CN118121609A - Application of Kenparone in preparation of medicines for preventing and treating coronavirus infection - Google Patents
Application of Kenparone in preparation of medicines for preventing and treating coronavirus infection Download PDFInfo
- Publication number
- CN118121609A CN118121609A CN202410261955.6A CN202410261955A CN118121609A CN 118121609 A CN118121609 A CN 118121609A CN 202410261955 A CN202410261955 A CN 202410261955A CN 118121609 A CN118121609 A CN 118121609A
- Authority
- CN
- China
- Prior art keywords
- cov
- kenpaullone
- group
- sads
- pedv
- 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
- 239000003814 drug Substances 0.000 title claims abstract description 40
- 229940079593 drug Drugs 0.000 title claims description 21
- 238000002360 preparation method Methods 0.000 title claims description 9
- 208000001528 Coronaviridae Infections Diseases 0.000 title abstract description 11
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 claims abstract description 53
- 241000711573 Coronaviridae Species 0.000 claims abstract description 37
- 206010012735 Diarrhoea Diseases 0.000 claims abstract description 28
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 25
- 201000010099 disease Diseases 0.000 claims abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 17
- 230000035755 proliferation Effects 0.000 claims abstract description 10
- 206010006451 bronchitis Diseases 0.000 claims abstract description 8
- 238000011282 treatment Methods 0.000 claims description 90
- 239000000243 solution Substances 0.000 claims description 60
- 239000003112 inhibitor Substances 0.000 claims description 22
- 230000002401 inhibitory effect Effects 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 13
- 230000002265 prevention Effects 0.000 claims description 11
- 150000003839 salts Chemical class 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 4
- 238000010253 intravenous injection Methods 0.000 claims description 4
- 206010035664 Pneumonia Diseases 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 230000002335 preservative effect Effects 0.000 claims description 3
- 241000792859 Enema Species 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000000443 aerosol Substances 0.000 claims description 2
- 239000003963 antioxidant agent Substances 0.000 claims description 2
- 230000003078 antioxidant effect Effects 0.000 claims description 2
- 239000011230 binding agent Substances 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 239000006184 cosolvent Substances 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000007884 disintegrant Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000003995 emulsifying agent Substances 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 239000007920 enema Substances 0.000 claims description 2
- 229940095399 enema Drugs 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000010255 intramuscular injection Methods 0.000 claims description 2
- 239000007927 intramuscular injection Substances 0.000 claims description 2
- 239000007928 intraperitoneal injection Substances 0.000 claims description 2
- 239000000314 lubricant Substances 0.000 claims description 2
- 230000003204 osmotic effect Effects 0.000 claims description 2
- 239000006187 pill Substances 0.000 claims description 2
- 238000010254 subcutaneous injection Methods 0.000 claims description 2
- 239000007929 subcutaneous injection Substances 0.000 claims description 2
- 239000000829 suppository Substances 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 239000000080 wetting agent Substances 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 44
- 238000002474 experimental method Methods 0.000 abstract description 33
- 208000015181 infectious disease Diseases 0.000 abstract description 30
- 241000282887 Suidae Species 0.000 abstract description 21
- 231100000956 nontoxicity Toxicity 0.000 abstract description 9
- 201000009840 acute diarrhea Diseases 0.000 abstract description 6
- 230000002458 infectious effect Effects 0.000 abstract description 6
- 208000011580 syndromic disease Diseases 0.000 abstract description 6
- 241000271566 Aves Species 0.000 abstract description 4
- 241001361508 Porcine deltacoronavirus Species 0.000 abstract description 4
- QQUXFYAWXPMDOE-UHFFFAOYSA-N kenpaullone Chemical compound C1C(=O)NC2=CC=CC=C2C2=C1C1=CC(Br)=CC=C1N2 QQUXFYAWXPMDOE-UHFFFAOYSA-N 0.000 description 174
- 210000004027 cell Anatomy 0.000 description 99
- 210000001519 tissue Anatomy 0.000 description 75
- 230000010076 replication Effects 0.000 description 54
- 241000700605 Viruses Species 0.000 description 50
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 37
- 238000011529 RT qPCR Methods 0.000 description 33
- 239000000523 sample Substances 0.000 description 33
- 241000711450 Infectious bronchitis virus Species 0.000 description 31
- 230000000968 intestinal effect Effects 0.000 description 30
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 29
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 28
- 241000287828 Gallus gallus Species 0.000 description 27
- 235000013330 chicken meat Nutrition 0.000 description 27
- 239000002609 medium Substances 0.000 description 27
- 239000013642 negative control Substances 0.000 description 27
- 241000282898 Sus scrofa Species 0.000 description 23
- 210000003405 ileum Anatomy 0.000 description 23
- 210000001630 jejunum Anatomy 0.000 description 23
- 210000001198 duodenum Anatomy 0.000 description 22
- 230000003612 virological effect Effects 0.000 description 22
- 238000006243 chemical reaction Methods 0.000 description 20
- 239000006228 supernatant Substances 0.000 description 19
- 208000024891 symptom Diseases 0.000 description 19
- 239000007788 liquid Substances 0.000 description 17
- 108020004999 messenger RNA Proteins 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 210000003837 chick embryo Anatomy 0.000 description 16
- 239000013641 positive control Substances 0.000 description 16
- 238000004113 cell culture Methods 0.000 description 15
- 239000006285 cell suspension Substances 0.000 description 14
- 230000037396 body weight Effects 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 230000008859 change Effects 0.000 description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 230000003902 lesion Effects 0.000 description 10
- 238000010839 reverse transcription Methods 0.000 description 10
- 230000004083 survival effect Effects 0.000 description 10
- 230000001988 toxicity Effects 0.000 description 10
- 231100000419 toxicity Toxicity 0.000 description 10
- 239000003053 toxin Substances 0.000 description 10
- 231100000765 toxin Toxicity 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- 238000000227 grinding Methods 0.000 description 9
- 231100000915 pathological change Toxicity 0.000 description 9
- 230000036285 pathological change Effects 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 230000001225 therapeutic effect Effects 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- 231100000135 cytotoxicity Toxicity 0.000 description 8
- 230000003013 cytotoxicity Effects 0.000 description 8
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 7
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 210000003734 kidney Anatomy 0.000 description 7
- 210000003292 kidney cell Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 231100000957 no side effect Toxicity 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 7
- 241000282552 Chlorocebus aethiops Species 0.000 description 6
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
- 108091000080 Phosphotransferase Proteins 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 6
- 238000009395 breeding Methods 0.000 description 6
- 230000001488 breeding effect Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 230000018044 dehydration Effects 0.000 description 6
- 238000006297 dehydration reaction Methods 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000012188 paraffin wax Substances 0.000 description 6
- 230000001575 pathological effect Effects 0.000 description 6
- 102000020233 phosphotransferase Human genes 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 238000010257 thawing Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 5
- 206010047700 Vomiting Diseases 0.000 description 5
- 210000000621 bronchi Anatomy 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 210000000813 small intestine Anatomy 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 230000008673 vomiting Effects 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 229910000831 Steel Inorganic materials 0.000 description 4
- 238000007605 air drying Methods 0.000 description 4
- 230000000840 anti-viral effect Effects 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 239000000919 ceramic Substances 0.000 description 4
- 238000005336 cracking Methods 0.000 description 4
- 231100000263 cytotoxicity test Toxicity 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000032798 delamination Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 239000006196 drop Substances 0.000 description 4
- 241001493065 dsRNA viruses Species 0.000 description 4
- 210000002257 embryonic structure Anatomy 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000003903 intestinal lesions Effects 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 230000003340 mental effect Effects 0.000 description 4
- 230000006996 mental state Effects 0.000 description 4
- 238000004806 packaging method and process Methods 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 238000012257 pre-denaturation Methods 0.000 description 4
- 230000001376 precipitating effect Effects 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 230000000241 respiratory effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000010959 steel Substances 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 241000004176 Alphacoronavirus Species 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 3
- 241001461743 Deltacoronavirus Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000583907 Taraka Species 0.000 description 3
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000002784 cytotoxicity assay Methods 0.000 description 3
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 231100000614 poison Toxicity 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 235000013594 poultry meat Nutrition 0.000 description 3
- 210000002345 respiratory system Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000000451 tissue damage Effects 0.000 description 3
- 231100000827 tissue damage Toxicity 0.000 description 3
- 239000003440 toxic substance Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- 208000005156 Dehydration Diseases 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- 101500025255 Severe acute respiratory syndrome coronavirus 2 3C-like proteinase nsp5 Proteins 0.000 description 2
- 101500025527 Severe acute respiratory syndrome coronavirus 2 3C-like proteinase nsp5 Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 239000003889 eye drop Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108091005981 phosphorylated proteins Proteins 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000025721 COVID-19 Diseases 0.000 description 1
- 101100495324 Caenorhabditis elegans cdk-7 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000742982 Homo sapiens Serine/threonine-protein kinase WNK3 Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 102100038115 Serine/threonine-protein kinase WNK3 Human genes 0.000 description 1
- 101001024637 Severe acute respiratory syndrome coronavirus 2 Nucleoprotein Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003684 drug solvent Substances 0.000 description 1
- 230000000369 enteropathogenic effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 230000035611 feeding Effects 0.000 description 1
- 206010016766 flatulence Diseases 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 208000037806 kidney injury Diseases 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- -1 oral preparation Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 238000009374 poultry farming Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Abstract
The invention discloses application of Kenparone in preparing a medicine for preventing and treating coronavirus infection. The invention discovers that Kenparone has activity of resisting coronavirus for the first time, and the coronavirus comprises SADS-CoV, PEDV, PDCoV, IBV and SARS-CoV-2; experiments prove that the kenstring has the activity of resisting SADS-CoV, PEDV, PDCoV, IBV and SARS-CoV-2, can inhibit the proliferation of SADS-CoV, PEDV, PDCoV, IBV and SARS-CoV-2, and shows dose dependency, and shows the potential of the kenstring in treating acute diarrhea syndrome, porcine epidemic diarrhea, porcine delta coronavirus disease, avian infectious bronchitis and novel coronavirus infection of pigs. And the Kenparone has definite structure, no toxicity or side effect and good application prospect in the aspect of preventing and treating various coronaviruses.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of Kenparone in preparation of medicines for preventing and treating coronavirus infection.
Background
Porcine acute diarrhea syndrome coronavirus (swine acute diarrhea syndrome coronavirus, SADS-CoV) is the etiology of porcine acute diarrhea syndrome, SADS-CoV belongs to the genus coronavirus (Coronavirus) of the family coronaviridae (Coronaviridae), and is a enveloped single-stranded positive-strand RNA virus. SADS-CoV can cause diarrhea, vomiting, dehydration and death in newborn piglets, and is also a potential threat to human health and public health as an RNA virus with high variability and ability to spread across species. Aiming at SADS-CoV, no effective vaccine and medicine can be put into production practice at present, and pig breeding enterprises mainly depend on biosafety prevention and control to prevent the transmission and infection of the virus at present. The strict biological safety prevention and control greatly increases the cost of the breeding enterprises, and the virus still continuously causes huge economic loss in the pig breeding industry at present.
