close that the donor molecule absorbs a photon of a certain frequency and is excited to a higher electron energy state before the electron returns to the ground state. In short, when two fluorescent proteins are in close proximity, the emitted light emitted by one fluorescent protein under excitation of the excitation light may be used as the excitation light of the other fluorescent protein, which is excited to emit the emitted light. The schematic diagram of the fluorescence resonance energy transfer principle is shown in fig. 10. In the system of the invention, the group of fluorescent proteins CFP (ex: 405nm; em:440/50 nm) and YFP (ex: 48nm; em:530/30 nm) is selected. In the case where both fluorescent proteins are present but not in close proximity, the two fluorescent proteins can only be excited by the respective excitation light, respectively, and emit the respective emission light (fig. 10 a). However, when two fluorescent proteins are sufficiently close to each other by an external force, after excitation by the excitation light Violet (405 nm) of the CFP, not only the emission light VL1 (440/50 nm) of the CFP but also the emission light VL2 (620/15 nm) of the YFP can be excited. Thus, the present invention constructs a fusion protein of CFP and YFP (CFP-YFP) (FIG. 10 b) as a positive control for detecting FRET signals. Meanwhile, plasmids encoding CFP-TRIM54 and YFP-STAT2 were constructed, and the interaction efficiency of TRIM54 and STAT2 was evaluated by detecting the fluorescence intensity of intracellular FRET channels, respectively (FIG. 10 c).
In the experimental process, the compound to be screened is added into cells co-expressing CFP-TRIM54 and YFP-STAT2 to a final concentration of 10 mu M, and is detected by an enzyme-labeled instrument and a flow cytometer after 24 hours of drug action, and the inhibition effect of the drug on the interaction of TRIM54 and STAT2 is analyzed by the intensity of fluorescence resonance energy transfer.
The effect of the compound in reversing the degradation of STAT2 by TRIM54 was analyzed by immunoblotting (Western Blot, WB) by adding the compound to 293T cells overexpressing TRIM 54. The effect of the compounds in blocking TRIM54/STAT2 interaction was analyzed by Co-Immunoprecipitation (Co-IP) by adding the compounds to a cell lysate containing TRIM54 and STAT2. An immune complex formed by DENV2 and a monoclonal antibody 4G2 (specific monoclonal antibody of DENV 1E protein) is infected with K562 cells to construct an ADE model, and a compound is acted on the ADE model, and the inhibition effect of the compound on the ADE is analyzed by detecting the viral load in the K562 cells through a flow cytometer.
EXAMPLE 1 construction of plasmids encoding CFP-YFP, CFP-TRIM54 and YFP-STAT2
First, we performed PCR amplification of DNA fragments encoding TRIM54 (left in FIG. 1 b) and YFP (left in FIG. 1C) using human monocytic mRNA, pCMV-C-YFP plasmid, respectively, as templates. The PCR product was digested with EcoRI and XhoI and inserted into the expression vector pCMV-N-CFP (FIG. 1 a). Plasmids pCMV-N-CFP-TRIM54 (hereinafter abbreviated as CFP-TRIM 54) and pCMV-N-CFP-YFP (hereinafter abbreviated as CFP-YFP) were obtained. After enzyme digestion identification (right side of FIG. 1 bc), sequencing is carried out, and sequence alignment is correct. Next, we performed PCR amplification of the DNA fragment encoding STAT2 using human monocyte mRNA as template (FIG. 1e left). The PCR product was digested with BamHI and HindIII and inserted into the expression vector pCMV-C-YFP (FIG. 1 d). The plasmid pCMV-C-YFP-STAT2 (hereinafter abbreviated as YFP-STAT 2) was obtained. After enzyme digestion identification (right in FIG. 1 e), sequencing is carried out, and sequence alignment is correct.