Porcine Epidemic Diarrhea Virus (PEDV) is a causative agent of Porcine epidemic diarrhea, PEDV belongs to the family coronaviridae (Coronaviridae), genus alphacoronavirus (Alphacoronavirus), and is a enveloped single-stranded positive-strand RNA virus. PEDV brings a huge economic loss to the pig industry worldwide and has become one of the most threatening viral diseases of pigs. The prevention and control means of PEDV mainly depend on vaccine to generate mucosal immunity, however, the main victim group of PEDV is a suckling piglet with a mucosal immune system which is not yet developed completely, so the current immune effect is not ideal.
Porcine delta coronavirus (Porcine deltacoronavirus, PDCoV) is a novel enteropathogenic coronavirus belonging to the genus Deltacoronavirus (Deltacoronavirus) of the family Coronaviridae (Coronaviridae) and is a single-stranded positive strand RNA virus with an envelope. PDCoV can infect pigs of different ages, is more susceptible to newborn piglets, and has clinical symptoms such as diarrhea, vomiting, dehydration, appetite reduction and the like. Histopathology manifests as intestinal lesions, proximal jejunum to ileum multifocal to diffuse villus atrophy. PDCoV has higher morbidity and mortality, causes diarrhea of large-scale piglets, and causes great economic loss for the global pig industry. Scientists have detected and isolated PDCoV from three febrile children serum samples at sea as reported by Nature, indicating that PDCoV can spread across species. At present, no effective PDCoV vaccine exists, and for PDCoV prevention and control, biological safety prevention and control is mainly relied on in production to prevent the transmission and infection of the virus, so that the cost of a breeding enterprise is greatly increased.
Avian infectious bronchitis (Infectious bronchitis, IB) is one of the B/II infectious diseases of poultry regulated by International epidemic prevention and control agency and China, is caused by infection of avian infectious bronchitis virus (Infectious bronchitis virus, IBV), and is an acute and high-contact infectious disease of chickens which are widely popular at present. IBV mainly infects the respiratory tract, kidneys, reproductive system of birds, and can cause respiratory discomfort, kidney injury, and decreased laying rate in host animals. In recent years, IB has serious disease occurrence in laying hens and hens, and particularly under the condition of large-scale feeding, the infectious bronchitis has higher infection rate, more complicated disease type, larger prevention and control difficulty and more severe harm. Aiming at IBV, the vaccine has poor immune effect at present, and no effective medicine is available. IBV has a large number of variant strains, and only part or all of serotypes have no cross protection effect, so that great difficulty is brought to the prevention and treatment of the disease. Poultry farming enterprises currently rely mainly on biosafety to prevent the transmission and infection of this virus. The strict biosafety prevention and control greatly increases the cost of the breeding enterprises, and the virus still continuously causes huge economic loss in the poultry breeding industry at present.
The novel coronavirus infection (COVID-19) is a highly transmitted acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV-2). The transmission mode of SARS-CoV-2 is mainly the close contact of respiratory tract spray and crowd, and its clinical symptoms are fever, cough, force, dyspnea, etc. The virus has been found to be a variety of variants since its emergence, including Alpha, beta, omnicron, gamma, della. With continuous variation of SARS-CoV-2, new variants are frequent. In view of the above, in order to cope with the evolution mutation of viruses, research on prevention, control and treatment of novel coronaviruses and variants thereof is still significant.
Therefore, there is an urgent need for a drug capable of effectively inhibiting SADS-CoV, PEDV, PDCoV, IBV, SARS-CoV-2.
Disclosure of Invention
The invention aims to provide application of Kenparone in preparing medicines for preventing and/or treating diseases caused by coronaviruses; wherein the structure of the Kenparone is as follows:
The technical scheme adopted by the invention is as follows:
In a first aspect of the invention there is provided the use of Kenparone in any of the following:
(a) Preparing a medicament for preventing and/or treating diseases caused by coronaviruses;
(b) Preparing coronavirus inhibitor.
Preferably, the coronavirus comprises at least one of SADS-CoV, PEDV, PDCoV, IBV, SARS-CoV-2.
Preferably, the SARS-CoV-2 also includes, for example, SARS-CoV-2Delta mutant, SARS-CoV-2Alpha mutant, SARS-CoV-2Beta mutant, SARS-CoV-2Gamma mutant, SARS-CoV-2Omicron mutant, etc.
Preferably, the disease caused by coronavirus comprises diarrhea, bronchitis and/or pneumonia.
Preferably, the medicament or inhibitor further comprises pharmaceutically acceptable excipients.
Preferably, the pharmaceutically acceptable excipients include: at least one of a diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, co-solvent, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, bacteriostat or buffer.
Preferably, the dosage form of the drug or inhibitor comprises at least one of suspension, granule, capsule, powder, tablet, emulsion, solution, drop pill, injection, oral preparation, suppository, enema, aerosol, patch or drop.
Preferably, the route of administration of the drug or inhibitor comprises at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration or transdermal administration.
Preferably, the medicament or inhibitor further comprises an active ingredient for preventing and/or treating diseases associated with coronavirus infection.
Preferably, the medicament or inhibitor contains an effective dose of Kenparone or a pharmaceutically acceptable salt thereof.
In a second aspect the invention provides a product comprising as its major component Kenparone or a pharmaceutically acceptable salt thereof; the function of the product is to prevent and/or treat diseases caused by coronaviruses and/or inhibit the proliferation of coronaviruses; the coronavirus comprises at least one of PEDV, PDCoV, IBV, SARS-CoV-2 and SADS-CoV.
Preferably, the disease caused by coronavirus comprises diarrhea, bronchitis and/or pneumonia.
Preferably, the product comprises a drug or inhibitor.
In a third aspect of the invention, there is provided a method for preventing or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject.
Preferably, the subject may be poultry (e.g., pigs, chickens, etc.), but is not so limited. The subject includes a human subject.
Preferably, the medicament of the invention may be administered to a subject by any suitable route of administration. Such routes of administration include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.
In the present invention, a suitable daily dosage range of Kenparone or a pharmaceutically acceptable salt thereof is 1 μg to 1g/kg body weight; the above-mentioned dosages may be administered in one dosage unit or in several dosage units, depending on the clinical experience of the physician and the dosage regimen involved in the application of other therapeutic means.
In the present invention, kenparone or a pharmaceutically acceptable salt thereof may be used to inhibit replication of coronavirus in a cellular assay; wherein, the working concentration of the Kenparone or the pharmaceutically acceptable salt thereof comprises 0.1-100 mu m; preferably 1 to 50. Mu.m.
The beneficial effects of the invention are as follows:
The invention discovers that Kenparone Kenpaullone has activity of resisting coronaviruses for the first time, wherein the coronaviruses comprise SADS-CoV, PEDV, PDCoV, IBV and SARS-CoV-2; experiments show that Kenpaullone has the activity of resisting SADS-CoV, PEDV, PDCoV, IBV and SARS-CoV-2, can inhibit the proliferation of SADS-CoV, PEDV, PDCoV, IBV and SARS-CoV-2, and shows dose dependence, and shows Kenpaullone potential for treating acute diarrhea syndrome of pigs, porcine epidemic diarrhea, porcine delta coronavirus disease, avian infectious bronchitis and novel coronavirus infection; and Kenpaullone has definite structure, no toxicity or side effect, and has good application prospect in the aspect of preventing and treating coronavirus.
Drawings
FIG. 1 is a graph showing the results of proteomics and phosphoproteomics analysis by Vero-E6 cells 12h, 24h, and 36h after infection and non-infection with SADS-CoV. Wherein A is the establishment of SADS-CoV infected Vero-E6 cell model and the analysis flow chart of proteomics and phosphorylation proteomics; the left graph in B is the PCA scattergram of the proteomic sequencing data, and the right graph is the PCA scattergram of the phosphorylated proteomic sequencing data; the left graph in C is protein with obvious difference screened by Fold change >1.5 and P-value <0.05, and the total number is 674, and the right graph is phosphorylated protein with obvious difference screened by Fold change >2 and P-value <0.05, and the total number is 709; d is a statistical analysis of phosphokinase upregulated expression at least 1 time point post-infection, co-identifying 19 phosphokinase enzymes including WNK3, MARK2 and CDK 7; upstream kinases of phosphorylated peptide were predicted and counted, E being the top 10-position phosphorylated kinase.
FIG. 2 is a graph showing the effect of Kenpaullone on SADS-CoV replication in Vero-E6 cells. Wherein A is Kenpaullone cytotoxicity detection result of Vero-E6; b is Kenpaullone IC 50 results that inhibit SADS-CoV; c is the effect of IFA assay Kenpaullone on SADS-CoV replication; d is RT-qPCR to detect the influence of different concentrations Kenpaullone on SADS-CoV replication; e is RT-qPCR to detect the effect of identical concentration Kenpaullone on SADS-CoV replication at different time points after SADS-CoV infection; f is WB to detect the effect of different concentrations Kenpaullone on SADS-CoV replication; g is RT-qPCR to examine the effect of identical concentrations Kenpaullone on SADS-CoV replication at different time points after SADS-CoV infection.
Fig. 3 is a graph of piglet treatment experiment clinical score data statistics and viral load determination results. Wherein A is a survival curve of piglets; b is the weight change condition of piglets after 7 days of toxin expelling; c is a piglet clinical scoring data statistical graph; d is detection of piglet enterotissue viral load of the virus attacking group and the virus attacking treatment group.
Fig. 4 is the results of the pathological section of the piglet treatment experiment tissue.
FIG. 5 is a graph showing the effect of Kenpaullone on PEDV replication in Vero-E6 cells. Wherein A is Kenpaullone cytotoxicity detection result of Vero-E6; b is the IC 50 result of Kenpaullone inhibiting PEDV; c is TCID 50 to detect the effect of different concentrations of Kenpaullone on PEDV replication; d is RT-qPCR to detect the influence of different concentrations Kenpaullone on PEDV replication; e is RT-qPCR to examine the effect of the same concentration Kenpaullone on PEDV replication at different time points after PEDV infection.
Fig. 6 is a graph of piglet treatment experiment clinical score data statistics and viral load determination results. Wherein A is a piglet survival curve; b is the weight change condition of piglets after 7 days of toxin expelling; c is a piglet clinical scoring data statistical graph; d is detection of piglet enterotissue viral load of the virus attacking group and the virus attacking treatment group.
Fig. 7 shows the results of the pathological section of the tissue of the piglet treatment experiment.
FIG. 8 is a graph showing the effect of Kenpaullone on PDCoV replication in LLC-PK1 cells. Wherein A is Kenpaullone to LLC-PK1 cytotoxicity detection result; b is RT-qPCR to detect the influence of Kenpaullone of different concentrations on PDCoV replication; c is TCID 50 to detect the effect of different concentrations Kenpaullone on PDCoV replication.
Fig. 9 is a graph of clinical data statistics and viral load measurement results of piglet treatment experiments. Wherein A is a piglet clinical scoring data statistical graph; b is weight change; c is a survival curve; d is the detection of the jejunum, ileum and duodenum tissue viral loads of piglets in the virus-attack group and the virus-attack treatment group.
Fig. 10 is a graph showing the results of pathological sections of the tissues of the piglet treatment experiment.
FIG. 11 is a graph showing the effect of Kenpaullone on IBV replication in chicken embryos, specifically the effect of RT-qPCR on IBV replication at different concentrations Kenpaullone.
Fig. 12 is a graph of chicken treatment experiment clinical scores, survival curve statistics, and tissue viral load determination results. Wherein A is the clinical score of the chicks; b is weight change; c is a survival curve; and D is the detection of viral load in kidney and bronchus tissues of the toxicity attacking group and the toxicity attacking treatment group.
FIG. 13 is a graph of the effect of Kenpaullone on SARS-CoV-2 replication in Vero cells, the effect of different concentrations of Kenpaullone on SARS-CoV-2 replication was detected by RT-qPCR.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The experimental procedure of this example is summarized as follows:
1. First, SADS-CoV was infected with Vero-E6 cells at moi=0.1, cells were collected at 12, 24 and 36 hours after infection (early post-infection, exponential growth phase and plateau phase), proteomics and phosphorylated proteomics were studied using a super-resolution liquid chromatography-mass spectrometer (Q Exactive HF-X), proteomics and phosphorylated proteomics data were analyzed by bioinformatics methods, and phosphorylated proteins significantly up-regulated at least at one time point and kinases upstream of phosphorylated peptide fragments were predicted and counted. Subsequently, the antiviral effects of these kinase inhibitors were explored and one inhibitor Kenpaullone was screened from them, which was found to significantly inhibit the viral replication of SADS-CoV. Therefore, kenpaullone was tested on passable African green monkey kidney cell line (Vero) for inhibition of SADS-CoV replication, and animal experiments were performed to administer treatment of SADS-CoV infected piglets and test Kenpaullone for clinical therapeutic effect.
2. The inhibition of PEDV replication by Kenpaullone was verified on passable african green monkey kidney cell line (Vero-E6) and animal experiments were performed to administer piglets infected with PEDV and examine the clinical therapeutic effect of Kenpaullone.
3. The inhibition of PDCoV replication by Kenpaullone was verified on a passable LLC-PK1 cell line, and animal experiments were performed to administer piglets infected with PDCoV and to examine the clinical therapeutic effect of Kenpaullone.
4. The inhibitory effect of Kenpaullone on IBV replication was verified on chick embryos, and animal experiments were performed to administer treatment to chicks infected with IBV and examine the clinical therapeutic effect of Kenpaullone.
5. The inhibitory effect of Kenpaullone on SARS-CoV-2 replication was demonstrated on passable African green monkey kidney cell line (Vero-E6).
The materials used in this experiment were as follows:
(1) Cells and viruses
SADS-CoV, PEDV, PDCoV, IBV, vero, LLC-PK1 cells of African green monkey kidney cells are preserved by the laboratory, and the strain and the cells are conventional strains and cells which are disclosed in the prior art, and SARS-CoV-2 strain refers to SARS-CoV-2Mpro inhibitors with antiviral activity in a transgenic mouse model; SPF chickens and 1 day old SPF chick embryos were purchased from the SPF laboratory animal center of Wen Shida Hua nong Biotechnology Co., ltd.
(2) Main reagent
Kenpaullone for in vivo and in vitro experiments were purchased from MedChemExpress; the enhanced CCK8 kit was purchased from bi yun-tian biotechnology limited; dimethyl sulfoxide (DMSO) was purchased from Sigma; EZ-press RNA Purification Kit kit was purchased from EZBioscience; /(I) V C58P2Multiplex One Step RT-qPCR Probe Kit (UDG Plus) was purchased from the following holy organism; other reagents are prepared by methods conventional in the art.
Example 1Kenpaullone use in the preparation of a medicament for inhibiting SADS-CoV replication
Vero-E6 cells were divided into control and infected groups, 3 replicates each, vero-E6 cells were infected with SADS-CoV strain at moi=0.1, cells were collected at 12, 24 and 36 hours post infection (early post infection, exponential growth phase and plateau phase), proteomic and phosphoproteomic assays were performed using a super resolution liquid chromatography-mass spectrometer (Q Exactive HF-X), and the results are shown in fig. 1.
Statistical analysis of phosphorylated kinases up-regulated at least 1 time point after infection, while predicting and counting upstream kinases of phosphorylated peptide fragments using GPS 5.0 software, found that members of multiple kinase families were significantly up-regulated, likely playing an important role in the infectious replication process of SADS-CoV. Through screening the inhibitors of these kinases, kenpaullone was found to have a significant antiviral effect.
Example 2Kenpaullone Effect on SADS-CoV replication
(1) Kenpaullone test for detecting cytotoxicity of Vero-E6
Cytotoxicity of Kenpaullone to Vero-E6 was determined using CCK8 cytotoxicity assay, as follows:
A96-well plate was seeded with a 6X 10 5 cells/mL cell suspension (100. Mu.L/well) leaving a column of uninoculated cells. The cells were cultured at 37℃under 5% CO 2 until they were completely confluent. The medium was aspirated, washed 3 times with PBS, kenpaullone dissolved in DMSO and diluted with DMEM medium to different concentrations (0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M), 3 replicates per concentration, 100. Mu.L/well.
After incubation at 37℃for 24h with 5% CO 2, the drug solution was aspirated, washed 3 times with PBS, 10% (v/v) CCK8 solution was added to 96-well plates (100. Mu.L/well) with DMEM medium, incubated at 37℃for 1h with 5% CO 2, absorbance (lambda=450 nm) was measured with a microplate reader and cell viability was calculated after 24h treatment with different concentrations of drug solution.
As a result, the maximum safe concentration of Kenpaullone to Vero-E6 was 10. Mu.M, as shown in FIG. 2A.
(2) Kenpaullone detection of IC 50 inhibiting SADS-CoV replication
1ML of the cell suspension was inoculated at a cell concentration of 3X 10 5 cells/mL in a 12-well cell culture plate, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The culture medium in the 6-well plate was discarded, washed 3 times with PBS, and the cells were treated with DMEM medium at Kenpaullone concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, and 10. Mu.M for 1 hour, and the treated solution was discarded, and SADS-CoV virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and cultured at 37℃under 5% CO 2 for 24 hours.
UsingEZ-press RNA Purification Kit kit extracts RNA according to the instruction, and then according to the/>, of the following holothuriansV C58P2Multiplex One Step RT-qPCR Probe Kit (UDG Plus) protocol RT-qPCR was performed to detect SADS-CoV-N mRNA expression levels in cells. The primer and probe sequences are as follows
SADS-CoV-N-F：5’-CTGACTGTTGTTGAGGTTAC-3’(SEQ ID NO.1)；
SADS-CoV-N-R：5’-TCTGCCAAAGCTTGTTTAAC-3’(SEQ ID NO.2)；
SADS-CoV-N-Probe：5’-FAM-TCACAGTCTCGTTCTCGCAATCA-TAMRA-3’(SEQ ID NO.3)。
The reaction is according to the following holy living beingsV C58P2Multiplex One Step RT-qPCR Probe Kit (UDG Plus) instruction manual, the reaction system is: 15 mu L/>V C58P2MP Buffer、1.2μL/>VC58P2 Enzyme Mix, 3. Mu.L Primer/Probe Mix (2.5. Mu.M), 1. Mu.L template RNA, 9.8. Mu. L RNASE FREE H 2 O. The system was assembled in 3 replicates per sample, 30 μl per well was added to 384 well plates and the reaction was performed on an ABI 7300Real time PCR instrument, the reaction procedure being: reverse transcription is carried out at 50 ℃ for 20min; pre-denaturation at 95℃for 5min; the amplification reaction was carried out at 95℃for 15s and at 60℃for 30s for 40 cycles.
The data were processed using Graphpad software to give an IC 50 value of 0.15 μm and the results are shown as B in figure 2.
(3) IFA determination of the fluorescence intensity of SADS-CoV-N protein after treatment of Vero cells with Kenpaullone at 10. Mu.M
1. 1ML of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 12-well cell culture plate, 4 wells were inoculated, and the cells were cultured at 37℃under 5% CO 2 until the cells were completely confluent.
2. The medium in the 12-well plate was discarded, washed 3 times with PBS, and after treatment with Kenpaullone. Mu.M (negative control), 0. Mu.M (positive control), 10. Mu.M (drug-loaded control) and 10. Mu.M (drug-loaded control) DMEM medium for 1 hour, SADS-CoV (MOI=0.1) was added to each of the 4 wells with cells, and the wells were incubated at 37℃under 5% CO 2 for 24 hours.
3. The treatment solution was discarded, washed 3 times with PBST for 5min each, and incubated with 4% paraformaldehyde pre-chilled at 4℃for 15min at room temperature at 500. Mu.L per well.
4. Washing with PBST for 3 times each for 5min, adding 0.4% Triton X-100 solution, and incubating at room temperature for 10-20min.
5. Wash 3 times with PBST for 5min each, add 3% BSA, 500 μl per well, incubate for 1h at room temperature.
6. The SADS-CoV-N protein primary antibody was diluted 1:1000 in PBST solution with 1% BSA 3 times with PBST for 5min, and incubated at 37℃for 1h at 300. Mu.L per well.
7. The primary antibody was recovered and washed 3 times with PBST for 5min each, FITC fluorescent secondary antibody was diluted 1:500 with PBST and incubated at 37℃in the dark for 1h at 300. Mu.L per well.
8. The DAPI fluorochromes were diluted 1:1000 with PBST 3 times each for 5min under light-protected conditions, 300. Mu.L per well incubated at 37℃for 7min in light-protected conditions.
9. Under the condition of avoiding light, PBST is used for washing 5-7 times, 500 mu L of PBST is added to each hole, and fluorescence intensity is observed by a fluorescence microscope and photographed.
As shown in FIG. 2C, the proliferation of SADS-CoV virus after Kenpaullone treatment was detected by indirect Immunofluorescence (IFA), and the specific fluorescence of SADS-CoV-N protein was gradually decreased in comparison with that of the positive control group after 24 hours of drug treatment.
(4) Kenpaullone Effect on SADS-CoV replication on Vero-E6
1. Effect of different concentrations Kenpaullone on SADS-CoV replication
2ML of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 6-well cell culture plate, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The culture medium in the 6-well plate was discarded, washed 3 times with PBS, and the cells were treated with Kenpaullone M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, and 10. Mu.M DMEM medium for 1 hour, and the treated solution was discarded, and SADS-CoV virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and incubated at 37℃under 5% CO 2 for 24 hours.