Example 2 verification of fusion expressed proteins CFP-TRIM54 and YFP-STAT2
Two plasmids encoding CFP-TRIM54 and YFP-STAT2 were separately transfected into 293T cells and cultured for 48h, and Western blot analysis and flow analysis were performed. The theoretical molecular weight of CFP-TRIM54 is 67kDa, the theoretical molecular weight of YFP-STAT2 is 121kDa, and the result of Western Blot is consistent with the theoretical value, as shown in FIG. 2a, indicating that the fusion protein expression was successful. The fluorescence intensities of the fluorescent proteins CFP and YFP of the two fusion expression proteins were further detected by flow cytometry. As shown in FIG. 2b, fluorescence of CFP protein and fluorescence of YFP protein were detected in VL1 channel and BL1 channel, respectively, and fluorescence intensity was in accordance with the requirement of the subsequent experiment.
Example 3 flow cytometry detection of intracellular TRIM54 interaction with STAT2
To determine the intensity of FRET signal with a flow cytometer, we prepared FRET negative controls as well as positive controls. Plasmids pCMV-N-CFP (CFP) and pCMV-C-YFP (YFP) were simultaneously transfected into 293T cells as negative controls (CFP+YFP), which were able to detect fluorescence in both the CFP channel (ex: 405nm; em:440/50 nm) and YFP (ex: 48nm; em:530/30 nm) channels, FRET channels (ex: 405nm; em:512/25 nm) should be non-signalled because CFP and YFP were not able to interact. Plasmid pCMV-N-CFP-YFP (CFP-YFP) was transfected into 293T cells as a positive control, and fluorescence was detected in both the CFP channel (ex: 405nm; em:440/50 nm) and YFP (ex: 48nm; em:530/30 nm) channels of the control group, and since the spatial distance of the two fluorescent proteins in CFP-YFP was sufficiently close, all cells expressing CFP-YFP could theoretically generate FRET signals. The flow cytometer analysis results are shown in FIG. 3, in which after dead cells and adhesion-removed cells, CFP and YFP double positive cells were circled and the gate of FRET signal channel was adjusted, the negative control group (CFP+YFP) was 0% (0.07% in the figure) and the positive control group (CFP-YFP) was 100% (93.16% in the figure). Meanwhile, we co-transfected the plasmids pCMV-N-CFP-TRIM54 (CFP-TRIM 54) and pCMV-C-YFP-STAT2 (YFP-STAT 2) into 293T cells (CFP-TRIM 54+YFP-STAT 2) and examined with a flow cytometer using gates set up for negative (CFP+YFP)/positive control (CFP-YFP). As shown in FIG. 3, the proportion of FRET channel positive cells in the CFP-TRIM54+YFP-STAT2 group is 42.98%, that is, the binding rate of TRIM54 and STAT2 is about 42.98%, and by using the system, the interaction degree of TRIM54 and STAT2 can be quantified, so that the subsequent quantitative analysis of the interaction efficiency of small molecular drugs for inhibiting TRIM54 and STAT2 is facilitated.
Example 4 fluorescence enzyme-labeled instrument for detecting intracellular TRIM54 interactions with STAT2
The flow cytometer detection can evaluate the interaction of TRIM54/STAT2 from a single cell level and can eliminate the influence caused by the difference of transfection efficiency, but the detection efficiency is lower as a drug screening means, so we also discuss a drug screening method for detecting FRET signals by a fluorescence enzyme labelling instrument.