UsingEZ-press RNA Purification Kit kit extracts RNA according to the instruction, and then according to the/>, of the following holothuriansV C58P2Multiplex One Step RT-qPCR Probekit (UDG Plus) protocol RT-qPCR was performed, and the levels of SADS-CoV-N mRNA expression in cells were detected as in (2).
RNA copy number was calculated from the standard curve and the results are shown in FIG. 2D.
2. Effect of identical concentrations Kenpaullone on SADS-CoV replication at different time points after SADS-CoV infection
2ML of the cell suspension was simultaneously inoculated at a cell concentration of 6X 10 5 cells/mL in 3 6-well cell culture plates, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. At the same time, the medium in the 6-well plate was discarded, washed 3 times with PBS, and the cells were treated with Kenpaullone. Mu.M (negative control), 0. Mu.M (positive control) and 10. Mu.M DMEM medium for 1 hour in each of 3 wells of each plate, the treated solution was discarded, and SADS-CoV virus solution (MOI=0.1) was added to the wells excluding the negative control, and cultured under conditions of 37℃and 5% CO 2. And respectively collecting samples at 12h, 24h and 36h after the virus liquid is added.
RNA extraction and RT-qPCR were performed as described above.
Finally, RNA copy number was calculated from the standard curve, and the results are shown in FIG. 2E.
(5) Kenpaullone Effect on SADS-CoV replication on Vero-E6-western blot
1. Effect of different concentrations Kenpaullone on SADS-CoV replication
2ML of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 6-well cell culture plate, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The culture medium in the 6-well plate was discarded, washed 3 times with PBS, and the cells were treated with Kenpaullone M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, and 10. Mu.M DMEM medium for 1 hour, and the treated solution was discarded, and SADS-CoV virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and incubated at 37℃under 5% CO 2 for 24 hours.
Discarding the treatment solution, washing 3 times with PBS, adding 200 mu L of RIPA lysate into each hole, lysing for 10min on ice, blowing and mixing cell precipitates, transferring to a 1.5mL sterile EP tube, centrifuging for 5min at 12000r/min, sucking 180 mu L of supernatant, mixing with 36 mu L of 5×loading buffer (5-fold concentration loading buffer), and performing water bath at 100deg.C for 10min to obtain a western blot protein sample.
Protein samples were obtained in the same manner at 0. Mu.M (negative control), 0. Mu.M (positive control), 10. Mu. M Kenpaullone, pretreated and infected with SADS-CoV (MOI=0.1) for 12h, 24h, 36 h.
Electrophoresis was performed with 10-well, 10% pre-cast gel, loading sequence: samples were treated with Marker, 0. Mu.M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, 10. Mu. M Kenpaullone.
The program of the electrophoresis apparatus is set for 80V electrophoresis for 30min and 120V electrophoresis for 1h. After electrophoresis, a semi-dry film transfer system is used for assembling the film transfer system, and the assembling sequence is as follows: after the black surface (negative electrode) -sponge-filter paper-albumin glue-PVDF film-filter paper-sponge-white surface (positive electrode) is assembled, bubbles are removed by using a clean test tube or a glass rod, and a film transferring instrument program is set to 15V film transferring for 40min. And taking out the PVDF film after the film transfer, washing for 5min by using TBST, and sealing for 1h at room temperature in 5% skimmed milk powder.
The blocking solution was decanted and washed three times with TBST for 5min each. Then taking out the membrane, wrapping the membrane with a clean preservative film, cutting the membrane containing 46kDa SADS-CoV-N protein and 36kDa GAPDH, placing the membrane in a corresponding primary antibody, placing the primary antibody on a shaking table for 40r/min, and incubating the primary antibody at 4 ℃ overnight. After the incubation, primary antibodies were recovered and washed three times with TBST for 5min each. Two membranes were placed in HRP-labeled goat anti-mouse IgG secondary antibody solution diluted with TBST (dilution ratio 1:10000), placed on a shaking table, 40r/min, and incubated for 1h at room temperature. Recovering secondary anti-dilution liquid, washing the membrane with TBST for 3 times and 5min each time, soaking the membrane in TBST, preparing ECL color development liquid under the condition of avoiding light, and developing and photographing by using a gel scanning system.
The results are shown as F in FIG. 2, and the proliferation of SADS-CoV virus after Kenpaullone treatment was detected using Western Blotting (WB): after Kenpaullone treatments, the corresponding SADS-CoV-N protein size bands gradually fade with increasing drug concentration, and show dose dependence, indicating that Kenpaullone has an inhibitory effect on SADS-CoV proliferation.
2. Effect of identical concentrations Kenpaullone on SADS-CoV replication at different time points after SADS-CoV infection
2ML of the cell suspension was simultaneously inoculated at a cell concentration of 6X 10 5 cells/mL in 3 6-well cell culture plates, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. At the same time, the medium in the 6-well plate was discarded, washed 3 times with PBS, and the cells were treated with Kenpaullone. Mu.M (negative control), 0. Mu.M (positive control) and 10. Mu.M DMEM medium for 1 hour in each of 3 wells of each plate, the treated solution was discarded, and SADS-CoV virus solution (MOI=0.1) was added to the wells excluding the negative control, and cultured under conditions of 37℃and 5% CO 2. Samples are collected at 12h, 24h and 36h after the virus liquid is added respectively.
The procedure for protein extraction and western blot was the same as above. The results are shown as G in fig. 2.
The above results demonstrate that Kenpaullone successfully inhibited the replication of SADS-CoV in passable african green monkey kidney cell line (Vero) and exhibited a dose-dependent, showing the potential of Kenpaullone for the treatment of acute diarrhea syndrome in pigs.
EXAMPLE 3Kenpaullone therapeutic Effect on SADS-CoV infected piglets
(1) Piglet treatment experiment design
Commercial piglets (purchased from Wenshi food group Co., ltd.) of 20 days old 3 days old were tested by ELISA and PCR, and were negative for PEAV, PEDV, PDCoV and TGEV antibodies and antigens, respectively. The random groups were 4 groups (5 heads per group), specifically:
The first group is a blank control group (mock), and each pig orally attacks an equal volume of DMEM with an agent amount, and the administration group starts treatment and simultaneously carries out intravenous injection of an equal volume of empty solvent (DMSO);
The second group is a dosing control group (Kenpaullone), and each pig orally attacks an equal volume of DMEM with an equal volume of Kenpaullone solution in the treatment stage;
The third group is an attack virus group (SADS-CoV), each pig is orally administered with SADS-CoV virus solution of 1X 10 7TCID50 dose, and the administration group is simultaneously and intravenously injected with equal volume of empty solvent (DMSO);
The fourth group was the challenge treatment group (SADS-CoV+ Kenpaullone), each pig was orally administered 1X 10 7TCID50 doses of SADS-CoV virus solution, and a dose of Kenpaullone solution of 6mg/kg body weight was intravenously injected on the first and third days after challenge.
Kenpaullone solution formula: 3.2ml DMSO is dissolved in 300mg Kenpaullone.
Experiment and sampling arrangement:
After the piglets are transported to an experimental site after disinfection, the piglets are randomly divided into 4 groups and placed on different sites, oral stomach-filling and toxin-counteracting are carried out on a third group and a fourth group, and simultaneously, the first group and the second group are orally treated with the same volume of DMEM. Kenpaullone dosing treatments were performed on the first and third days after challenge, and the second and fourth groups of piglets were given intravenous Kenpaullone solutions at a dose of 6mg/kg body weight. The clinical symptoms of each group of piglets, including body weight, diarrhea status, feeding status, mental status, etc., were continuously monitored.
Immediately dissecting the dead pigs after the toxin is attacked, and completely dissecting the rest pigs only on the seventh day after the toxin is attacked:
① Observing intestinal lesions of piglets in an experimental group and a control group, checking whether intestinal wall thinning and transparency occur in intestinal tracts or not, and recording clinical scoring conditions when pathological changes such as water sample contents occur;
② Different piglet small intestine tissues (jejunum, ileum, duodenum) were collected:
One part was kept at 4 degrees after 48h fixation with 10% formaldehyde and sent to the company for paraffin embedded sections for HE staining and immunohistochemistry. Lesions of intestinal tissue were observed under a microscope, including intestinal villus morphology, villus shedding, villus height and crypt depth ratio.
100Mg of intestinal tissue sample is weighed, 1ml of PBS and 2 grinding steel balls are added, balanced, and fully ground in a grinder. After finishing grinding, centrifuging at 4deg.C and 12000r/min for 10min, collecting supernatant, sub-packaging into 200 μl/tube, and preserving at-80deg.C for subsequent extraction of RNA, and detecting viral load in tissue.
(2) Clinical symptoms
One dead pig was present on each of the third, fourth and fifth days during the experiment, and one dead pig was present on the third day during the treatment group, and the survival curves are shown in fig. 3 a. The weight change is shown as B in fig. 3, and the weight loss of piglets in the toxin-attacking group is caused by diarrhea, while the weight of piglets in the toxin-attacking administration group is obviously improved. The clinical scoring results are shown as C in fig. 3, and the results show that Kenpaullone administration treatment significantly weakens diarrhea symptoms of piglets in the treatment group, and the mental states of the piglets are superior to those of piglets in the toxicity attack group.
(3) Ileum, jejunum and duodenum tissue sample viral load detection
Immediately dissecting the dead pigs after the toxicity attack, completely dissecting the rest pigs only on the seventh day after the toxicity attack, collecting three tissue samples of ileum, jejunum and duodenum which are easy to be infected by SADS-CoV and generating lesions, and measuring the mRNA expression level of the SADS-CoV-N by adopting a qRT-PCR method, wherein the method comprises the following steps of:
After tissue thawing, transferring about 1g of tissue sample to a special tube of a tissue breaker, adding 1mL of Trizol, adding sterilized ceramic beads, placing the tissue breaker, carrying out severe shaking and breaking for 3 times and 20 s/time, and then placing the tissue breaker on ice for cracking for 20min; centrifuging at 12000rpm for 5min, transferring supernatant to a clean RNase-free EP tube, adding 200 μl of chloroform, shaking with vigorous vortex, standing for delamination, and centrifuging at 12000rpm for 10min; transferring the supernatant to a new RNase-free EP tube, adding equal volume of isopropanol, mixing well, and precipitating at-20deg.C for 1 hr; centrifuging at 12000rpm for 15min, discarding supernatant to obtain RNA precipitate; rinsing with 75% alcohol for 2 times, air-drying for 5min, adding 50 μl RNase-FREE WATER for dissolving, and measuring concentration and OD value. According to the next holy living things V C58P2Multiplex One Step RT-qPCR Probekit (UDG Plus) protocol RT-qPCR was performed and the levels of SADS-CoV-N mRNA expression in tissue samples were measured as described above.
The results are shown in D in FIG. 3, and the significant reduction of SADS-CoV-N mRNA in the ileum, jejunum and duodenum of piglets after Kenpaullone treatment compared to the challenge group indicates that Kenpaullone has an in vivo inhibitory effect on SADS-CoV replication.
(4) Pathological changes of tissue
Removing tissue blocks not more than 4mm multiplied by 2cm 2 from the collected ileum, jejunum and duodenum, and fixing with 4% paraformaldehyde; alcohol gradient dehydration; the dimethylbenzene is transparent; embedding paraffin; repairing the sheet; slicing; hematoxylin, eosin staining. Intestinal tissue sections were visualized under a mirror.
The results are shown in FIG. 4: the ileum, jejunum and duodenum tissue sections of the administration control group have no lesions, which shows that Kenpaullone has no obvious influence on host organs; compared with a control group, the intestinal tissue section of the toxicity attack group can be seen to have obvious thinning of intestinal walls, intestinal villus Mao Tuola and the like, and belongs to the characteristic pathological phenomenon after acute coronavirus infection of pigs; the thickness of the intestinal wall of the treatment group for counteracting toxic substances is recovered to be normal, and no obvious intestinal villus shedding condition exists. These results indicate Kenpaullone treatment effectively reduced the damage to tissue by SADS-CoV.
In summary, kenpaullone is taken as an inhibitor molecule in a piglet treatment experiment, and has the advantages of no toxicity and no side effect in a dosing control group; in the toxin-counteracting piglets, kenpaullone obviously relieves the symptoms of diarrhea, vomiting and the like of the piglets; and Kenpaullone treatment greatly reduces the viral load in jejunum, ileum and duodenum of infected piglets, and reduces the tissue damage of small intestine. And Kenpaullone has definite structure, no toxicity and no side effect.
Example 4Kenpaullone use of the preparation of a medicament for inhibiting replication of PEDV
(1) Kenpaullone test for detecting cytotoxicity of Vero-E6
Cytotoxicity of Kenpaullone to Vero-E6 was determined using CCK8 cytotoxicity assay, as follows:
a96-well plate was seeded with a 6X 10 5 cells/mL cell suspension (100. Mu.L/well) leaving a column of uninoculated cells. The cells were cultured at 37℃under 5% CO 2 until they were completely confluent. The medium was aspirated, washed 3 times with PBS, kenpaullone dissolved in DMSO and diluted with DMEM medium to different concentrations (0.1. Mu.M, 1. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M), 3 replicates per concentration, 100. Mu.L/well. After incubation at 37℃for 24h with 5% CO 2, the drug solution was aspirated, washed 3 times with PBS, 10% (v/v) CCK8 solution was added to 96-well plates (100. Mu.L/well) with DMEM medium, incubated at 37℃for 1h with 5% CO 2, absorbance (lambda=450 nm) was measured with a microplate reader and cell viability was calculated after 24h treatment with different concentrations of drug solution.
As a result, the maximum safe concentration of Kenpaullone to Vero-E6 was 10. Mu.M, as shown in FIG. 5A.
(2) Kenpaullone IC 50 assay to inhibit PEDV replication
1ML of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 12-well cell culture plate, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The culture medium in the 6-well plate was discarded, washed 3 times with PBS, and cells were treated with DMEM medium at Kenpaullone concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, and 10. Mu.M for 1 hour, and the treated solution was discarded, and PEDV virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and incubated at 37℃under 5% CO 2 for 24 hours.
UsingEZ-press RNA Purification Kit kit extracts RNA according to the instruction, and then according to the/>, of the following holothuriansV C58P2Multiplex One Step RT-qPCR Probe Kit (UDG Plus) protocol RT-qPCR was performed to detect PEDV-N mRNA expression levels in tissue samples. The primer and probe sequences were as follows:
PEDV-N-F：5’-CAGGACACATTCTTGGTGGTCTT-3’(SEQ ID NO.4)；
PEDV-N-R：5’-CAGGACACATTCTTGGTGGTCTT-3’(SEQ ID NO.5)；
PEDV-N-Probe：5’-FAM-ACGCGCTTCTCACTAC-MGB-3’(SEQ ID NO.6)；
the reaction is according to the following holy living beings V C58P2Multiplex One Step RT-qPCR Probe Kit (UDG Plus) instruction manual, the reaction system is: 15 mu L/>V C58P2MP Buffer、1.2μL/>VC58P2 Enzyme Mix, 3. Mu.L Primer/Probe Mix (2.5. Mu.M), 1. Mu.L template RNA, 9.8. Mu. L RNASE FREE H2O. The system was assembled in 3 replicates per sample, 30 μl per well was added to 384 well plates and the reaction was performed on an ABI 7300Real time PCR instrument, the reaction procedure being: reverse transcription is carried out at 50 ℃ for 20min; pre-denaturation at 95℃for 5min; the amplification reaction was carried out at 95℃for 15s and at 60℃for 30s for 40 cycles.
The data were processed using Graphpad software to give an IC 50 value of 0.2 μm and the results are shown as B in figure 5.
(3) Kenpaullone Effect on PEDV replication on Vero-E6-TCID 50
1ML of cells were inoculated per well at a cell concentration of 6X 10 5 cells/mL in a 12-well cell culture plate, and DMEM medium having Kenpaullone concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, 10. Mu.M was added to each of the 5 wells; the treatment liquid is discarded after the medicine treatment for 1 h. PEDV virus solution (moi=0.1) was added to the wells excluding the negative control, and incubated at 37 ℃ under 5% co 2 for 24 hours. Taking out the cell culture plate, sealing with sealing film, freezing and thawing at-80deg.C for 3 times, collecting the frozen and thawed culture in sterile 1.5mL EP tube, centrifuging at 4 deg.C for 5min at 10000r/min with high speed centrifuge, collecting supernatant in new 1.5mL EP tube, and preserving at-80deg.C.
The cells are planted in 5 96-well plates according to the cell concentration of 6 multiplied by 10 5 cells/mL, 100 mu L of each well is cultivated to complete confluence of the cells under the conditions of 37 ℃ and 5 percent CO 2, the 96-well plates with the well cells are washed 3 times by PBS, the collected samples are diluted by 10 times for 0 to 10 -7 times, the diluted samples are added into the cell wells with the corresponding dilution times, 8-well of each gradient is repeated, 100 mu L of each well is cultivated under the conditions of 37 ℃ and 5 percent CO 2 for 5 to 7d.
The virus titer of PEDV infection for 24h after treatment of Vero-E6 cells with different concentrations of PEDV and its inhibitor Kenpaullone was calculated by IFA assay in combination with Reed-Muench method.
As shown in FIG. 5C, kenpaullone treated Vero-E6 cells were able to reduce the viral titer of PEDV and were dose dependent.
(4) Kenpaullone Effect on PEDV replication on Vero-E6
1. Effect of different concentrations Kenpaullone on PEDV replication
2ML of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 6-well cell culture plate, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The culture medium in the 6-well plate was discarded, washed 3 times with PBS, and cells were treated with Kenpaullone cells in each of the 5 wells at a concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, and 10. Mu.M in DMEM medium for 1 hour, the treated solution was discarded, and PEDV virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and incubated at 37℃under 5% CO 2 for 24 hours.
UsingEZ-press RNA Purification Kit kit extracts RNA according to the instruction, and then according to the/>, of the following holothuriansV C58P2Multiplex One Step RT-qPCR Probekit (UDG Plus) protocol RT-qPCR was performed, and the experimental procedure was the same as (2), detecting the level of PEDV-N mRNA expression in cells.
RNA copy number was calculated from the standard curve and the results are shown in FIG. 5D.
2. Effect of identical concentrations Kenpaullone on PEDV replication at different time points after PEDV infection
1ML of the cell suspension was simultaneously inoculated at a cell concentration of 6X 10 5 cells/mL in 3 6-well cell culture plates, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. At the same time, the medium in the 6-well plate was discarded and washed 3 times with PBS, and the cells were treated with Kenpaullone. Mu.M (negative control), 0. Mu.M (positive control) and 10. Mu.M DMEM medium for 1 hour in each of 3 wells of each plate, and the treated solution was discarded, and then PEDV virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and incubated at 37℃under 5% CO 2. And (5) respectively collecting samples at 12h, 24h and 36h after the virus liquid is added.
RNA extraction and RT-qPCR were performed as described above.
Finally, RNA copy number was calculated from the standard curve, and the results are shown in FIG. 5E.
The results of the above experiments demonstrate Kenpaullone that having anti-PEDV activity, using Kenpaullone successfully inhibited PEDV replication in passable african green monkey kidney cell line (Vero-E6) and exhibited dose dependence.
Example 5Kenpaullone therapeutic Effect on PEDV infected piglets
(1) Piglet treatment experiment design
Commercial piglets (purchased from Wen food Co., ltd.) of 20 days old, were tested by ELISA and PCR, and were negative for PEDV, PEDV, TGEV and SADS-CoV antibodies and antigens, respectively. The random groups were 4 groups (5 heads per group), specifically:
The first group is a blank control group (mock), and each pig orally attacks an equal volume of DMEM with an agent amount, and the administration group starts treatment and simultaneously carries out intravenous injection of an equal volume of empty solvent (DMSO);
The second group is a dosing control group (Kenpaullone), and each pig orally attacks an equal volume of DMEM with an equal volume of Kenpaullone solution in the treatment stage;
The third group is a virus attack group (PEDV), 2mL of PEDV virus solution with the titer of 1x10 6TCID50/mL is orally taken by each pig, and the same volume of empty solvent (DMSO) is simultaneously injected intravenously during treatment of the administration group;
The fourth group is a treatment group for challenge, 2mL of PEDV virus solution with a titer of 1x10 6TCID50/mL is orally administered to each pig, and Kenpaullone solution with a dose of 6mg/kg body weight is intravenously injected on the first and third days after challenge.
Kenpaullone solution formula: 3.2mL of DMSO was dissolved in 300mg Kenpaullone.
Experiment and sampling arrangement:
after the piglets are transported to an experimental site after disinfection, the piglets are randomly divided into 4 groups and placed on different sites, oral stomach-filling and toxin-counteracting are carried out on a third group and a fourth group, and simultaneously, the first group and the second group are orally treated with the same volume of DMEM. Kenpaullone dosing treatments were performed on the first and third days after challenge, and the second and fourth groups of piglets were given intravenous Kenpaullone solutions at a dose of 6mg/kg body weight. The survival, clinical symptoms including weight, diarrhea, feeding, mental status, etc. of each group of piglets are continuously monitored.
Immediately dissecting the dead pigs after the toxin is attacked, and completely dissecting the rest pigs only on the seventh day after the toxin is attacked:
① Observing intestinal lesions of piglets in an experimental group and a control group, checking whether intestinal wall thinning, transparency and flatulence occur in intestinal tracts or not, and recording clinical scoring conditions when pathological changes such as water sample contents occur;
② Different piglet small intestine tissues (jejunum, ileum, duodenum) were collected:
One part was fixed with 10% formaldehyde for 48h and stored at 4℃and sent to the company for paraffin embedded sections for HE staining and immunohistochemistry. Lesions of intestinal tissue were observed under a microscope, including intestinal villus morphology, villus shedding, villus height and crypt depth ratio.