We transfected the plasmids pCMV-N-CFP (CFP), pCMV-C-YFP (YFP), pCMV-N-CFP and pCMV-C-YFP (CFP+YFP), pCMV-N-CFP-YFP (CFP-YFP), pCMV-N-CFP-TRIM54 (CFP-TRIM 54), pCMV-C-YFP-STAT2 (YFP-STAT 2), pCMV-N-CFP-TRIM54 and pCMV-C-YFP-STAT2 (CFP-TRIM 54+YFP-STAT 2) into 293T cells, and after 48h of culture, the cells were plated into whole black 96-well cell culture plates, three duplicate wells per cell group, 5X 10 per well 4 Individual cells. Wherein CFP, YFP and cfp+yfp expressing cells are used as a test to calculate the binding rates of CFP and YFP in cfp+yfp expressing cells; cells expressing CFP, YFP, CFP-YFP were used as a test to calculate the binding rates of CFP and YFP in cells expressing CFP-YFP; cells expressing CFP-TRIM54+His-STAT2, YFP-STAT2+flag-TRIM54 and CFP-TRIM54+YFP-STAT2 were used as assays to calculate the binding rates of CFP-TRIM54 and YFP-STAT2 in cells expressing CFP-TRIM54+YFP-STAT 2. Then we scan the fluorescence intensity with the microplate reader on CFP channel (ex: 430nm; em:480 nm), YFP channel (ex: 4815 nm; em:530 nm), FRET channel (ex: 430nm; em:530 nm), and calculate the interaction efficiency of two fluorescent tagged proteins using the formula. The formula is: e% = 1- (D) DA /(D DA +(F DA -(D DA ×d)-(A DA ×a))×Q D /Q A ))。(d＝F D /D D ，a＝F A /A A 。D D : read values of expressed CFP protein detected in the donor channel; d (D) DA : read values of CFP and YFP proteins were simultaneously expressed as detected in the donor channel; a is that A : a read of the detected YFP protein expression of the receptor channel; a is that DA : fluorescence values of expressed CFP and YFP proteins detected in the receptor channel; f (F) A : fluorescence value of the expressed YFP protein detected in the fret channel; f (F) D : fluorescence values of CFP-expressing proteins detected in the fret channel; f (F) DA : fluorescence values detected in the fret channel for simultaneous expression of CFP and YFP proteins; q (Q) D =0.4 and Q A Quantum yields of fluorescence donor and fluorescence acceptor respectively =0.61). The result of detection by the microplate reader shown in FIG. 4 was calculated to have a binding rate of about 0 for the negative control CFP+YFP; the binding rate of the positive control CFP-YFP was about 50%; the binding rate of CFP-TRIM54+YFP-STAT2 was about 30%.
Example 5 screening of Small molecule Compounds targeting TRIM54/STAT2 interaction
The small molecule library of targeting protein interactions we selected was purchased from Med Chem Express company and contained 167 small molecule compounds. The medicines are dissolved in corresponding solvents (DMSO, ethanol and water) in the form of lyophilized powder, and the concentration is 10mM, and stored in a refrigerator at-80 ℃ for a long time. Drugs in the small molecule drug library were diluted to 1mM with DMEM at a ratio of 1:10 and frozen in a-20℃refrigerator for subsequent experiments.
We transfected the plasmids pCMV-N-CFP (CFP), pCMV-C-YFP (YFP), pCMV-N-CFP and pCMV-C-YFP (CFP+YFP), pCMV-N-CFP-YFP (CFP-YFP), pCMV-N-CFP-TRIM54 (CFP-TRIM 54), pCMV-C-YFP-STAT2 and p3xflag-cmv-10-TRIM54 (YFP-STAT 2), pCMV-N-CFP-TRIM54 and pCMV-C-YFP-STAT2 (CFP-TRIM 54+YFP-STAT 2) into 293T cells, and after 48h of culture, the cells were plated into whole black 96-well cell culture plates, three wells per cell group, 5X 10 per well 4 Individual cells. Subsequently, we added the compound to cells co-expressing CFP-TRIM54 and YFP-STAT2 to a final concentration of 10. Mu.M as the experimental group, and added the same dilution of DMSO to cells co-expressing CFP-TRIM54 and YFP-STAT2. After 24h incubation, scanning with an enzyme-labeled instrument, and reading the values according to the fluorescence intensity of each group using the formula E% = 1- (D) DA /(D DA +(F DA -(D DA ×d)-(A DA ×a))×Q D /Q A ) Calculating the binding efficiency between CFP-TRIM54 and YFP-STAT2 after treatment with different compounds. And substituting the value of the binding rate into the formula: the "(control group-experimental group)/control group" calculates the inhibition efficiency of the compound so as to more intuitively exhibit the compound inhibition effect.