100Mg of intestinal tissue sample is weighed, 1mL of PBS and 2 grinding steel balls are added, balanced, and fully ground in a grinder. After finishing grinding, centrifuging at 4deg.C and 12000r/min for 10min, collecting supernatant, sub-packaging into 200 μL/tube, and preserving at-80deg.C for subsequent extraction of RNA and protein, and detecting viral load in tissue.
(2) Clinical symptoms
One dead pig was in each of the third, fourth, fifth, sixth and seventh days of the challenge group during the experiment, and two dead pigs were in the fifth day of the challenge group. The survival curves are shown in figure 6 a. Weight change as shown in fig. 6B, the weight loss of piglets in the challenge group resulted from diarrhea, while the weight of piglets in the challenge administration group was significantly improved. The clinical scoring results are shown in fig. 6C. The results show that Kenpaullone administration treatment significantly reduces diarrhea symptoms of piglets in the treatment group, and the mental states of the piglets are superior to those of piglets in the toxicity attack group.
(3) Ileum, jejunum and duodenum tissue sample viral load detection
Immediately dissecting the dead pigs after the virus attack, completely dissecting the rest pigs only on the seventh day after the virus attack, collecting three tissue samples of ileum, jejunum and duodenum which are easy to be infected by PEDV and lesions, and measuring the expression level of PEDV-NmRNA by adopting a qRT-PCR method, wherein the following specific steps are as follows:
After tissue thawing, transferring about 1g of tissue sample to a special tube of a tissue breaker, adding 1mL of Trizol, adding sterilized ceramic beads, placing the tissue breaker, carrying out severe shaking and breaking for 3 times and 20 s/time, and then placing the tissue breaker on ice for cracking for 20min; centrifuging at 12000rpm for 5min, transferring supernatant to a clean RNase-free EP tube, adding 200 μl of chloroform, shaking with vigorous vortex, standing for delamination, and centrifuging at 12000rpm for 10min; transferring the supernatant to a new RNase-free EP tube, adding equal volume of isopropanol, mixing well, and precipitating at-20deg.C for 1 hr; centrifuging at 12000rpm for 15min, discarding supernatant to obtain RNA precipitate; rinsing with 75% alcohol for 2 times, air-drying for 5min, adding 50 μl RNase-FREE WATER for dissolving, and measuring concentration and OD value. According to the next holy living things V C58P2 Multiplex One Step RT-qPCR Probekit (UDG Plus) protocol RT-qPCR was performed and the level of PEDV-N mRNA expression in tissue samples was measured as described above.
The results are shown in panel D of fig. 6, and the significant reduction of PEDV-N mRNA in the ileum, jejunum, and duodenum of piglets after Kenpaullone treatment compared to the challenge group, indicates that Kenpaullone has an in vivo inhibitory effect on PEDV replication.
(4) Pathological changes of tissue
Removing tissue blocks not more than 4mm multiplied by 2cm 2 from the collected ileum, jejunum and duodenum, and fixing with 4% paraformaldehyde; alcohol gradient dehydration; the dimethylbenzene is transparent; embedding paraffin; repairing the sheet; slicing; hematoxylin, eosin staining. Intestinal tissue sections were visualized under a mirror.
The results are shown in FIG. 7: the ileum, jejunum and duodenum tissue sections of the administration control group have no lesions, which shows that Kenpaullone has no obvious influence on host organs; compared with a control group, the intestinal tissue section of the toxicity attack group can be seen to have obvious thinning of intestinal walls, intestinal villus Mao Tuola and the like, and belongs to the characteristic pathological phenomenon of pigs after epidemic diarrhea infection; the thickness of the intestinal wall of the treatment group for counteracting toxic substances is recovered to be normal, and no obvious intestinal villus shedding condition exists. These results indicate that Kenpaullone treatment effectively reduced the damage to tissue by PEDV.
The experimental results show that Kenpaullone is taken as an inhibitor molecule in a piglet treatment experiment, and has the advantages of no toxicity and no side effect in a dosing control group; in the toxin-counteracting piglets, kenpaullone obviously relieves the symptoms of diarrhea, vomiting and the like of the piglets; and Kenpaullone treatment greatly reduces the viral load in jejunum, ileum and duodenum of infected piglets, and reduces the tissue damage of small intestine; and Kenpaullone has definite structure, no toxicity and no side effect.
Example 6Kenpaullone use of A medicament for inhibiting PDCoV replication
(1) Kenpaullone cytotoxicity test against LLC-PK1
Cytotoxicity of Kenpaullone to LLC-PK1 was determined using the CCK8 cytotoxicity assay, as follows:
Cell suspensions of 6X 10 5 cells/mL (100. Mu.L/well) were seeded in 96-well plates leaving a list of uninoculated cells. The cells were cultured at 37℃under 5% CO 2 until they were completely confluent. The medium was aspirated, washed 3 times with PBS, kenpaullone dissolved in DMSO and diluted with DMEM medium to different concentrations (0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M), 3 replicates per concentration, 100. Mu.L/well. After incubation at 37℃for 24h with 5% CO 2, the drug solution was aspirated, washed 3 times with PBS, 10% (v/v) CCK8 solution was added to 96-well plates (100. Mu.L/well) with DMEM medium, incubated at 37℃for 1h with 5% CO 2, absorbance (lambda=450 nm) was measured with a microplate reader and cell viability was calculated after 24h treatment with different concentrations of drug solution.
As a result, the maximum safe concentration of Kenpaullone to LLC-PK1 was 10. Mu.M as shown in A in FIG. 8.
(2) Influence of Kenpaullone on LLC-PK1 on PDCoV replication-RT-qPCR
2ML of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 6-well cell culture plate and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The culture medium in the 6-well plate was discarded, washed 3 times with PBS, and cells were treated with Kenpaullone cells in concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, 10. Mu.M DMEM medium for 1 hour, the treated solution was discarded, PDCoV virus solution (MOI=0.1) was added to all wells except the wells of the negative control, and cultured at 37℃under 5% CO 2 for 24 hours.
UsingThe EZ-press RNA Purification Kit kit extracts RNA according to the requirements of the specification,
According to TarakaReverse transcription was performed by RT Master Mix (PERFECT REAL TIME) instruction manual, and the product was frozen at-80℃and stored for further use.
And detecting the mRNA expression level of PDCoV-N in the tissue sample by using the cDNA as a template through real-time fluorescence quantitative PCR. The primer sequences were as follows:
PDCoV-N-F：5’-CGTCGTAAGACCCAGCATCAAGC-3’(SEQ ID NO.7)；
PDCoV-N-R：5’-GATTATGCTGTACCCTCGATCGT-3’(SEQ ID NO.8)；
The PCR reaction was performed according to PERFECTSTART GREEN QPCR Supermix specification of full gold, the reaction system was: 1. Mu.L of cDNA, 5. Mu.L of 2X PERFECTSTART GREEN QPCR Supermix, 0.2. Mu. L PASSIVE REFERENCE DYE (50X), 10. Mu.M upstream and downstream primers each 0.2. Mu.L, 3.4. Mu. L RNASE FREE WATER. The system was assembled in 3 replicates per sample, 10 μl per well was added to 384 well plates and the reaction was performed on an ABI 7300Real time PCR instrument, the reaction procedure being: pre-denaturation at 95℃for 2min;95 ℃ for 15s and 61 ℃ for 31min, and 40 cycles are total. RNA copy number was calculated from the standard curve and the results are shown in FIG. 8B.
(3) Effect of Kenpaullone on LLC-PK1 on PDCoV replication-TCID 50
1ML of cells were inoculated per well at a cell concentration of 6X 10 5 cells/mL in a 12-well cell culture plate, and cells were treated with DMEM medium at Kenpaullone concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 1. Mu.M, 5. Mu.M, 10. Mu.M, respectively, for 1 hour in 5 wells with cells; the treatment liquid is discarded after the medicine treatment for 1 h. Then PDCoV virus solution (MOI=0.1) was added to all wells except the wells of the negative control, and the cells were incubated at 37℃under 5% CO 2 for 24 hours. Taking out the cell culture plate, sealing with sealing film, freezing and thawing at-80deg.C for 3 times, collecting the frozen and thawed culture in sterile 1.5mL EP tube, centrifuging at 4 deg.C for 5min at 10000r/min with high speed centrifuge, collecting supernatant in new 1.5mL EP tube, and preserving at-80deg.C.
The cells are planted in 5 96-well plates according to the cell concentration of 6X 10 5 cells/mL, 100 mu L of each well is cultivated to complete confluence under the conditions of 37 ℃ and 5% CO 2, the 96-well plates with the well cells are washed 3 times by PBS, the collected samples are diluted by 10 times for 0 to 10 -7 times of gradient, the diluted samples are added into the cell wells with the corresponding dilution times, 8-well of each gradient is repeated, 100 mu L of each well is cultivated under the conditions of 37 ℃ and 5% CO 2 for 3 to 5d. The virus titer was calculated using the Reed-Muench method.
As shown in FIG. 8C, kenpaullone was able to reduce PDCoV virus titer and was dose dependent after treatment of LLC-PK1 cells.
The above results demonstrate that Kenpaullone has anti-PDCoV activity, successfully inhibits PDCoV replication in passable porcine kidney cells (LLC-PK 1) using Kenpaullone, and exhibits dose dependence.
Example 7Kenpaullone therapeutic Effect on PDCoV infected piglets
(1) Piglet treatment experiment design
The 20 first 3-day-old commercial piglets (purchased from Wen food group Co., ltd.) were tested by ELISA and PCR, respectively, and were negative for SADS-CoV, PEDV, TGEV and PDCoV antibodies and antigens. The random groups were 4 groups (5 heads per group), specifically:
the first group is a blank control group (Mock), and each pig orally attacks an equal volume of DMEM with an equal volume of empty solvent (DMSO) simultaneously injected intravenously when the administration group starts treatment;
The second group is a dosing control group (Kenpaullone), and each pig orally attacks an equal volume of DMEM with an equal volume of Kenpaullone solution in the treatment stage;
The third group is a virus-attacking group (PDCoV), each pig is orally administered with 2mL of PDCoV virus liquid with the titer of 1x10 6 TCID50/mL, and the administration group is simultaneously injected with equal volume of no-load solvent (DMSO) in an intravenous way during treatment;
the fourth group was a challenge treatment group (PDCoV + Kenpaullone) with 2mL of PDCoV virus solution with a titre of 1X10 6TCID50/mL administered orally to each pig, and a dose of Kenpaullone solution of 6mg/kg body weight was intravenously injected on the first and third days after challenge.
Kenpaullone solution formula: 3.2mL of DMSO was dissolved in 300mg Kenpaullone.
Experiment and sampling arrangement:
After the piglets are transported to the experimental site after disinfection, the piglets are randomly divided into 4 groups and placed on different sites, the third group and the fourth group are subjected to oral stomach-filling and toxin-eliminating, and the first group and the second group are subjected to oral DMEM with the same volume. Kenpaullone dosing treatments were performed on the first and third days after challenge, and a second group, fourth group, piglets were given intravenous doses of Kenpaullone solution at a dose of 6mg/kg body weight. Clinical symptoms of each group of piglets, including body weight, diarrhea status, feeding status, mental status, etc., were continuously monitored, and clinical scores were recorded.
Immediately dissecting the dead pigs after the toxin is attacked, and completely dissecting the rest pigs only on the seventh day after the toxin is attacked:
③ Observing intestinal lesions of piglets in an experimental group and a control group, and checking whether pathological changes such as intestinal distension, mesenteric congestion, thinning of intestinal walls, yellow-filled liquid and the like appear in intestinal tracts;
④ Different piglet small intestine tissues (jejunum, ileum, duodenum) were collected:
One part was fixed with 10% formaldehyde for 48h and stored at 4℃and sent to the company for paraffin embedded sections for HE staining and immunohistochemistry. Lesions of intestinal tissue were observed under a microscope, including intestinal villus morphology, villus shedding, villus height and crypt depth ratio.
100Mg of intestinal tissue sample is weighed, 1mL of PBS and 2 grinding steel balls are added, balanced, and fully ground in a grinder. After finishing grinding, centrifuging at 4deg.C and 12000r/min for 10min, collecting supernatant, sub-packaging into 200 μL/tube, and preserving at-80deg.C for subsequent extraction of RNA, and detecting viral load in tissue.
(2) Clinical symptoms
Clinical scores for the treatment group and the challenge group during the experiment are shown as a in fig. 9, with significant reduction in clinical symptoms in the treatment group. The weight loss of the offending group was significantly reduced due to diarrhea and vomiting, and the change in the body weight of each treatment group was shown as B in fig. 9. One dead pig was found in the challenge group on the third, fourth, fifth and sixth days, and one dead pig was found in the challenge treatment group on the third day, and the survival curve is shown as C in fig. 9. The results show that Kenpaullone administration treatment significantly weakens diarrhea symptoms of piglets in treatment groups, and the mental states of the piglets are superior to those of piglets in toxin-attacking groups.
(3) Ileum, jejunum and duodenum tissue sample viral load detection
Immediately dissecting the dead pigs after the virus attack, completely dissecting the rest pigs only on the seventh day after the virus attack, collecting tissue samples of ileum, jejunum and duodenum PDCoV infected with pathological changes, and measuring the mRNA expression level of PDCoV-N by adopting a qRT-PCR method, wherein the method comprises the following steps:
after tissue thawing, transferring about 1g of tissue sample to a special tube of a tissue breaker, adding 1mL of Trizol, adding sterilized ceramic beads, placing the tissue breaker, carrying out severe shaking and breaking for 3 times and 20 s/time, and then placing the tissue breaker on ice for cracking for 20min; centrifuging at 12000rpm for 5min, transferring supernatant to a clean RNase-free EP tube, adding 200 μl of chloroform, shaking with vigorous vortex, standing for delamination, and centrifuging at 12000rpm for 10min; transferring the supernatant to a new RNase-free EP tube, adding equal volume of isopropanol, mixing well, and precipitating at-20deg.C for 1 hr; centrifuging at 12000rpm for 15min, discarding supernatant to obtain RNA precipitate; rinsing with 75% alcohol for 2 times, air-drying for 5min, adding 50 μl RNase-FREE WATER for dissolving, and measuring concentration and OD value. Reverse transcription was performed according to the PRIMESCRIPT RT MASTER Mix (Perfect Real Time) instructions of Taraka, the reverse transcription system being: 2. Mu.L of 5X PRIMESCRIPT RT MASTER Mix,500ng of total RNA, RNASE FREE DDH 2 O to 10. Mu.L of total RNA were added and mixed well, and then reverse transcription was performed on a PCR apparatus, the reaction procedure being: 15min at 37 ℃; and 5s at 85 ℃. Freezing the product at-80deg.C, and storing for use.
And detecting the mRNA expression level of PDCoV-N in the tissue sample by using the cDNA as a template through real-time fluorescence quantitative PCR. The test procedure was as described in example 1 (2), and the copy number of PDCoV-NRNA was calculated for each tissue sample based on the standard curve.
The results are shown in figure 9D, with significantly reduced PDCoV-N mRNA levels in the ileum, jejunum and duodenum of piglets after Kenpaullone treatment compared to the challenge group, indicating that Kenpaullone has an in vivo inhibitory effect on PDCoV replication.
(5) Pathological changes of tissue
Removing tissue blocks not more than 4mm multiplied by 2cm 2 from the collected ileum, jejunum and duodenum, and fixing with 4% paraformaldehyde; alcohol gradient dehydration; the dimethylbenzene is transparent; embedding paraffin; repairing the sheet; slicing; hematoxylin, eosin staining. Intestinal tissue sections were visualized under a mirror.
The results are shown in FIG. 10: the ileum, jejunum and duodenum tissue sections of the administration control group have no lesions, which shows that Kenpaullone has no obvious influence on host organs; compared with a control group, the intestinal tissue section of the toxicity attack group can be seen in pathological phenomena after the infection of pig delta coronavirus such as obvious intestinal villus shedding, shortening and the like; the wall thickness of the intestines of the treatment group for counteracting toxic substances is recovered to be normal, and the conditions of shortening, fusing, falling-off and the like of the intestinal villi are avoided obviously. These results indicate that Kenpaullone treatment effectively reduced PDCoV damage to the tissue.
The results show that Kenpaullone is taken as an inhibitor molecule in a piglet treatment experiment, and has the advantages of no toxicity and no side effect in a administration control group; in the treatment of Kenpaullone of the toxin-counteracting piglets, the symptoms of diarrhea, dehydration and the like of the piglets are obviously relieved; and Kenpaullone treatment greatly reduces the viral load in jejunum, ileum and duodenum of infected piglets, reduces intestinal tissue damage, has definite Kenpaullone structure, and has no toxic or side effect.
Example 8Kenpaullone use of the preparation of a medicament for inhibiting IBV replication
Kenpaullone Effect on IBV replication on chick embryo
(1) Kenpaullone determination of safe concentration on chick embryo
The method comprises the following steps:
Taking 60 9-day-old chick embryos, randomly dividing the chick embryos into 6 groups, namely 50 mu M group, 10 mu M group, 1 mu M group and 0.1 mu M group, injecting a solvent control group (DMSO group) and a blank group; 10 for each group.
Kenpaullone (50. Mu.M, 10. Mu.M, 1. Mu.M, 0.1. Mu.M) was formulated at various concentrations.
Each group of chick embryos is injected with the liquid medicine with the corresponding concentration in the allantoic cavity according to 0.2 mL/piece, the control group is injected with the drug solvent with the same volume, the blank group is not treated, the chick embryos are incubated in a constant temperature incubator at 37 ℃, dead chick embryos in 24h are discarded, and the number of the live chick embryos in each group is recorded for 24h-72h and during the incubation period.
As a result, the maximum safe concentration of Kenpaullone on chick embryos was 10. Mu.M, as shown in Table 1.
TABLE 1
(2) Kenpaullone effect of inhibiting IBV proliferation in chick embryo
60 9-Day-old chick embryos are randomly divided into 6 groups, namely, a virus group, a 10 mu M Kenpaullone group, a 10 mu MKenpaullone + virus group, a 1 mu M Kenpaullone + virus group, a 0.1 mu M Kenpaullone + virus group and a blank group, wherein 10 chick embryos are used for each group.
Kenpaullone (10. Mu.M, 1. Mu.M, 0.1. Mu.M) was formulated at various concentrations.
The virus group is injected with virus liquid in the allantoic cavity according to 0.2 mL/piece, kenpaullone groups are injected with Kenpaullone with equal volume, kenpaullone + virus groups, the medicine and the virus liquid are firstly mixed and then injected into the allantoic cavity, the blank groups are not treated, dead embryos occurring in 24 hours are discarded, and the number of each group of living embryos during 24 hours to 72 hours and incubation period is recorded and observed.
As shown in Table 2, kenpaullone can significantly inhibit IBV viral replication in chicken embryos.
TABLE 2
(3) Kenpaullone Effect on IBV replication in chick embryo-RT-qPCR
Collecting allantoic fluid of 72h in (2), extracting RNA according to specification requirements by using EZBioscience EZ-press RNA Puriification Kit kit, performing reverse transcription according to PRIMESCRIPT RT MASTER Mix (Perfect Real Time) specification operation of Taraka, and freezing the product at-80deg.C for later use.
Using the cDNA as a template, detecting Kenpaullone influence on IBV-NmRNA expression in chicken embryo by real-time fluorescence quantitative PCR, and the primer sequences are as follows:
IBV-N-F：5’-CGAACCGAGACCAAAGTCAC-3’(SEQ ID NO.9)；
IBV-N-R：5’-TCCTTCTTTGGGCGTTGTTG-3’(SEQ ID NO.10)；
The PCR reaction was performed according to PERFECTSTART GREEN QPCR Supermix specification of full gold, the reaction system was: 1. Mu.L of cDNA, 5. Mu.L of 2X PERFECTSTART GREEN QPCR Supermix, 0.2. Mu. L PASSIVE REFERENCE DYE (50X), 10. Mu.M upstream and downstream primers each 0.2. Mu.L, 3.4. Mu. L RNASE FREE WATER. The system was assembled in 3 replicates per sample, 10 μl per well was added to 384 well plates and the reaction was performed on an ABI 7300Real time PCR instrument, the reaction procedure being: pre-denaturation at 95℃for 2min;95 ℃ for 15s and 61 ℃ for 31min, and 40 cycles are total. . RNA copy number was calculated from the standard curve and the results are shown in FIG. 11.
Results show that Kenpaullone can significantly inhibit viral replication of IBV in chick embryos and are dose dependent.
The above results demonstrate Kenpaullone has the activity of inhibiting IBV proliferation, successful inhibition of IBV replication in chick embryos using Kenpaullone, and shows dose dependence.
EXAMPLE 9Kenpaullone therapeutic Effect on IBV infected chicks
(1) Design of chick treatment experiment
80 Chicks with the age of 10 days are respectively detected by ELISA and PCR, and the IBV, the NDV antibodies and the antigens are negative. The random number was divided into 4 groups (20 per group), specifically:
the first group is a blank control group, each chicken is inoculated with sterile PBS with the same dosage as the virus liquid by eye drop and nose drop, and the administration group is simultaneously injected with equal volume of empty solvent (DMSO) by muscle when the treatment is started;
The second group is a dosing control group, each chicken is inoculated with sterile PBS with the same dosage as the virus liquid by eye drop and nose drop, and the same volume of Kenpaullone solution is injected into muscle in the treatment stage;
The third group is a virus attacking group, each chicken is inoculated with 10 3.5EID50 IBV virus liquid by eye and nose dropping, and the same volume of no-load solvent (DMSO) is injected into the muscle during the treatment of the administration group;
The fourth group is a treatment group for challenge, each chicken was inoculated with 10 3.5EID50 IBV virus solution by eye and nose drops, and 7.5mg/kg body weight of Kenpaullone solution was intramuscular injected on the first and third days after challenge.