Drugs screened in the FRET screening system for inhibition greater than 20% were considered effective drugs, and the initial screening results showed (fig. 5 a) that there were 4 compounds with inhibition greater than 20%, i.e., compounds No. 9, 64, 86, 159, with inhibition of 159 exceeding 40%.
And then, detecting the cells screened by the enzyme-labeled instrument by using a flow cytometer to obtain the binding efficiency between the CFP-TRIM54 and the YFP-STAT2 after different compound treatments. And substituting the value of the binding rate into the formula: the "(control group-experimental group)/control group" calculates the inhibition efficiency of the compound to more intuitively demonstrate the inhibition effect of the compound. As shown in FIG. 5b, 6 compounds with an inhibition ratio higher than 20%, namely compounds No. 9, 64, 66, 86, 87, 159. Wherein, the inhibition rate of the compounds No. 9 and 159 exceeds 40 percent.
EXAMPLE 6 Small molecule Compound double Screen
The primary screening results of the small molecule drugs show that compounds 9, 64, 66, 86, 87 and 159 have obvious inhibition effect on the interaction of TRIM54 and STAT2. Since we detected the fluorescence intensity of FRET channels, compounds that inhibited cell viability also inhibited FRET signals, and corresponding compounds were screened as false positives. We therefore excluded false positive compounds by looking at the inhibition of the FRET channels of CFP-YFP fusion protein sets by compounds. We added the above compound to the experimental group (cells transfected with CFP-TRIM54 and YFP-STAT2 plasmids) to a final concentration of 10. Mu.M, and added the same compound to the control group (cells transfected with fusion expression of CFP-YFP plasmid) to a final concentration of 10. Mu.M, and read the fluorescence intensities of each channel with a microplate reader after culturing for 24 hours, and calculate the effect of the compound on the protein binding rate of the experimental group as well as the control group (FIG. 6 a). The effect of the compound on the FRET channel positive rate was further examined with a flow cytometer (fig. 6 b). The results indicate that the screened drug does not affect FRET signal of the control group, which is specific for inhibition of TRIM54 and STAT2.
Example 7 Compounds reverse the performance of TRIM54 in degrading STAT2
To verify whether the compounds can inhibit TRIM 54-induced degradation of STAT2. We transfected the Flag-TRIM54 encoding plasmid into 293T cells and added the compounds (9, 64, 66, 86, 87, 159) to a final concentration of 10. Mu.M after 9 h. After 24 hours, the cells are collected and lysed, and the cell lysate is taken by centrifugation for Western blot. By examining the protein expression level of intracellular STAT2, the inhibition effect of the medicine on TRIM 54-mediated degradation of STAT2 is verified. As shown in fig. 7, TRIM54 can induce degradation of STAT2, while compounds 9, 64, 66, 87, 159 can reverse degradation of STAT2.
EXAMPLE 8 Compounds blocking the Performance of TRIM54/STAT2 interaction
To verify the true inhibitory effect of the compounds on TRIM54/STAT2 interaction, we validated using co-immunoprecipitation experiments. The plasmid encoding Flag-TRIM54 was transfected into 293T cells, the cells were lysed after 24 hours, the cell lysates were centrifuged and added to compound (9, 64, 66, 86, 87, 159) to a final concentration of 10. Mu.M, the control group was added with DMSO at the same dilution concentration, incubated with Anti-Flag Affinity gel at4℃for 12 hours, and Western blot was performed on proteins attached to the gel. As shown in FIG. 8, flag-TRIM54 can bind to endogenous STAT2, and after a compound targeting TRIM54/STAT2 interaction is added into a Co-IP system, the compound can inhibit interaction between TRIM54 and STAT2.
EXAMPLE 9 inhibition of Denv2 and antibody 4G2 infection with K562 by Compounds
K562 cells are human monocyte lines expressing fcγriia and are conditioned for ADE modeling. We infected monocytes with immune complexes formed by DENV2 and monoclonal antibody 4G2 (specific mab to DENV 1E protein). First, we incubated DENV2 and 4G2 antibodies (final concentration 1 μg/ml) at 25 ℃ for 1h, respectively, to form complexes. K562 cells were infected at moi=1 (5×10 5 ). After incubation for 72h with equal amounts of RPMI, 4G2 or DENV2 respectively added to the control, cells were collected, fixed, broken, intracellular DENV2 labeled with Alexa Fluor 647-labeled 4G2 antibody, and then the proportion of cells infected with DENV2 and viral load were detected using a flow cytometer. The efficiency of virus infection of K562 cells was examined on RL1 channel after performing cell death, de-adhesion. As shown in FIG. 9a, the control (RPMI, 4G2 or DENV2 addition) had 1% (0 theoretical), 1% (0 theoretical) and 10% infection, respectively. Whereas the infection rate of the experimental group (DENV 2+4G 2) was 70%. The above results indicate that immune complexes formed by DENV2 and 4G2 can promote DENV2 infection, and that the ADE model was successfully constructed.
The invention explores the inhibitory effect of compounds on ADE constructed by K562 cells. We added K562 cells of ADE model to 10 μm compound. Cells were collected after incubation for 72 h. After fixation, membrane rupture, staining we analyzed the cells by flow cytometry. As shown in fig. 9b, the viral infection rate of K562 cells to which the compound was added tended to decrease. Suggesting that it may be a potential drug to inhibit the effects of viral ADE. Compounds with greater inhibition (64, 86, 159) were further selected to investigate their dose dependence on ADE effect inhibition. We infected K562 cells (moi=1) after incubation of DENV2 and antibody 4G2 (final concentration of 1 μg/ml) for 1h at 25 ℃ and compounds (final concentrations of 1,3,10 and 30 μm) were added to the cells, respectively, to explore the inhibition of ADE effect by different doses of compounds, and the control group was added with the same Dilution of Med (DMSO). After fixation, membrane rupture, staining we analyzed the cells by flow cytometry. The results are shown in FIG. 9c, where the inhibition of ADE effect by 64 (Baricitinib) and 159 (Rivaroxaban) was more pronounced and dose dependent.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. An antiviral ADE drug screening method with TRIM54 ubiquitination degradation STAT2 inhibition as a target spot is characterized by comprising the following steps:
s1, respectively constructing a first plasmid and a second plasmid, introducing the first plasmid and the second plasmid into cells, and detecting FRET signals; the first plasmid and the second plasmid respectively contain a gene for encoding TRIM54 and a gene for encoding STAT2, the first plasmid and the second plasmid respectively contain a gene for encoding a first fluorescent protein and a gene for encoding a second fluorescent protein, and the first fluorescent protein and the second fluorescent protein can generate fluorescence resonance energy transfer;
s2, incubating the compound to be detected with the cells in the step S1, and screening out the drug for inhibiting the ADE effect of the virus according to the change of the FRET signal.
2. The screening method according to claim 1, wherein: the nucleotide sequence of the gene for encoding TRIM54 is shown as SEQ ID NO. 1.
3. The screening method according to claim 1, wherein: the nucleotide sequence of the gene for encoding STAT2 is shown as SEQ ID NO. 2.
4. The screening method according to claim 1, wherein: and taking the cells into which the third plasmid is introduced as a positive control, wherein the third plasmid contains a gene encoding the first fluorescent protein and a gene encoding the second fluorescent protein.
5. The screening method according to claim 1, wherein: and taking cells into which a fourth plasmid and a fifth plasmid are introduced as negative controls, wherein the fourth plasmid and the fifth plasmid respectively contain a gene encoding a first fluorescent protein and a gene encoding a second fluorescent protein.
6. The screening method according to claim 1, wherein: FRET signals are detected by flow cytometry or fluorogenic enzyme labeling.
7. Use of a substance inhibiting the interaction of TRIM54 and STAT2 in the preparation of a medicament for the antiviral ADE effect.
8. Use of a substance characterising the interaction of TRIM54 and STAT2 in antiviral ADE effect drug screening.
Application of Baricitinib or Rivaroxaban in preparing medicine for inhibiting virus ADE effect.
10. An antiviral ADE effect drug, characterized by: the medicine comprises Baricitinib or Rivaroxaban.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211060667.1A CN116445573A (en) | 2022-08-30 | 2022-08-30 | Antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as target point |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211060667.1A CN116445573A (en) | 2022-08-30 | 2022-08-30 | Antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as target point |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116445573A true CN116445573A (en) | 2023-07-18 |
Family
ID=87118901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211060667.1A Pending CN116445573A (en) | 2022-08-30 | 2022-08-30 | Antiviral ADE drug screening method taking TRIM54 ubiquitination degradation STAT2 inhibition as target point |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116445573A (en) |
-
2022
- 2022-08-30 CN CN202211060667.1A patent/CN116445573A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nechiporuk et al. | The TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition in AML cells | |
| Mar et al. | LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step | |
| Kim et al. | Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression | |
| Campbell Dwyer et al. | The lymphocytic choriomeningitis virus RING protein Z associates with eukaryotic initiation factor 4E and selectively represses translation in a RING-dependent manner | |
| US8334101B2 (en) | Intracellular DNA receptor | |
| Kawase et al. | PH domain-only protein PHLDA3 is a p53-regulated repressor of Akt | |
| JP4511033B2 (en) | Method for screening for bioactive agents capable of altering cell phenotype using FACS | |
| King et al. | Inhibition of human adenovirus replication by the importin α/β1 nuclear import inhibitor ivermectin | |
| Yasunaga et al. | Ubiquitin-specific peptidase 20 targets TRAF6 and human T cell leukemia virus type 1 tax to negatively regulate NF-κB signaling | |
| Andrejeva et al. | The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5 | |
| Martinez-Gil et al. | Exploring the human-Nipah virus protein-protein interactome | |
| Kim et al. | Association of Tat with promoters of PTEN and PP2A subunits is key to transcriptional activation of apoptotic pathways in HIV-infected CD4+ T cells | |
| Courtney et al. | Identification of novel host cell binding partners of Oas1b, the protein conferring resistance to flavivirus-induced disease in mice | |
| Yatherajam et al. | Export of adenoviral late mRNA from the nucleus requires the Nxf1/Tap export receptor | |
| US20130178382A1 (en) | Identification of Modulators of Autophagy | |
| Yuen et al. | Suppression of type I interferon production by human T-cell leukemia virus type 1 oncoprotein Tax through inhibition of IRF3 phosphorylation | |
| Tremblay et al. | Spliceosome SNRNP200 promotes viral RNA sensing and IRF3 activation of antiviral response | |
| Tolani et al. | NEMO is essential for Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13-induced gene expression and protection against death receptor-induced cell death, and its N-terminal 251 residues are sufficient for this process | |
| Desplancq et al. | Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells | |
| Wang et al. | Nuclear localization and cleavage of STAT6 is induced by Kaposi’s sarcoma-associated herpesvirus for viral latency | |
| EP2985347B1 (en) | Method for detecting protein stability and uses thereof | |
| Chen et al. | Comprehensive analysis of the host-virus interactome of SARS-CoV-2 | |
| Schweibenz et al. | The intrinsically disordered CARDs‐Helicase linker in RIG‐I is a molecular gate for RNA proofreading | |
| Luo et al. | CMPK2 accelerates liver ischemia/reperfusion injury via the NLRP3 signaling pathway | |
| CN107109457A (en) | For the method for the therapy for finding to change target protein stability |
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 |