Kenpaullone solution formula: 3.2mL of DMSO was dissolved in 300mg Kenpaullone.
Experiment and sampling arrangement:
Chickens are transported to the experimental site after disinfection, are randomly divided into 4 groups and placed on different sites, the third group and the fourth group are subjected to eye-dropping and nose-dropping and poison-tapping, and meanwhile, the first group and the second group are subjected to eye-dropping and nose-dropping with equal volume of sterile PBS. Kenpaullone doses of Kenpaullone solution at 7.5mg/kg body weight were administered to the second and fourth groups of chicks intramuscularly on the first and third days after challenge. The chicks of each group were continuously monitored for mental, feeding, drinking, excretion, etc., and clinical scores were recorded.
Immediately dissecting the dead chicken after the toxin is attacked, and totally dissecting the rest chicken on the seventh day after the toxin is attacked:
① Observing the respiratory tract and kidney pathological changes of the chicks in the experimental group and the control group;
② Different chicken lesion tissues (kidney, bronchi) were collected:
100mg of tissue sample is weighed, 1mL of PBS and 2 grinding steel balls are added, balanced, and the mixture is placed into a grinder for full grinding. After finishing grinding, centrifuging at 4deg.C and 12000r/min for 10min, collecting supernatant, sub-packaging into 200 μL/tube, and preserving at-80deg.C for subsequent extraction of RNA, and detecting viral load in tissue.
(2) Clinical symptoms
Clinical scores of chickens according to their clinical symptoms are shown in fig. 12a, and fig. 12B shows the weight change of chickens, and the weight gain of the challenge group is significantly slower. During the experiment, the administration control group and the blank control group have no dead chicken, and the toxicity attack group dies 8 animals from the fourth day; one dead chicken was present on each of the fourth and fifth days of the challenge treatment group, and the survival curve results are shown as C in fig. 12. The results show that Kenpaullone administration treatment significantly reduces respiratory symptoms of the chickens in the treatment group, and the mental state of the chickens in the treatment group is superior to that of the chickens in the toxin-attacking group.
(3) Viral load detection for lesion tissue samples
Immediately dissecting the dead chicken after the virus attack, totally dissecting the rest chicken on the seventh day after the virus attack, collecting three tissue samples of kidney and bronchus which are easy to be infected by IBV and generate pathological changes, and measuring the expression level of IBV-N mRNA by adopting a qRT-PCR method, wherein the following concrete steps are as follows:
After tissue thawing, transferring about 1g of tissue sample to a special tube of a tissue breaker, adding 1mL of Trizol, adding sterilized ceramic beads, placing the tissue breaker, carrying out severe shaking and breaking for 3 times and 20 s/time, and then placing the tissue breaker on ice for cracking for 20min; centrifuging at 12000rpm for 5min, transferring supernatant to a clean RNase-free EP tube, adding 200 μl of chloroform, shaking with vigorous vortex, standing for delamination, and centrifuging at 12000rpm for 10min; transferring the supernatant to a new RNase-free EP tube, adding equal volume of isopropanol, mixing well, and precipitating at-20deg.C for 1 hr; centrifuging at 12000rpm for 15min, discarding supernatant to obtain RNA precipitate; rinsing with 75% alcohol for 2 times, air-drying for 5min, adding 50 μl RNase-FREE WATER for dissolving, and measuring concentration and OD value. Reverse transcription was performed according to the PRIMESCRIPTTM MASTER Mix (Perfect Real Time) instructions of Taraka, the reverse transcription system being: 2. Mu.L of 5X PRIMESCRIPT RT MASTER Mix,500ng of total RNA, RNASE FREE WATER to 10. Mu.L of total RNA were added and mixed well, and then reverse transcription was performed on a PCR apparatus, the reaction procedure being: 15min at 37 ℃; and 5s at 85 ℃. Freezing the product at-80deg.C, and storing for use.
Using the cDNA as a template, real-time fluorescent quantitative PCR was performed to detect the mRNA expression level of IBV-N in tissue samples, and the copy number of IBV-N RNA in each tissue sample was calculated according to a standard curve as described in (3) of example 1.
The results are shown in D in fig. 12, and the IBV-N mRNA levels of Kenpaullone treated chicks kidney, bronchi were significantly reduced compared to the challenge group, indicating that Kenpaullone had an effect of inhibiting IBV replication in vivo.
The results show that Kenpaullone is taken as an inhibitor molecule in the chick treatment experiment, and has the advantages of no toxicity and no side effect in the administration control group; in the toxin-counteracting chickens, kenpaullone treatment obviously relieves the respiratory symptoms of the chickens; and Kenpaullone treatment greatly reduces viral load in kidneys and bronchi of infected chickens. And Kenpaullone has definite structure, no toxicity and no side effect.
Example 10Kenpaullone inhibition of SARS-CoV-2 replication
Effect of different concentrations Kenpaullone on SARS-CoV-2 replication:
1mL of the cell suspension was inoculated at a cell concentration of 6X 10 5 cells/mL in a 12-well cell culture plate, and cultured at 37℃under 5% CO 2 until the cells were completely confluent. The medium in the 12-well plate was discarded, washed 3 times with PBS, and the cells were treated with DMEM medium at Kenpaullone concentration of 0. Mu.M (negative control), 0. Mu.M (positive control), 0.16. Mu.M, 0.32. Mu.M, 0.63. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, and then treated with SARS-CoV-2 virus solution (MOI=0.1) was added to the wells from which the negative control was removed, and cultured at 37℃under 5% CO 2 for 24 hours.
UsingThe EZ-press RNA Purification Kit kit extracts RNA according to the requirements of the specification,
According to the next holy living thingsV C58P2Multiplex One Step RT-qPCR Probekit (UDG Plus) protocol RT-qPCR was performed, and the level of SARS-CoV-2-N mRNA expression in cells was detected (cf. SARS-CoV-2Mpro inhibitors with antiviral activity in a transgenic mouse model) as described above.
RNA copy number was calculated from the standard curve and the results are shown in FIG. 13. The results show Kenpaullone that inhibited SARS-CoV-2 replication.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. Use of kenPardone or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention and/or treatment of a disease caused by coronavirus; the coronavirus comprises at least one of PEDV, PDCoV, IBV, SARS-CoV-2 and SADS-CoV.
2. The use according to claim 1, wherein the disease caused by coronavirus comprises at least one of diarrhea, bronchitis, pneumonia.
3. Use of Kenparone or a pharmaceutically acceptable salt thereof in the preparation of a coronavirus inhibitor; the coronavirus comprises at least one of PEDV, PDCoV, IBV, SARS-CoV-2 and SADS-CoV.
4. Use according to any one of claims 1 to 3, wherein the medicament or inhibitor further comprises an active ingredient for preventing and/or treating diseases caused by coronaviruses and/or inhibiting the proliferation of coronaviruses.
5. The use according to any one of claims 1 to 3, wherein the medicament or inhibitor further comprises a pharmaceutically acceptable adjuvant.
6. The use according to claim 5, wherein the pharmaceutically acceptable excipients comprise: at least one of a diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, co-solvent, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, bacteriostat or buffer.
7. The use according to any one of claims 1 to 3, wherein the pharmaceutical or inhibitor dosage form comprises at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, an oral preparation, a suppository, an enema, an aerosol, a patch or a drop.
8. The use according to any one of claims 1 to 3, wherein the route of administration of the drug or inhibitor comprises at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration or transdermal administration.
9. A product comprising as a major component colpitone or a pharmaceutically acceptable salt thereof; the function of the product is to prevent and/or treat diseases caused by coronaviruses and/or inhibit the proliferation of coronaviruses; the coronavirus comprises at least one of PEDV, PDCoV, IBV, SARS-CoV-2 and SADS-CoV.
10. The product of claim 9, wherein the product comprises a drug or an inhibitor.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118121609A true CN118121609A (en) | 2024-06-04 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | Novel genotype of infectious bronchitis virus isolated in China | |
| CN103451158B (en) | The substance and method controlled for respiratory disease in canid | |
| An et al. | Novel goose-origin astrovirus infection in geese: the effect of age at infection | |
| Grgiæ et al. | Pathogenicity of infectious bronchitis virus isolates from Ontario chickens | |
| Wang et al. | Comparative study on physical–chemical and biological characteristics of grass carp reovirus from different genotypes | |
| KR20110038181A (en) | Infectious bronchitis vaccines derived from ib-qx-like strains | |
| CN114794018A (en) | Mumps virus counteracting animal model and establishment method thereof | |
| Zhou et al. | Characterization of an avian infectious bronchitis virus isolated in China from chickens with nephritis | |
| US20120198578A1 (en) | Viral assay | |
| WO2021196666A1 (en) | Use of homoharringtonine in preparation of human body beta-coronavirus replication inhibitor | |
| Feng et al. | Combined effect of Shegandilong granule and doxycycline on immune responses and protection against avian infectious bronchitis virus in broilers | |
| CN105802921B (en) | Recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein and construction method and application thereof | |
| Gao et al. | Inhibitory effects of recombinant porcine interferon-α on porcine transmissible gastroenteritis virus infections in TGEV-seronegative piglets | |
| CN114796193B (en) | Traditional Chinese medicine monomer for resisting bovine viral diarrhea virus | |
| CN118121609A (en) | Application of Kenparone in preparation of medicines for preventing and treating coronavirus infection | |
| Hewson et al. | The presence of viral subpopulations in an infectious bronchitis virus vaccine with differing pathogenicity–a preliminary study | |
| Sunjian et al. | Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis | |
| He et al. | Attenuation of a Highly Pathogenic Porcine Deltacoronavirus Strain CZ2020 by a Serial Passage In Vitro | |
| CN105624122A (en) | Avian infectious bronchitis virus natural mutant | |
| Tsai et al. | Duck hepatitis | |
| Cowley et al. | Molecular biology and pathogenesis of roniviruses | |
| WO2014061914A1 (en) | Infectious bronchitis virus k40/09 strain, and vaccine for infectious bronchitis using same | |
| Rafique | Seroprevalence and molecular characterization of Infectious bronchitis virus variants from poultry in Pakistan | |
| CN114410594B (en) | Avian infectious bronchitis virus suitable for cell replication and proliferation and application thereof | |
| Rasool et al. | Novel Coronavirus (2019-nCoV) Outbreak in China: From Local Epidemics to Global Pandemics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication |